ES2393930T3 - TALAROMYCES EMERSONII strain and uses thereof - Google Patents

Abstract

A thermophilic strain of Ta / aromyces emersonii, with the deposit ND IMI 393751 or a mutant thereof with a growth temperature range of 30 to 90 'C and that actively secretes enzymes at temperatures above 55' C.

Description

Ta strain / aromyces emersonii and uses thereof

Introduction��

The present invention relates to a strain of Ta / aromyces emersonii and enzymes and enzymatic systems that

5 can be isolated from it, for use in the environment and waste treatment, chemical and biochemical production and processing, biotechnological procedures, in test or diagnostic kits, prebiotics and symbiotics, sanitary products, new and functional foods and beverages production of surfactants, agricultural and horticultural applications.

Europe is currently facing a crisis in terms of waste treatment producing 2,000 million

10 tons of waste every year. Many of these wastes are organic, derived from plant materials (biomass), are rich in carbohydrates (sugars) and therefore represent a valuable resource when they are broken down into their component sugars. Other types of waste, such as fruit waste, rot and pose an environmental risk. These wastes are usually combined with pig feed to extract some value from them, however, they are considered low cost waste.

15 Biomass represents a very varied and variable raw material, however it is the most renewable energy raw material on earth. If taken advantage of, it could provide a sustainable alternative to the increasingly depleted fossil fuel reserves. There is a global interest in converting the biomass energy reserve into usable forms of energy. Although an important focus of attention has been the production of biofuels, such as bioethanol, for transportation purposes, markets have also been identified for co

20 valuable products generated during the production of biofuels (for example CO2, residues rich in lignin, chemical raw materials). Currently, in the United States, approximately annually occur

11,360 million liters of ethanol from corn, mainly for use in the transport fuel sector. This represents only approximately 1% of total engine fuel consumption, and it is expected that in 2010, bioethanol production will increase more than 7 times, and will include other biomass substrates 25 such as woody debris. If the woody remains derive from sources of rapidly renewable "wastes" generally considered "weeds" or brushwood, considerable value can be derived both in terms of the costs of the procedure, and in the fulfillment of production targets and local environmental problems. The advantages of bioethanol as a clean and ecological alternative to oil and other fossil fuels are obvious. The global adoption of bioethanol as a main engine fuel would compensate for many of the problems of

30 air pollution that are a characteristic of densely populated urban areas. Modern cars can be easily adapted to work with ethanol / gasoline mixtures ("gasohol") and new engines are available that, as the sole source of fuel, can use pure ethanol.

Vegetable biomass, including species of conifers, is rich in complex carbohydrates (polysaccharides) that can be broken down by chemical or enzymatic means into simple fermentable sugars. For example, 35 softwoods such as those of Sitka spruce and pine contain (% dry weight) about 41-43% cellulose (a polymer of glucose units linked by β-1,4), 20-30% of hemicellulose (a mixture of polysaccharides containing mannose, galactose, xylose and arabinose) and 25-30% lignin, a non-carbohydrate polyphenolic polymer of high calorific value. Therefore, approximately 65-70% of the dry weight of the woody debris are complex carbohydrates that can be used to provide sugar-rich raw materials for

40 fermentation to bioethanol.

Fungi represent one of the most important microbial life forms that break down such materials. Ta / aromyces emersonii is an aerobic thermophilic fungus found naturally in compost piles and in other ecosystems that degrade biomass-rich materials. Thermal stability is a typical feature of many of the enzymatic systems of Ta / aromyces emersonii isolated so far. Considered a kind of

45 "soft rot", which can be directed to all parts of the plant material, this euascomycete produces extensive carbohydrate modifying enzyme systems, including cellulolytic, hemicellulitic, pectinolytic and amylolytic enzymes, as well as a series of oxidase / oxide reductase and proteolytic activities. Therefore Ta / aromyces emersonii can access complex growth substrates found in their natural habitat.

McCarthy et al. (2005) J. Ind. Microbiol. Biotechnol 32: 125-134 reveal a comparison of strains of

50 Ta / aromyces emersonii producing beta-glucanase mutants by UV and wild type with potential in brewing applications. Moloney et al. (1983) Enzyme Microb. Technol 5: 260-264 disclose the isolation of CBS 814.70 mutants from Ta / aromyces emersonii with enhanced cellulase activity. Maheswari et al. (2000) Microbiol. Mol. Biol. Rev. 64: 461-488 reveal that the optimum and maximum growth temperature for Ta / aromyces emersonii is respectively 40-45 and 55 DC.

55 PCT Publications Nos WO 01/70998 and WO 02/24926 disclose the isolation of Ta / aromyces emersonii cellulases and in particular cellulases having β-glucanase and xylanase activity. The disadvantage of these enzymes is that they can only target one type (or a limited number) of a constituent (or constituents) of a substrate. Therefore, to metabolize a specific substrate, it is necessary to identify the components of that

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substrate, produce polypeptides that have adequate enzymatic activity to metabolize these components, produce a necessary amount of the polypeptide by routine procedures, such as, recombinant techniques, modify the polypeptides, if necessary, to modify the thermostability or the optimum pH, for example, of the enzymes, express the polypeptide and use the resulting enzyme to target that constituent of that substrate. These procedures are very expensive and time consuming.

In addition, these procedures do not take into account that some enzymes may show more than one activity or, when used in combination with another enzyme, may have modified efficacy. Therefore, these procedures could lead to overproduction of enzymes that are not required, thus also leading to excessive spending and time costs. There is therefore a need for enzymes and enzymatic systems isolated from Ta / aromyces emersonii and a method for isolating such enzymes and enzyme systems that overcomes the aforementioned disadvantages.

There is also a need for optimized enzyme compositions, particularly thermostable enzyme compositions, to generate "syrups" rich in fermentable sugars, for use in biomass to bioethanol projects. Since the substrates of biomass or target raw materials for the production of bioethanol can vary from waste streams (for example VFCW and OFMSW (Vegetable Fraction of Waste Collected and Organic Fraction of Municipal Solid Waste) to agricultural crops (for example, corn, sugar beet, herbs, etc.) to woody biomass, the achievement of the correct enzymatic preparation, at a low process cost, to obtain maximum yield of fermentable sugars is a significant challenge.For many years, the cost of enzymes used in The conversion of biomass before the production of bioethanol by microbial fermentation has been an important negative factor, therefore it is an object to produce low cost enzymatic preparations for these purposes.

Enzymatic preparations are derived from thermophilic fungal sources, generally considered safe (GRAS), while fungal enzymes in use so far in commercial and research bioethanol applications are mainly from mesophilic sources (for example Trichoderma sp. G / ioc / adium sp ., Aspergi // us sp. And Penici // ium sp.).

There is also a need for enzymatic preparations that act at higher reaction temperatures, that is, thermostable enzymes allow shorter reaction times / enzymatic treatment steps, allow simultaneous pasteurization of the hydrolyzate, result in significant global hydrolysis / saccharification , have a potential to reduce the enzymatic load and / or a potential to recycle the enzyme preparation, all of which can serve to reduce costs associated with the use of these enzymes.

Decayation�d� �aa�invention

The present invention relates to a strain of Ta / aromyces emersonii that was deposited with the International Micological Institute (CABI Bioscience United Kingdom) on November 2, 2005 with the number IMI 393751.

The advantage of using Ta / aromyces emersonii as the enzymatic source is that it is a "generally considered safe" (GRAS) microorganism and has a long history of use in the food, beverage, agricultural and pharmaceutical feed sectors.

The advantage of this new strain of Ta / aromyces emersonii is that the enzymes derived from it have an optimal activity at temperatures between 54 DC and 85 DC, keeping some enzymes activity at temperatures up to 95 DC. These enzymes will therefore retain activity even when high processing temperatures are desired or required, for example, production of raw materials rich in sugars for biochemical, biopharmaceutical, chemical or biofuel production (ethanol and methane), in which the Reaction temperatures of 65-90 DC facilitate higher simultaneous reaction rates, faster substrate conversion and simultaneous raw material pasteurization, which improves storage and transportation potential. Additionally, since with these enzymes higher temperatures can be used, each procedure has a shorter reaction time so that there is a saving of both time and costs. An additional advantage is that if the temperature is high enough, pasteurization will occur destroying any undesirable microorganism that cannot withstand such high temperatures. Additionally it has been shown that purified enzymes from this strain have a longer shelf life.

The strain of the invention can provide an enzymatic system comprising a cellobiohydrolase I or a cellobiohydrolase II or a mixture thereof, a β-glucosidase 1, a xylanase and an endo-β- (1,3) 4-glucanase. The invention also provides a method for using this microorganism for plant bioconversion.

or materials derived from plants, such as materials from virgin plants of terrestrial and marine origin, and waste streams thereof, including remnants of coffee, tea, fermentation of beer and beverages, fruit and fruit skins / peeling, vegetable peeling , food and hospitality processing, leaf / vegetable waste, floral waste, cereals and cereal processing remains, other agricultural and garden waste, production waste and bakery shops, paper products such as satin and newspaper magazines in color, black and white newspapers, white, colored and recycled paper, brown paper, paper bags, cardboard and cardboard, paper cups and plates, paper cupcakes, wipes, cellophane and Sellotape® (adhesive tape), packaging biodegradable,

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hospital waste rich in cellulose, such as bandages, papers, wipes, bandages, masks, textiles such as pajamas and towels and for later use in the production of biofuel (bioethanol and biogas).

The strain of the invention can produce an enzymatic system comprising a cellobiohydrolase I or a cellobiohydrolase II or a mixture thereof, a β-glucosidase 1, a xylanase and an endo-β- (1,3) 4-glucanase. The invention also provides a method for using this strain in the production of raw materials rich in monosaccharides from plant residues for the generation of high value products, for example antibiotics, antibiotics and antivirals, carotenoids, antioxidants, solvents and other chemical compounds. and biochemicals, including ingredients for food use, additives for cosmetics, oligosaccharides and glycopeptides for research and functional glycolics.

The invention provides a thermophilic strain of Ta / aromyces emersonii, which has a growth temperature range of 30 to 90 DC, with an optimal range of 30 to 55 DC and that actively produces enzymes at temperatures above 55 DC. The strain of Ta / aromyces emersonii was deposited with the deposit number IMI 393751. The invention relates to a mutant thereof that also encodes thermostable enzymes, an enzyme produced by the strain that retains activity at temperatures above 55 DC. The enzyme can be selected from the group consisting of carbohydrate modifying enzymes, proteolytic enzymes, oxidases and oxidoreductases.

The strain of the invention can produce an enzymatic composition comprising a cellobiohydrolase I or a cellobiohydrolase II or a mixture thereof, a β-glucosidase 1, a xylanase and an endo-β- (1,3) 4-glucanase. The enzymatic composition may comprise 0.5 to 90% cellobiohydrolase I or a cellobiohydrolase II or a mixture thereof, 0.1 to 33% β-glucosidase 1, 0.6 to 89% xylanase and 0.4 to 68% of endoβ- (1,3) 4-glucanase.

The strain of the invention can also produce an enzyme system comprising CBH I (10-30%), CBHII (1015%), β- (1,3) 4-glucanase (20-45%), β-glucosidase (2 -15%) and xylanase (18-55%). The composition may further comprise one or more of the following: β-xylosidase, a-glucuronidase, exoxylanase, α-L-arabinofuranosidase, pectinolytic enzymes, hemicellulases, starch modifying enzymes, oxidoreductase oxidase and esterases; and proteases.

The invention also provides a method for using this microorganism in the processing and recycling of construction wood, wood and wood products. It is effective against highly lignified woody materials, and is resistant to possible inhibitory molecules present in woody debris and processed materials (for example, extractants, resins, lignin decomposition products, hydroxifurfural and furfural derivatives).

The strain of the invention can also produce an enzyme system comprising CBH I (15-30%), CBH II (1040%), β (1,3) 4-glucanase (15-40%), β-glucosidase (2 -15%), xylanase (15-30%) and 1-8% of β-xylosidase. The composition may also comprise one or more of the following: exoxylanase; α-glucuronidase; α-Larabinofuranosidase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogolacturonase and galactanase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase oxidase and esterases. The invention also provides a method for using this strain in textile processing and recycling. The strain of the invention can also produce an enzyme system comprising CBH I (5-55%), CBH II (8-50%), β (1,3) 4-glucanase (10-30%), β-glucosidase (0.5-30%), xylanase (5-30%) and β-xylosidase (0.1-10%). The composition may also comprise one or more of the following: α-Larabinofuranosidase; α-glucuronidase; other hydrolases, including selected pectinolytic enzymes, esterases; protease; and oxidases. The invention also provides a method for using this strain system in saccharification of paper waste.

The strain of the invention can also produce an enzyme system comprising CBH I (2-10%), CBH II (210%), β (1,3) 4-glucanase (10-45%), β-glucosidase (5 -10%) and Xylanase (1-30%). The composition may additionally comprise one or more of the following: N-acetylglucosaminidase; chitinase; β (1,3) 6-glucanase; β-xylosidase, αglucuronidase; α-L-arabinofuranosidase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase / oxidase and esterases; and protease. The invention also provides a method for using this strain in antifungal, biocontrol and sludge control strategies in the environmental, medical and construction sectors (for example combating dry rot), and in the pulp and pulp industry. paper (eg mud control).

The strain of the invention can also produce an enzyme system comprising CBHI (1-20%), CBH II (128%), β (1,3) 4-glucanase (15-40%), β-glucosidase (2- 15%), xylanase (18-55%), β-xylosidase (0.1-10%) and α-Larabinofuranosidase (0.5-5.0%). The composition may also comprise one or more of the following; αglucuronidase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase / oxidase and esterases; protease; exoxylanase; other hydrolases, including pectinolytic enzymes, phenolic acid and acetyl (xilan) esterases; protease; and lignin modifying oxidase activities. The invention also provides a method for using this strain in horticultural applications, for example, for the production of new

Four. Five

Bioactive compounds for the promotion of growth and disease resistance. For example, this strain can be used for the release of bioactive flavonoid glycosides, for the production of oligogalacturonides from materials rich in pectin, xyloglucooligosaccharides from xyloglucans, galactooligosaccharides from galactans, substituted xylooligosaccharides from plant xylanes, or 1,3-glucooligosaccharide-dehydrocarbonacerosides fungal cell wall glucan, or algal laminaran, to promote plant growth, activation of plant defense response mechanisms against plant pathogens and increase disease resistance, since some of these oligosaccharides (for example, laminaryl oligosaccharides) can initiate and mediate the bioactive properties that are antifungal, antibacterial and antinematode.

The strain of the invention can also produce an enzyme system comprising CBH I (5-30%), CBHII (115%), β (1,3) 4-glucanase (10-40%), β-glucosidase (2- 15%), xylanase (18-48%), 0.1-20% of β-xylosidase, 1-10%, αglucuronidase and 0.1-5.0% of α-L-arabinofuranosidase. The composition may also comprise one or more of the following: exoxylanase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase oxidase and esterases and protease. The invention also provides a method for using this strain in the production of animal feed to enhance the digestibility of cereal-based feed. This strain degrades the fibrous components of cereal-based feed to oligosaccharides and monosaccharides (simple sugars), some of which are absorbed in the intestine and metabolized. The strain also produces 'prebiotic' oligosaccharides (e.g., mixed-bond glucooligosaccharides of non-cellulosic β-glucans) that reinforce the growth of prebiotic bacteria, and can release antioxidants, for example, ferulic acid, and produce oligosaccharides (glucurono- xylooligosaccharides substituted from cereal xylanes) that have an antibacterial effect on species of the intestinal microflora that are known to produce carcinogenic molecules (for example phenols, amines, etc.).

The strain of the invention can also produce an enzyme system comprising CBH I (0.5-10%), CBH II (0.5-10%), β (1,3) 4-glucanase (15-43%) , β-glucosidase (2-10%), xylanase (30-88%), 0.1-2.0% of β-xylosidase, 0.13.0% of α-glucuronidase, 0.1-4.0 % of α-L-arabinofuranosidase. The composition may also comprise one or more of the following: pectonolytic enzymes; modified starch activity; oxidoreductase / oxidase and esterases; and protease. The invention also provides a method for using this strain in the production of cereal-based feed containing low pentose content for monogastric animals with improved digestibility and low non-cellulosic β-glucan content. Non-cellulosic β-glucans and cereal arabinoxylans have a high capacity to bind water and generate highly viscous solutions. Monogastric animals (for example pigs and poultry) cannot degrade these carbohydrates, which alter the uptake and bioavailability of important nutrients, increase the diffusion of digestive enzymes, alter the proper mixture of intestinal contents and act as a physical barrier to degradation of the proteins and starch present in these feeds. In general, these polysaccharides can lead to poor growth performance characteristics. The strain in the present invention can catalyze the degradation of cereal arabinoxylans and non-cellulosic β-glucans to produce oligosaccharides (mainly DP3-10), some of which have possible probiotic properties, without the release of pentose sugars (arabinose and xylose ), that monogastric animals metabolize poorly and may have antinutritional effects. A procedure is provided for using alternative cereals such as feed, for example, sorghum and corn.

The strain of the invention can produce an enzyme system comprising CBH I (3-15%), CBH-II (3-15%), (1,3) 4-glucanase (25-45%), α-glucosidase ( 2-15%), xylanase (18-55%), 0.5-7.0% β-xylosidase, 0.5-10% of αglucuronidase and 0.1-5.0% of α-L-arabinofuronosidase. The composition may also comprise one or more of the following: exoxylanase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other amicellulases, including galactosidase; oxidoreductase / oxidase and esterases; and protease. The invention also provides a method for using this strain in the production of functional feeds with bioactive potential for use in human and veterinary health care. This strain can produce bioactive oligosaccharides from raw materials with GRAS status for use in animal health care (companion and larger animals), including β-glucooligosaccharides immunostimulators of plants and terrestrial and marine fungi, xylo-oligosaccharides with prebiotic properties and antimicrobials of terrestrial and marine plants, chitooligosaccharides with growth promotion, antimicrobial and antiviral potential of fungal and crustacean cell walls, and phenolic compounds with antioxidant potential.

The strain of the invention can produce an enzyme system comprising CBH I (1-15%), CBHII (1-15%), β (1,3) 4-glucanase (10-45%), α-glucosidase (2 -10%), xylanase (1-55%), 0.5-12% of β-xylosidase. The composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuronosidase; β (1,3) 6glucanase; N-acetylglucosaminidase; chirinase, pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase / oxidase and esterases; protease The invention also provides a method for using this strain in the production of specialized dairy or dietary products, for example, food products and beverage formulations for geriatric and children's health care. For example, this strain can be used for the production of easily digestible food products, rich in prebiotics, for geriatric and infant nutrition, and also for the production of lactose-free products for

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individuals with galactosemia or for those who are lactose intolerant.

The strain of the invention can produce an enzyme system comprising CBH I (1-10%), CBH II (5-15%), β (1,3) 4-glucanase (15-40%), α-glucosidase ( 2-30%), xylanase (15-55%), 1-12% of β-xylosidase, 1-8% of αglucuronidase and 0.5-5.0% of α-L-arabinofuronosidase. The composition may also comprise one or more of the following; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase / oxidase and esterases; protease The invention also provides a method for using this strain in the bakery and confectionery sectors, and in the formulation of new healthy food bakery products. The strain is suitable for the use of new raw materials / cereals, such as rye, corn and sorghum. The strain can increase the volume of the loaf of bread and enhance expiration by selectively modifying the arabinoxylan components of cereal flours, generating prebiotic oligosaccharides and immunostimulators and effecting the release of antioxidant molecules (for example ferulic and coumaric acids), and modifying the texture, aroma and sensory properties of bakery and confectionery products.

The strain can also produce an enzyme system comprising CBH I (1-20%), CBH II (140%), (1-40%), β (1,3) 4-glucanase (15-45%), α -glucosidase (2-30%), xylanase (10-55%), 0.5-10% of β-xylosidase, 0.1-5% of α-Larabinofuranosidase. The composition may also comprise one or more of the following: β (1,3) 6-glucanase; N-acetylglucosaminidase; chitinase α-glucuronidase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; oxidoreductase / oxidase and esterases; protease A procedure is provided for using this strain in the generation of flavor, aroma and sensory precursor compounds in the food industry by releasing monosaccharides and disacarids that can be fermented in a variety of products including citric acid, vanillin, etc., releasing aromatic glycoconjugates (precursors of the aroma) of fruit / fruit pulps (for example, glycosylated aromatic alcohols found in various fruits, including melon, as well as, geraniol, limonene, etc.), peptides with salty, bitter and sweet flavors, amino acids for the transformation into sweeteners (for example, phenylalanine) and phenolic molecules (cinnamic acids, flavonoid glycosides such as quercetin-3-Oramnoside), which have flavor, aroma or sensory properties or are precursors of such products, for example, rhamnose, which can be bioconverted to furaneol , a strawberry flavored molecule. In addition, some of these molecules, such as flavonoid glycosides have antioxidant and antimicrobial (anti-protozooary) activity.

The strain can also produce an enzyme system comprising CBH I (1-15%), CBH II (1-15%), β (1,3) 4glucanase (10-45%), β-glucosidase (2-30% ), xylanase (1-55%), 0.5-12% of β-xylosidase. The composition may also comprise one or more of the following: α-L-arabinofuronosidase; β (1,3) 6-glucanase; Nacetylglucosaminidase; chitinase α-glucuronidase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases, including galactosidases; oxidoreductase / oxidase and esterases; and protease. The invention also provides a method for using this strain for the generation of functional foods, specifically, the modification of vegetable carbohydrates (terrestrial and some marine) to generate food products with enhanced health promoting properties, for example, food products enriched in glycooligosaccharides. immunostimulators, xylooligosaccharides, soy oligosaccharides, (arabino) gatactooligosaccharides (galacto) and / or (gluco) manooligosaccharides, gentiooligosaccharides, isomaltooligosaccharides and galatinose oligosaccharides, and promote the release of antioxidant molecules such as carotenoids and kinetic carotenoids (caotenoids, carotenoids, and carotenoids) flavonoid glycosides).

The strain can produce an enzyme system comprising CBH I (1-15%), CBH II (1-15%), β (1,3) 4-glucanase (10-45%), β-glucosidase (1-15 %), xylanase (1-30%), 0.5-20% of β-xylosidase. The composition may also comprise one or more of the following: α-L-arabinofuronosidase; β (1,3) 6-glucanase; N-acetylglucosaminidase; chitinase; α-glucuronidase; pectinolytic enzymes, including galactosidases, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; oxidoreductase oxidase and esterases; protease The invention also provides a method for using this strain for the production of new alcoholic and non-alcoholic design beverages, fruit juices and healthy drinks. This system can modify β- (1,3) 4-glucans, pectic substances, arabinos, xylans, lactose, proteins and phenolic substances to generate low-calorie alcoholic and non-alcoholic blond beers / beers, fruit juices and healthy drinks with potential sensory, antioxidant, immunoreformer, antibacterial and improved antiviral, for example, a "light" beer with low residual β-glucan content to prevent turbidity formation (but sufficient βglucan to provide organoleptic characteristics) containing bioactive mixed-linked glucooligosaccharides and cinnamic acids (antioxidants), or a fruit juice rich in pectin fragments (oligosaccharides), which have a prebiotic effect, and cinnamic acids and flavonoid glycosides that provide antioxidant potential.

The strain can produce an enzyme composition CBH I (1-25%), CBH II (1-28%), β (1,3) 4-glucanase (18-40%), βglucosidase (2-30%), xylanase (15-55%) and β-xylosidase (0.7-20%). The composition may further comprise one or more of the following: α-glucuronidase (1-10%), α-L-arabinofuronosidase (0.1-5.0%), 1-15% exoxylanase, 5-25% pectinolytic enzymes, 2-12% starch modifying activity, 2-11% hemicellulases, 1-15% oxidoreductase / oxidase and esterases and 2-15% protease. The composition can be used in the production of

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raw materials rich in monosaccharides from plant residues.

The strain can produce an enzymatic composition comprising CBH I (12-55%), CBH II (15-30%), β (1,3) 4glucanase (12-26%), β-glucosidase (5-12%) , xylanase (5-30%), β-xylosidase (0.1-10%) and α-L-arabinofuronosidase (0.5-3.0%). The composition may further comprise one or more of the following: α-glucuronidase, other hydrolases including pectinolytic enzymes, phenolic acid and acetyl (xylan) esterases, protease and lignin modifying oxidase activities, proteases and oxidases. The composition can be used in the processing and recycling of wood, paper and paper products.

The strain can produce an enzymatic composition comprising CBH I (3-15%), CBH II (3-15%), β (1,3) 4glucanase (15-45%), β-glucosidase (1-15%) , xylanase (16-55%) and β-xylosidase (0.5-7%). The composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuronosidase, exoxylanase, pectinolytic enzymes, enzymes with starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases and proteases. The composition can be used in the production of biopharmaceutical compounds, such as bioactive oligosaccharides (including 1,3 (4) and 1,3 (6) -glucooligosaccharides mixed bond, galactooligosaccharides, xyloglucooligosaccharides, pectical oligosaccharides, branched xylooligosaccharides, (galacto ) glucomanooligosaccharides, glycopeptides and flavonoid glycosides of terrestrial and marine plants, plant residues, fungi and wastewater flows or secondary products rich in simple sugars.

The strain can produce an enzymatic composition comprising CBH I (1-20%), CBH II (1-40%), β (1,2) 4glucanase (15-45%), β-glucosidase (2-12%) , xylanase (1-35%) and β-xylosidase (1-5%). The enzymatic composition may also comprise one or more of the following; α-glucuronidase, α-L-arabinofuronosidase, exoxylanase, pectinolytic enzymes, starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases and proteases. The composition can be used to increase the bioavailability of biomolecules with natural antibacterial and antiviral activity, including flavonoid and cyanogenic glycosides, saponins, oligosaccharides and phenolics (including ferulic and p-coumaric acids, epicatechin, catechin, pyrogallic acid and the like).

The strain can produce an enzymatic composition comprising CBH I (3-15%), CBH II (3-15%), β (1,3) 4glucanase (25-45%), β-glucosidase (2-15%) , xylanase (10-30%) and β-xylosidase (0.5-8%). The enzymatic composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuronosidase, exoxylanase, pectinolytic enzymes, starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases and proteases. The composition can be used to increase the bioavailability of natural antioxidant biomolecules, for example, carotenoids, lycopenes, xanthophylls, anthocyanins, phenolics and glycosides of all materials, residues, plant residues, including various fruits and berries.

The strain can produce an enzyme composition comprising CBH I (1-25%), CBH II (1-40%), β (1,3) 4glucanase (15-40%), β-glucosidase (2-15%) , xylanase (18-35%) and β-xylosidase (0.5-12%). The enzymatic composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuronosidase, pectinolytic enzymes, starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases and protease. The composition can be used for feed generation from plant raw materials, plant debris and waste for use in the microbial production of antibiotics by fungi and bacteria, including Penici // ium sp. and Streptomyces sp.

The strain can produce an enzymatic composition comprising CBH I (1-30%), CBH II (1-40%), β (1,3) 4glucanase (15-40%), β-glucosidase (2-15%) , xylanase (18-35%) and β-xylosidase (0.5-8%). The enzymatic composition may also comprise one or more of the following; α-glucuronidase, α-L-arabinofuronosidase, pectinolytic enzymes, starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases; exoxylanase and proteases. The composition can be used in feed generation from plant raw materials, plant debris and waste for use in the microbial production of citric acid.

The strain may produce an enzymatic composition comprising CBH I (2-15%), CBH II (2-15%), β (1,3) 4glucanase (20-45%), β-glucosidase (2-25%) , xylanase (1-30%) and β-xylosidase (0.5-8%). The enzymatic composition may also comprise one or more of the following; α-glucuronidase, α-L-arabinofuronosidase, pectinolytic enzymes, starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases, exoxylanase and proteases. The composition can be used in the production of oligosaccharides from algae polysaccharides (for example, laminaran and fucoidane) and additives derived from plant extracts, by procedures generally considered safe, in the formulation of cosmetics.

The strain can produce an enzymatic composition comprising CBH I (3-30%), CBH II (1-10%), β (1,3) 4glucanase (10-45%), β-glucosidase (2-12%) , xylanase (1-48%) and β-xylosidase (0.1-8%). The enzymatic composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuronosidase, pectinolytic enzymes, starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases, exoxylanase and proteases. The composition can be used in the production of oligosaccharides and glycopeptides for use as research reagents, in the production of biodetectors and as tools in functional glycolics to explore receptor-ligand-type interactions and in the production of substrate libraries to perform profiles. of enzyme-substrate specificity.

7 5

The strain can produce an enzyme composition comprising CBH I (5-15%), CBH II (5-30%), β (1,3) 4glucanase (20-45%), β-glucosidase (1-12%) , xylanase (10-30%) and β-xylosidase (0.5-8%). The enzymatic composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, pectinolytic enzymes, enzymes with starch modifying activity, hemicellulases, oxidoreductase / oxidase and esterases and proteases. The composition can be used for the production of modified cellulose and β-glucans, celooligosaccharides, modified starches and maltooligosaccharides, lactulose and polyols (for example, mannitol, glucitol or dulcitol, xylitol, arabitol).

As a raw material, a substrate produced by any of the above processes can be used in the production of biofuel and bioethanol or biogas such as methane or carbon dioxide, provided that it is produced by that process. The advantage of using this strain to produce bioethanol or biogas, is that this is a cost-effective process to produce, from a non-valuable residual product, a valuable product that can be used in many industries. While producing a valuable product, there is a reduction in waste, which in turn has a positive impact on the environment.

All the procedures described above could be carried out with the enzymatic compositions as defined herein or with the microbial strains described herein.

The invention also provides the use of the strain or mutants thereof which also includes the use of enzymes derived from other fungal species including Chaetomium thermophi / e and Thermoascus aurantiacus.

The strain of the microorganism is Ta / aromyces emersonii IMI 393751 or a mutant thereof that is also capable of producing enzymes capable of having activity at temperatures of, or above, 55 'C.

A method for obtaining a suitable enzymatic system for converting a target substrate comprising the procedure is also described: obtaining a sample of the target substrate; allow an inoculum of a strain of microorganism to grow on the target substrate and secrete enzymes; recover secreted enzymes during growth on the target substrate; determine enzymatic activities and properties; build a profile of genetic expression; Identify enzymatic proteins and build a protein expression profile; compare the genetic expression with the protein expression profile; Purify the enzymes. The enzymes can then be preserved. The method may further comprise analyzing enzymes and / or designing an enzymatic system.

Description of the invention

The invention will be more clearly understood from the following description, provided only as an example with reference to the attached drawings, in which:

Figure 1: Process scheme for a design process of an enzymatic system suitable for converting a target substrate. Figure 2: Generation of a raw material rich in sugar for the production of biofuel by thermoenzymatic treatment of apple pulp / pomace. Figure 3: Thin layer chromatogram of the products generated during the hydrolysis of paper cups. Figure 4: Electron microscopy of paper cups before (0 h) and after (24 h) of treatment with the enzymatic cocktail of Ta / aromyces emersonii induced in paper cups demonstrating extensive hydrolysis of the target substrate. Figures 5A-D: Comparison of xylanase production by strain 393751 and wild type CBS strain

549.92 (formerly CBS 814.70). Figures 6A-E: Comparison of glucanase and (galacto) mannanase production by strain 393751 and CBS 549.92 wild type strain. Figure 7: Reduction in the volume of sterile cellulose-rich clinical waste catalyzed by cocktails of 10 enzymes at 50'C after 24 hours. Figure 8: Reduction of the volume of cellulose-rich clinical waste sterilized by STG catalyzed by 10 enzyme cocktails at 70'C after 24 hours. Figures 9A-B: Untreated cellulose-rich wastes (A) and residues treated with enzymes (B). Figures 10A-B: Ethanol production by S. cerevisiae in hydrolysates obtained by treatment of clinical residues rich in cellulose sterilized at 70'C for 24 h (60% humidity) with cocktail 5 (A) and Cocktail 8 (B). Figure 11A: effect of pH on cellulase activity in T. emersonii cocktails. Figure 11B: effect of pH on xylanase activity in T. emersonii cocktails. Figure 12: Effect of temperature on cellulase activity in T. emersonii cocktails. Figure 13: Temperature effect on xylanase activity in T. emersonii cocktails. Figure 14: Activity of the new purified xylanase against different xylanes. OSX, xylan husks oatmeal (Oat Spe / ts Xy / an); WSX, wheat straw xylan (Wheat Straw Wylan); LWX, wood xylan Larch (LarchWood Xy / an); BWX, birch wood xylan (BirchWood Xylan); RM, Rodimenano (1.3; 1,4-β-D-xylan of red algae). The activity is expressed as a% relative to the xylan of oatmeal (100%) Figure 15A-B: Activity of (A) Xyn IV and (B) Xyn VI purified against different xylanes. The activity is

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Expresses as a% relative to the xylan of oatmeal (100%). Figure 15 C-D: Activity of (C) Xyn VII and (D) Xyn VIII purified against different xylanes. The activity is Expresses as a% relative to the xylan of oatmeal (100%). Figure 15E-F: Activity of (E) Xyn IX and (F) Xyn X purified against different xylanes. The activity is Expresses as a% relative to the xylan of oatmeal (100%). Figure 15G: Activity of purified Xyn XI, against different xylanes. The activity is expressed as a% relative to the xylan of oatmeal husks (100%). Figure 16:% relative activity of purified Xyn XII against a variety of purified polysaccharides. OSX, oatmeal husk xylan (Oat Spe / ts Xy / an); BBG, barley β-glucan (Bar / e and β-g / ucan); LIC, lichen (Lichenan); CMC, carboxymethyl cellulose; LAM, laminarin; D1, dextran; D2, dextrin; INU, inulin; ARA, arabinano; GAL L, Galactan (Lupine); GAL P, Galactan (Potato); RG, Ramnogalacturonano; LWAG, Larch wood arabino-galactane (LarchWood Arabino-Ga / actano) Figure 17: Specific activity of purified Xyn XII against aryl β-xylosides and aryl β-glucosides. Figure 18: Comparison of the specific activities of selected xylanases expressed by IMI393751 and CBS549.92 against OSX, oatmeal husk xylan, as a test substrate.

Referring to Figure 1, a process scheme is provided to design an enzyme system to convert a target substrate. In step 1 a target substrate is obtained. In step 2, the target substrate is inoculated with the microorganism that is grown in the target substrate to provide a culture. The microorganism secretes enzymes and these enzymes are recovered in step 3, obtaining culture samples. The culture samples are separated in cell fraction and culture filtrate in step 4. The cell fraction (mRNA) is analyzed in step 5, to determine the enzymatic activities and properties. In stage 6, a gene expression profile is constructed based on the analysis of stage 5. In stage 7, the culture filtrate is explored with respect to protein activity and a protein expression profile is constructed in stage 8 In stage 9 the protein and genetic expression profiles are compared. In step 10 the enzymes are purified. The enzymes can be conserved in stage 11 and in stage 12 the enzymes can be analyzed further and in stage 13 an enzymatic system is designed.

It has been found that when a fungus is used as a microorganism, better results are obtained when the substrate is inoculated with the mycelium of the fungus. This culture is preferably carried out in a fermenter and the reaction conditions will vary depending on the type of microorganism and the substrate used. The culture may be in the form of fermentation in a liquid or solid state. For Ta / aromyces emersonii, a culture temperature in the region of 45 'to 65' is preferable, enzymes being optimally active up to 85-90 'C.

Enzymes are recovered from the target substrate by separation of the cellular (fungal) biomass from the extracellular culture filtrate using centrifugation in the case of enzymes produced by liquid fermentation, or with the help of a cell separation system for larger cultures. The enzymes are recovered after the fraction of cellular biomass by homogenization of a known weight of the biomass in two volumes of buffer. Suitable buffers include 50 mM ammonium acetate, pH 4.5-6.0 or 50 mM sodium phosphate, pH 7.0-8.0. For the extraction of enzymes for solid state cultures, the cultures were mixed with 10 volumes of 100 mM citrate phosphate buffer, pH 5.0 containing 0.01% Tween 80 (v / v), homogenized and extracted with stirring during 2 hours at 140 rpm at room temperature. An enzyme-rich extract is then recovered by centrifugation.

It is essential to make a comparison of the enzyme system at both genome and proteome levels, that is, genes, expression products (mRNA) and identified proteins will be purchased. This is because there may be genetic information present that is expressed at the mRNA level, but that does not translate into functional protein at the protein level. To determine the relative abundance of certain enzymes, transcriptomic, genomic and proteomic analyzes are important.

An isolate of the filamentous fungus strain, from which some of the enzymes have been isolated, has been deposited at the International Micological Institute (IMI) (CABI Bioscience United Kingdom), Bakeham Lane, Englefield Green, Egham, Surrey TW20 9TY, Kingdom United for the purposes of the patent procedure on November 22, 2005, with the deposit ID IMI 393751. After the purification of the enzymes these can, optionally, be conserved or analyzed directly to obtain (a) detailed information on the thermal activity / individual thermostability in general catalytic and functional properties, (b) detailed information on its mode of action and catalytic potential, individually and in combination, (c) information on synergistic interactions, (d) partial sequence information that would help in the cloning of the genes and (e) in some cases, get enough protein to facilitate the collection of three-dimensional structural data. The analysis of these enzymes is then used to identify key enzyme systems and optimal collection times for these systems. The systems can be optimized with respect to key activity levels and combinations of key enzymes, characteristics and performance conditions (at laboratory scale) for target applications.

Execution 1 T. emersonii IMI393751, grounding

Cuttings of freshly picked grass (∼ 200 g) and other mixed plant biomass were placed in a closed container to simulate a composting environment, and incubated in a chamber at a constant temperature, at 65 'C, for approximately 2 hours (combined pasteurization and balance of the substrate). The moisture / moisture content was maintained at ∼ 65-70%. The vessel was equipped with a line that provided a pulse

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low humid air, filtered, at intervals. After 2 hours, a spore suspension (1 x 108 spores in 2% sterile water) of T. emersonii (laboratory reserves of a 12-year-old isolate, originally from CBS814.70) was used to inoculate the region biomass plant. The temperature was maintained at 65 'C for 2 days, and was increased at 2' C intervals every 24 hours thereafter until an air temperature of 70 'C was reached inside the chamber. The culture was allowed to grow for another 7 days before an inoculated "hot spot" sample or central region was removed aseptically and transferred to agar plates (Emerson agar medium for thermophilic fungi), and subculture, to ensure the purity of the crop; Purity was verified by microscopic analysis. Liquid medium containing basic nutrients was inoculated (Tuohy et al. 1992; Moloney et al., 1983) and 2% glucose (w / v) with 1 cm2 pieces of mycelium tapestry from cultures in agar agar plates 36 hours of life The liquid cultures were allowed to grow at 55 'C for 36 hours in 250 ml Erlenmeyer flasks (containing 100 ml of growth medium), with agitation at 220 rpm. After 36 hours, aliquots (2.0 ml) of mycelium suspensions were extracted, washed aseptically with sterile water, transferred to sterile Petri dishes (10 mm in diameter) and irradiated with UV (ultraviolet) light during timed intervals (10-60 seconds). Sterile agar media (noble agar containing 0.2% w / v of individual catabolite repressors, such as 2-deoxyglucose) was inoculated with samples of the irradiated fungal mycelium. Repeated cultures were incubated at 45 and 58 'C. Individual colonies (white spongy-looking) were carefully selected, aseptically transferred to Sabouraud dextrose agar plates and further purified by various transfers. IMI 393751 strain represents an isolate extracted from the plates incubated at 58 'C. The mutant was evaluated in a comparative study with the parental organism, with respect to the enhanced thermal stability and enzymatic production, which revealed clear differences between both strains in terms of crop appearance, ability to sporulate (IMI 393751 strain is non- sporulant), thermophilia and stability, enzymatic production patterns under identical growth conditions, differential expression and levels of individual enzymatic activities.

Differences� physiological / mycoagical �between�CBS ��14.70�e�IMI393751

Appearance of the quantum during growth in different means of aggregation. When strain CBS 814.70 is grown on Emerson agar, it has the typical characteristics of this species (Stolk and Samson, 1972), that is, beige / pale cream, with pale yellowish tones near the surface of the agar (around the surface of the agar). inoculation zone), which becomes a stronger beige / reddish brown color as the crop matures, and consists of a dense mycelium tapestry of many ascomas. On the contrary, the IMI393751 strain is much whiter and the crop looks very 'fluffy'.

Differences regarding sporulation. The strain CBS 814.70 is a sporulant strain of T. emersonii, which produces conidioforos, disgust and ascospores. The conidioforos appear as perpendicular branches in the hyphae (light yellow and septate). The disks, or sacks containing spores, have a somewhat ellipsoidal shape, and can often be found in chains; the ascospores are smooth and elliptical (they appear green on an electron micrograph). On the contrary, the mycelium of IMI393751 produces very few conidioforos, ascosporas and classic ascosporas. Quite the opposite of CBS814.70, strain IMI393751 only produces an insignificant number of spores under extreme and quite specific conditions, while strain CBS 814.70 produces spores in several different media.

Differences with respect to Thermophony and optimal growth temperature range. Strain CBS 814.70 has a strict growth interval of 35-54'C, with little growth at lower or higher temperatures and an optimal growth interval of 40-45'C. On the other hand, the IMI393751 strain shows a broader growth temperature range of 30-65 'C, with good growth at 80 DC, 85 DC and up to 90 DC, with an optimum between 4855' C. The IMI393751 strain grows very well at a temperature> 55 'C and continues to produce high levels of fungal biomass (mycelium) and actively secreting significant amounts of various proteins.

Differences with respect to growth at higher pH values - CBS strain 814.70 does not grow well (in fact it begins to die) at pH values above 6-6.5 (previous data, Tuohy and Coughlan, 1992), while that strain IMI393751 grows well and secretes substantial levels of enzymes at pH values of up to 9.0.

Growth in nutrient / depleted targeting conditions. IMI393751 survives well in crops with low nutrients for up to 55 days, while CBS 814.70 undergoes autolysis from 7 days onwards and after 15 days there are few surviving cells / mycelia. The mycelia collected from liquid cultures with scarce nutrients of 55 days of IMI393751 life could be resurrected by transfer to agar media with Sabouraud dextrose, while, through the same strategy, the remains of mycelia of strain CBS814.70 (55 days of life) could not revive.

Example 2: an enzymatic system of Talaromyces emersonii to convert hemiceaua6s materials. Complete hydrolysis of xylan requires the synergistic action of xylanases, β-xylosidase, α-glucuronidase, α-Larabinofuranosidase and esterases. Table 1 provides an example of selected target substrates (including residues / residues) and the percentage of the inductive carbon source used in this example. The induction percentage refers to the amount of carbon source per volume of medium (g / 100 ml)

Tabaa1: Growth substrates / inducing carbon sources for enzymatic production by T. emersonii

Source of Carbon Abbreviation% of Source of Carbon Inductor

Beech wood xylan BEX 1.0 Birch wood xylan BWX 1.0 Oatmeal husk xylan OSX 1.0 Larch wood xylan LWX 0.3 Wheat arabinoxylan WAX 1.0 Spruce chips SS 2.0- 6.0 Packaging material PK 0.5-6.0 Cereal straws CS 1.0-2.0 PC paper cups 2.0-6.0 White office paper WOP 2.0-6.0 Sheets TL 2.0-4.0 Methyl Xylose MEX 0.2 Xylose Xyl 1 Glucuronic Acid GlcA 1 Solka floc SF 2 A Glucose Glc 2 A

A included for comparative purposes

Based on the results obtained, an enzymatic system was designed using purified Ta / aromyces emersonii enzymes for the degradation of a hemicellulose (xylan) or of a product, residue or residue, derived from wood, rich in xylan. Table 2 shows the relative amounts of the key enzymes present in the enzyme system.

Tabaa 2: A system for the degradation of a rich heroic or a product of rich wood in the Indian.

ENZYM TO

% � REQUIRED

Xylanase Exoxylanase β-Xylosidase α-glucuronidase α-L-arabinofuranosidase Accessory biopolymer modifying activities (e.g. cellulase, non-cellulolytic β-glucanase, pectinolytic activities, mannanolytic activities, acetyl (xilan) esterase, oxidoreductase, protease)

10-50% 4.0-25.0% 0.1-5.0% 0.2-4.0% 0.1-5.0% 25-50%

The relative amounts of the main activities, the profile and the relative amounts of the accessory activities will vary depending on the composition of the target substrate. Table 3 summarizes the enzymatic systems 10 of Ta / aromyces emersonii that have been designed for specific applications.

Table 3: �Specific Enzymatic� Enzymatic Systems � for the �Degradation of Oligosaccharides �from Wood iaanos�, �Results � of � Newspaper Dash and �Aeooso.

Enzymatic system and composition

Appearances / substrate are selected Enzymatic dosage ** % of carbohydrate hydrolysis

TE wood cells (induced by LWX) *

Oligosaccharides of wood xylan xylan 15-30 nkat / g 91-100% (1-3 h)

Xylanase 15-30% A

β-xylosidase 0.2-1.0% α-glucuronidase 0.1-3.0% α-L-arabinofuranosidase 0.13.0%

Newspaper bioconversion newspaper paper $ 11.5 -46.0 nkat / g 70-85% carbohydrate present% 34-50% (70 'C / 24

7-10% cellulase

h) A A

Non-cellulosic glucanase, pectinolytic enzymes,

Bioconversion of woody remains (for example, sawdust, shavings, virgin wood, bark, etc.) Woody remains 46.0-150 nkat / g 35-55% (80 DC / 24h) A A

esterases) 20-35%

A Xylanase levels were measured with different xylan models as substrates * LWX, larch wood glucuronoarabinoxylan ** Xylanase levels: 45.2 IU / ml (753.5 nkat / ml), 40.9 IU / ml ( 681.8 nkatml) and 27.5 IU / ml (458.4 nkat / ml) with larch wood xylan, birch wood xylan and oat husk xylan as test substrates, respectively; at the time of harvest, in which nkat = nanokatales (16.67 IU / ml) $ Depending on the quality of the newspaper, that is, in color against black and white, polished versus coarse / recycled finish, etc. % of cellulose, 40-55%, and hemicellulose, 25-40% as the main types of carbohydrates present A A% Conifer substrate conversion (fractions not ground and ground into large pieces, untreated and previously treated with diluted acid). The% total conversion can be increased (up to 74%) using this enzymatic system in a combination with other enzymatic systems of T. emersonii or Chaetomium thermophi / e, or amplification of key enzymatic activities by the addition of recombinant enzyme.

Example 3: System of Talaromyces emersonii to convert cereaaes, beet puapa and other materials, including waste), rich in arabinoiians and �hemiceauaosas�acetiaadas.

5 Table 4 provides relative amounts of different enzyme activities in enzyme systems designed from Ta / aromyces emersonii, strain IMI 393751 and two previously identified mutant strains designed for the conversion of cereals, beet pulp and other materials (including residues) rich in arabinoxylans and acetylated hemicelluloses.

Table 4: Enzymatic activities of systems for the conversion of aematerial materials in arabinoiiaanos and 10 hemiceous aqueous

Enzyme preparation / composition

T. emersonii �iIMI 393751; �1: 1 WB / BP� as inductor) T. emersonii �iIMI 393751; Apoavo�de aagarroba as inductor) T. emersonii �iTC2 mutant; �WB as an inductor) T. emersonii �iTC5 mutant; `` Blades '' as `` inductor ''

Enzyme activity profile (%)

Enzyme activity profile (%)

Enzyme activity profile (%)

Enzyme activity profile (%)

Xylanase

20-30% 15-25% 35-50% 12-20%

Exoxylanase

5-10% 2-5% 10-15% 2-8%

β-xylosidase

0.5-2.0% 0.2-1.5% 0.5-1.5% 0.1-1.0%

α-glucuronidase

0.5-2.0% 0.2-1.5% 0.2-1.5% 0.2-1.5%

α-L-arabinofuranosidase

0.5-2.0% 0.2-2.0% 0.2-1.2% 0.1-1.5%

Biopolymer modifying enzymes including acetyl esterase and acetyl xilan esterase

30-45% 32-50% 20-35% 25-60%

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Example 4: Talaromyces emersonii system to convert non-ceaua6s, such as tea leaves and aagarroba poavo. Characterization of three new endoglucanases (EG), which are important both for the selective modification and for the degradation of a variety of cereal residues, including possible candidates for the manufacture of beer and animal feed, such as sorghum, corn and rye. These three enzymes have been purified from Ta / aromyces emersonii, strain IMI393751.

The total carbohydrate content of the purified enzyme preparations was determined by the phenolsulfuric acid procedure (Dubois et al) by reference to the standard curves of glucose or mannose (20-100 μg.ml-1).

Protein separation techniques. To obtain highly purified preparations of EG V, EG VI and EG VII, sequential gel filtration, ion exchange, hydrophobic interaction, lectin affinity chromatography and chromato-focus (pseudo-ion exchange) were needed. Non-denaturing and isoelectro-focusing gel electrophoresis was performed followed by coomassie blue staining by conventional methodology known by the expert in this field and provided in the relevant references (for example, Murria et al., 2001; Tuohy and Coughlan. 1992, Tuohy et al. 2002; Maloney et al., 2004). Initial experiments revealed that the pI values of EG V-VII were <pH 3.5. Therefore, chromato-focusing was used in PBE 94, pre-equilibrated with 0.025 M piperazine-HCl, pH 3.5, to determine the precise pI values for EG V-VII. It was determined that the pH corresponding to the elution peak for β-glucanase was pI. Differences of 0.01 pH units could be detected thereby producing precise pI values for each enzyme.

Optimum pH and temperature and stability. Optimum pH values were determined by monitoring the enzymatic activity at pH values between 2.5 and 9.0, at 50 'C, in citrate phosphate buffer of constant ionic strength. To evaluate the effects of pH on the activity of EG V-VII, purified samples of each enzyme were incubated at pH 4.0, 5.0 and 6.0 at 50 'C. Aliquots were extracted after 0, 1, 2, 3, 6, 9,12, 24, 36, 48 and up to 336 hours (14 days), diluted appropriately in 100 mM NaOAc buffer, pH 5.0 ( normal test pH) and were tested for residual activity according to the conventional test procedure. The stability of each purified enzyme was investigated at 50 'C, 60' C, 70 'C and 80' C, in the absence of substrate. EG V-VII were not modular proteins because none of the three enzymes adsorbed on cellulose (or other insoluble carbohydrates, that is, they did not contain carbohydrate binding).

Substrate specificity in various synthetic polysaccharides and glycosides was evaluated by measuring activity against a wide variety of carbohydrates (BBG, lichenano, CMC, other synthetic polysaccharides and glycosides) using the normal β-glucanase assay procedure.

Effects of metal ions, chemical modification reagents and possible inhibitors. A series of monovalent, divalent and heavy metal ions were investigated, at final test concentrations of 1.0 mM, with respect to their possible effects on the activities of EG V, EG VI and EG VII, incubating the enzymes with the compounds Chemicals and then performing the normal enzyme assay. Finally, the inhibitory effects of glycosides, such as disacarids, lactones and flavonoid glycosides on purified enzymes, were examined. Specific concentrations of each glycoside were included in normal test cocktails and the residual activity was determined by comparison with controls (no glycoside present).

A summary of the purification of EG V, EG VI and EG VII, and purification parameters such as yield (%) are provided in Table 5.

Tabaa 5: Summary of purification of enzymes gaendogaucanases from Talaromyces emersonii

Purification Stage

Totaa�iUI activity) Totaa protein img) Specific activity iUI.mg · 1) Purification factor (ii times) Yield%)

Gross Extract

54,806.2 573.1 95.6 1.00 100.00

Ultrafiltration (Amicon DC2)

50,613.4 452.2 111.9 1.17 92.30

Propan-2-ol pptn.

50,016.9 364.8 137.4 1.44 91.30

Anion Exchange

(DE-52, pH 5.5) Phenyl Sepharose

25,064.7 240.1 104.4 1.10 45.70

CL-4B

Peak 1

3,862.6 24.9 154.9 1.62 7.10

Peak 2

6,763.5 30.8 219.3 2.29 12.30

Peak 3

14,411.1 42.1 342.3 3.58 26.30

EG VII (Peak 1)

Concanavalin A

3,120.2 11.3 275.9 2.89 5.70

Sephacryl S-100 HR

670.1 3.2 212.5 2.22 1.20

13 5

(continuation)

Factor Factor Activity

Protein Performance Activity

Specific purification stage purification�ii

totaa�iUI) totaa�img) i%)

iUI.mg1) times)

Chromato Focus

400.4 0.53 756.9 7.92 0.71

EGV, EGVI (Peak 3)

Anion exchange (DE-52, pH 4.5)

4,662.3 10.40 447.4 4.68 8.50

Anion exchange (DE-52, pH 7.0)

3,339.2 5.77 579.1 6.06 6.10

BioGel P-60

3,043.8 3.60 855.7 8.95 5.50

Concanavalin A

Peak 1

1,115.6 1.60 679.1 7.10 2.10

Peak 2

863.50 1.20 738.7 7.73 1.60

PBE Chromato Focus

94,

pH 3.5-2.0

EG V

415.3 0.54 764.8 7.99 0.76

EGVI

168.7 0.18 947.6 9.09 0.31

The yields, as well as the final specific activity values, were similar for EG V and EG VII, while EG VI was obtained at a lower yield and had a higher specific activity.

Mr and pa values of enzymatic homogeneity. Positive staining with Schiff reagent (results not shown) indicated that the three enzymes were glycoproteins of a subunit. The homogeneity of each enzyme preparation was verified by IEF. The values of pl obtained were the following EG V, 2.45; EG VI, 3.00; EG VII, 2.85.

Tests of gaicosiaación and caacuao of coefficients of moaar eitinci6n. Total carbohydrate contents (w / w) were 22.6 p 0.1%, 14.7 p 0.1% and 65.9 p 0.04% for EG V, EG VI and EG VII, respectively. The calculated molar extinction coefficient values (ε280) (mol.1.cm-1) were 1.04 x 10-5 for EG V, 8.80 x 10-6 for EG VI and 7.05 x 10-6 for EG VII.

Optimum pH and temperature and stability. All three enzymes were active over relatively wide pH ranges but showed optimal values at acidic pH of 5.7, 5.4 and 5.7 for EG V, EG VI and EG VII, respectively. An optimum pH of 4.5 was obtained for BBGase activity in the crude extract of T. emersonii. Obvious result approximately 75% of the optimal activity between pH 3.0 and 7.0 (EG V), pH 2.5 and 7.5 (EG VI) and pH 2.8 and 7.5 (EG VII).

The release of BBG reducing sugars, at pH 5.0, for 10 min, at 30-90 AD. The optimum temperature values for the activity were 78.0 DC, 78.0 DC and 76.0 DC for EG V, EG VI and EG VII, respectively. The activation energies (Ea), calculated from Arrhenius representations, were 21.2 p 0.05 kJ.mol-1 for EG V, 23.5 p 0.02 kJ.mol-1 for EG VI, and 26.7 p 0.05 kJ.mol-1 for EG VII.

The effect of pH on enzyme stability was investigated at pH 5.0, pH 5.5 and pH 6.0 (at 50 DC and 70 DC). The pH stability was greater in the range of pH 4.0-7.0, during an incubation period of 1 hour, with a notable reduction observed at pH <4.0 and> 7.0. EG V did not lose activity at pH 5.0 and at 50 DC for a period of 15 days, while EG VI and EG VII lost minimal activity (13.0% and 11.5% respectively). However, at 70 DC and the same pH, EG V, EG VI and EG VII lost 42%, 35% and 33% respectively, of the original activity in each sample over a period of 60 minutes. At pH 5.5 (50 DC), EG V, EG VI and EG VII lost 30%, 22% and 8% of their respective original activities over a period of 15 days, but at 70 DC, EG V was further destabilized ( 58% reduction of the original activity after 60 minutes), while the activity of EG VI and EG VII after 60 minutes was very similar to that obtained after incubation at pH 5.5 and 70 DC. EG V was considerably less stable at pH 6.0 and at 50 DC losing 63% of its original activity in 15 days. EG VI and EG VII were considerably more stable at the last pH with similar stability to those determined at pH 5.5 (30% loss of activity for both enzymes). By increasing the incubation temperature to 70 DC, the activity of EG V was markedly reduced (65%) during a 60-minute incubation period (at pH 6.0), while EG VII remained remarkably stable losing only 22% of Your original activity The half-life (TY) values were greater than 15 days at pH 5.0 and at 50 DC, while at 70 DC values they varied from 67 minutes (EG V) to> 80 minutes (EG VI and EG VII.

Tests of the modular structure. EG V, EG VI, and EG VII were weakly adsorbed to Avicel (microcrystalline cellulose) at pH 5.0 and at 4 DC (8-15% adsorption); However, this degree of adsorption is not indicative of the presence of a carbohydrate binding module (CBM, Carbohydrate Binding Modules / e).

14 5

Effect of metallic ions, chemical modifiers and possible inhibitors on enzymatic activity. The effects of 1 mM final concentrations of a series of mono, di and multivalent cations on the activity of EG V, EG VI and EG VII were investigated. The activity was expressed as a% in relation to a control (no metal ion present in incubation mixture). In general, heavy metal ions are believed to inactivate enzymes forming covalent salts with cysteine, histidine or carboxyl groups. Although several of the metal ions, for example, Mg2 +, Zn2 + and Mo6 + enhanced the activity of the three enzymes and ions such as Ag +, Fe2 + and especially Hg2 + significantly reduced the activity of EG V-VII, notable differences were observed between enzymes with regarding the effects of other metal ions. The salts of Na + and K + chloride had no effect or slightly stimulated the activity of each enzyme, for example K + increased the activity of EG VII by 20%, while Na + potentiated the activity of EG V by> 39%. Ba2 + and Ca2 + reduced the activity of EG V by 16.5% and 21.5-24.6%, respectively, while both ions increased the activity of EG VI by 37.6% and 10%. Other divalent cations, such as, Cd2 +, Co2 + and Cu2 + exerted a remarkable inhibitory effect on EG V (∼26.5-77.4% loss of activity). The almost total inhibition of the activity of the three enzymes by Hg2 + suggests the presence of an essential thiol group (or groups) involved in the catalysis. It was observed that valence modulated activity, for example, Fe3 + exerted a stronger negative effect on the activity of the three enzymes (35.4-88.0% reduction in activity), unlike Fe2 +, which potentiates done the activity of EG V by 20%, it has minimal effects on EG VII and reduces the activity of EG VI by 38.6% (Fe3 + reduces the activity of EG VI by 53.6-59.3%). The nature of the counter-anion, that is, Cl-versus SO42-had a great effect on activity when both salts of a particular anion were investigated. The SO42- salts of Ca2 + and Fe3 + selectively potentiated the activity of EG VII ∼2 times and ∼4.5 times, respectively, in relation to the activity observed with the corresponding Cl-salt. On the contrary, the SO42- salt of Mg2 + significantly reduced the activity of EG V (54.2%) and EG VI (50.1%) in relation to the Cl-salt of the same cation. A selection of reagents that are known to modify R groups of amino acids with respect to their effects on EG V, EG VI and EG VII, both in the absence and presence of substrate were tested (in this way substrate protective effects could be observed) (see Table 6).

Table 6: `` Chemical '' modification studies to identify amino acid groups that are essential for catalysis in VEG, IV, and VI.

Chemical reagent

Reactive enzyme EGV EV�VI EG�VII EGV Substrate�r�reactivab EV�VI EG�VII

Control

100.0 100.0 100.0 100.0 100.0 100.0

DL-DTT

218.9 514.3 575.0 264.3 915.5 348.6

Dithioerythritol

270.6 657.2 773.8 271.9 917.90 360.9

Dimethylsfofoxide

125.5 124.3 194.0 93.6 141.7 100.0

Sodium borohydride

96.1 125.0 194.0 81.5 104.8 82.8

Sodium periodate

181.0 357.1 300.0 283.3 1001.2 469.7

Woodward reagent K

162.7 280.0 305.9 121.1 266.7 143.0

Thioglycolic acid

211.1 187.1 188.1 148.1 269.0 150.0

o-Ftaldialdehyde

106.1 100.0 155.0 94.6 139.0 98.8

N-Bromosuccinimide (NBS)

0.0 0.0 0.0 0.0 0.0 0.0

p-chloromercuribenzoate

125.4 115.4 105.4 89.7 107.1 91.6

p-hydroxymercuribenzoate

92.8 98.3 99.5 261.7 125.0 94.8

2,3,5-triphenyltetrazolium chloride

97.5 97.6 98.5 89.1 97.4 100.0

Iodoacetamide

103.6 99.9 101.6 87.4 122.6 83.7

Iodine

143.4 180.6 200.0 97.2 147.6 104.8

Cysteine

163.8 170.4 196.5 237.8 370.20 367.7

13 Incubation (30 minutes) Enzyme + 23 incubation EG V EGVI EG VII (30 minutes) + NBS Control X 100.0 100.0 100.0

Cysteine

' 117.0 217.0 167.0

DDT

' 204.3 140.0 94.6

NBS

' 0.0 0.0 0.0

a Pre-incubation of enzyme with reagent for 30 minutes (50 DC) before substrate addition b Pre-incubation of substrate with reagent for 30 minutes (50 DC) before substrate addition

The strong inhibitory effect of N-bromosuccinimide (NBS) suggests the involvement of tryptophan in binding and / or catalysis. However, o-phthaldialdehyde, another tryptophan modifying reagent has no net effect on the activity of EG V, EG VI or EG VII, therefore the NBS could modify another amino acid residue with which it is known to have secondary reactivity , for example, cysteine. In addition, the inability of the substrate to protect against enzyme inactivation would rule out a direct role for tryptophan in the binding or catalysis. Since both cysteine and dithiothreitol (DTT) protected against inactivation, the effect of NBS may be due to side reactions, for example, cysteine oxidation. The sulphydryl iodoacetamide, p-hydroxymercuribenzoate and N-ethylmaleimide reagents cause little or no inhibition in the presence or absence of substrate, which would suggest that EG V, EG VI and EG VII do not have essential thiol groups. However, cysteine, DTT and dithiothreitol activate the three enzymes, especially EG VI and EG VII, which may suggest the reduction of an oxidized disulfide perhaps during extraction and / or enzymatic purification, thus restoring the native conformation

15 5

from the region of the active site of the enzyme, or the whole enzyme molecule. Sodium borohydride, a strong reducing agent, inhibits all three enzymes (especially EG VI) in the presence of substrate. On the contrary, the oxidation of other reactive groups in the active site by means of the action of strong oxidizing agents, such as sodium periodate, iodine and thioglycolic acid, notably enhance the activity of the three enzymes, the most pronounced effects being with sodium periodate and EG VI, in the presence of a substrate. Woodward reagent K, a carboxylate modifying reagent, potentiated the activity of EG V, EG VI and EG VII, being more effective in the absence of substrate.

In general, phenolic substances, such as m-phenylphenol, (-) epicatechin, (+) catechin, o-coumaric acid, caffeic acid, ferulic acid, syringic acid and tanic acid, to name a few of the compounds tested, did not have a remarkable inhibitory effect on enzymatic activity (in the absence of substrate) with the exception of tanic acid which is a strong inhibitor due to its protein precipitating function. In fact, some of the compounds, for example, protocatechic acid, syringic acid, caffeic acid and polyvinyl alcohol remarkably activated all three enzymes (results obtained during enzyme pre-incubation with inhibitor in the absence of substrate). However, when they were incubated together with the enzyme and the substrate, it was observed that several of the phenolics made a reduction in activity of ∼7-32% (the substrate did not protect any of the enzymes against the strong effect of tanic acid ).

In general, most of the detergents tested did not result in loss of enzymatic activity. However, taurocolic acid, taurodeoxycholic acid, Tween 20 and Tween 80 all reduced the activity of EG V, by 47.7%, 22.7%, 12.7% and 30.8% respectively, when pre-incubated with enzyme before the addition of the substrate. With the exception of Tween 80 (loss of activity of 34.2%), simultaneous incubation with substrate restored full activity. Enhanced activity was observed when substrate, enzyme and detergent (deoxycholic acid, CHAPS, taurodeoxycholic acid and chenodeoxycholic acid) were incubated simultaneously, for example, the activity of EG VI increased> 2 times in the presence of substrate and deoxycholic acid.

Glycosides, such as salicin, esculin and arbutin, had no apparent effect on the activity of EG V-VII, as did the melodious, maltose, sucrose and alditol, sorbitol disacarids. However, cellobiose concentrations of 50-75 mM significantly inhibited all three enzymes, especially EG V, which was also inhibited by lactose at concentrations> 75 mM. Lactose also effected ∼50% inhibition of BBGase activity of EG VI and EG VII at concentrations of ∼120 mM. Glucono-δ-lactone and glucoheptono-1,4-lactone also inhibited EG V-VII but at much higher concentrations (from 500 to> 750 mM for 50% inhibition).

Substrate specificity. The crude extracts of T. emersonii, catalyze the hydrolysis of a variety of polysaccharides including cellulose, CMC, BBG, laminaran, lichen, xylan, pectin and a spectrum of synthetic glycoside derivatives. None of the three purified enzymes were active against 4-nitrophenyl derivatives of β-Dxilopyranoside or α-D-galactopyranoside (Table 7).

Tabaa 7: �Specificities of substrate� of EG �V, �EG �VI andEG �VII�purified� of Talaromyces emersonii

xylopyranoside

Substratum

% � of Activity�reaativaa

Enaace

"aiquenase" �de 40.7�DDa EGV EG�VI EG�VII

barley β-glucan

β- (1,3) (1,4) 100.0 100.0 100.0 100.0

DP BBG mixed

β- (1,3) (1,4) 159.0 165.0 230.0 107.6

DP BBG high CMC

β- (1,3) (1,4) β- (1,4) 118.0 0.5c 249.9 74.9 342.8 58.4 144.1 57.7

Lichenano

β- (1,3) (1,4) 118.9 204.3 177.7 166.9

Laminarano

β- (1,3) 0.0 0.0 21.3 18.1

Xylan

β- (1,4) 0.0 1.8 0.7 1.5

Rhodimenan (xylan)

β- (1,3) (1,4) 0.0 35.4 9.3 0.0

Pectin

α- (1,4) 0.0 0.0 0.0 0.0

Manano

β- (1,4) 0.0 0.0 0.0 0.0

Avicel

β- (1,4) 0.0 0.0 0.0 0.0

Filter paper

β- (1,4) 0.0 0.0 0.0 0.0

4NP-β-D-glucopyranoside

β- (1,4) 0.0 0.0 0.0 0.0

4NP-β-D-galactopyranoside

β- (1,4) 0.0 0.0 0.0 0.0

4NP-β-D-

β- (1,4) 0.0 0.0 0.0 0.0

a% of activity in relation to the activity against BBG, which is taken as 100%; purified Ta / aromyces emersonii bliquenase [6]; c After 24 h at 50 DC

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In addition, EG V, EG VI and EG VII did not show any activity against filter paper, Avicel, carob gum galactomannan and did not catalyze cellobiose oxidation, even in expanded incubation with substrate. The results are expressed in terms of% activity in relation to the control (the activity against BBG is assigned a value of 100%). The three enzymes show maximum activity against mixed-linked β-glucans, BBG and lichen, notably with more activity on the last substrate. The residual activity shown by the three enzymes with xylan in extended incubation periods can be explained by the fact that the used xylan preparation of oatmeal husks contained lower amounts of β-glucan contaminants.

After performing similar analyzes on each of the expressed enzymes, an enzyme system was designed using purified enzymes from Ta / aromyces emersonii for the degradation of non-cellulosic material, such as tea leaves, locust bean powder and other similar materials.

Table 8 provides an example of the relative amounts of different enzymatic activities for this enzyme system.

Tabaa �: �System�to�a�conversion.of.material.is not only oceanic, for example, jas leaves �de�te, poavo� aagarroba.

Enzymatic Composition

Perfume of enzymatic activity �i%)

β-glucanase

45.0-55.0

Xylanase

16.5-42.0

β-glucosidase

0.5-2.0

β-xylosidase

0.1-1.0

Protease

0.1-1.0

Additional hemicellulase enzymes that include β-galactosidase, esterases, αglucuronidase, etc.

10.0-18.0

Example 5: `` T. emersonii system '' to convert `` watery, '' wastewater into `` watery '' and `` watery ''

The IMI 393751 strain of Ta / aromyces emersonii was grown in a variety of paper waste and paper products as substrates. Excreted enzymes were extracted and enzymatic expression was monitored and quantified by proteome and transcriptome analysis and by a comprehensive spectrum of functional assays. Several paper wastes proved to be excellent cellulase inducers (and complementary activities, for example, enzymes that hydrolyze starch, in which coated / finished paper products were used). The differences were quite evident with respect to the relative amounts / types of cellulase enzymes induced by different waste / paper products. The data obtained confirmed that the isoenzymes cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II) were the most important cellulase activities and, when desired more complete saccharification / bioconversion of cellulose in the target substrate (eg generation of rich raw materials in monosaccharides for biofuel production), β-glucosidase I (BG I) was also very important.

The paper plates induced remarkable levels of degrading activity of filter paper (FP) (1573 IU / g, in which UI represents μmoles of product formed / minutes of reaction time / g of inducing substrate), low levels of endocellulase ( 27.5 IU / g) and low levels of β-glucosidase (6.05 IU / g). At the level of the transcriptome, CBH I was the most abundant / highly expressed cellulase, a complementary observation at a functional level, detecting activity of type CBH I 242.0 IU / g. Enzymes produced during growth in paper cups were significantly more exoactive, detecting activity FP 495.0 IU / g and β-glucosidase 53.9 IU / g. In the last example, the gene expression and functional tests indicated that CBH II was the key cellulase (transcript and enzyme levels for CBH II were veces2 times the corresponding levels for CBH I enzymes). The CBH II was again the key cellulase induced by brown paper, corrugated cardboard and white office paper. It was shown that individual enzyme systems, and combinations thereof (for example for the amplification of accessory / secondary or key external activities), were effective tools for the conversion of cellulose (and hemicellulose / other carbohydrates) into a wide variety of virgin materials, secondary and residual rich in cellulose.

Enzymes were isolated and analyzed by conventional procedures (Walsh, (1997) et al., 2002).

CBH IA, which contains xylanase residues as the only contaminating activity, eluted at a concentration of NaCl between 115 and 170 mM. Fractions 52-66 were pooled and dialyzed for 16 hours against 4 changes of 100 mM ammonium acetate buffer, pH 5.0, and subjected to affinity chromatography on a column (1.4 x 11.3 cm ) of CH Sepharose 4B substituted with p-aminobenzyl-1-thio-cellobioside. The residual pollutant xylanase activity was not bound and eluted in the application and wash buffers. Using 0.1 M lactose in 100 mM ammonium acetate buffer, pH 5.0, CBH IA was eluted. Fractions 13-21 were pooled, dialyzed against distilled water to remove lactose and stored at 4 DC until use.

17 5

CBH IB of the anion exchange stage (DE 52 at pH 5.5) was dialyzed against 100 mM ammonium acetate buffer, pH 5.5 and applied to the affinity column as for BCH IA. Residual pollutant activities, mainly endoglucanase, did not bind the affinity matrix and eluted in the wash. CBH IB was eluted specifically using 0.1 M lactose in affinity buffer. Fractions 43-48 were pooled and dialyzed against distilled water to remove lactose and stored at 4 'C until later use.

Based on the above analysis, the following enzyme system was designed using purified Ta / aromyces emersonii enzymes for the degradation of paper waste and paper products, and other residues containing cellooligosaccharides.

Tabaa 9: System for the conversion of aqueous, en residues en ua ua ua ua cea cea cea and car car car car car car car car car idos idos

Enzymatic Composition

Profile of enzymatic activity%)

Cellobiohydrolase 1A and 1B

5.0-80.0

Cellobiohydrolase II

6.0-45.0

Endoglucanase (Cel 45, EGV, EGVI, EGVII)

4.6-66.0

�-xylosidase

0.1-2.5

Xylanase

1.0-89.0

Other hydrolases (including pectin modifying enzymes, arabinofuranosidase, �-galactosidase)

1.2-20.5

Example 6: `` System of fungal species '', Chaetomium thermophile and Thermoascus aurantiacus.

The strategy described for the design of enzymatic systems / thermoenzymatic compositions of the IMI 393751 strain of T. emersonii and previously known mutants was adapted for the production of carbohydrate modifying compositions for more than twenty three fungal mesophilic and thermophilic species. Special attention was given to the production / induction of strong enzymatic systems rich in pectinase and hemicellulase (xylanase, mannanase) by these fungal species.

Chaetomium thermophi / e and Themoascus aurantiacus species were grown individually in liquid fermentation, as described above, in the nutrient medium of T. emersonii containing 1-6% inductive carbon source (enzymatic production by solid fermentation was also investigated ). A strong enzymatic system was obtained that degraded mannan by culture of C. thermophi / e for 96-120 hours in coffee residues. This composition of this system was characterized and shown to contain 45-60% of mannan hydrolysis activities, 0.7-4.0% of pectin modifying enzymes, 35.2-52.0% of xylan modifying activities the rest being attributed to cellulase activities (very low or residual CBH and β-glucosidase were observed).

The cultivation of the same fungus in soybean bran produced a strong xylanolytic enzymatic system (70.2-86.5% of the total activities being attributed to xylan modifying enzymes); ,610.6-28.0% and ,68.6-22.5% of the remaining carbohydrate modifying activities were attributed to cellulase, pectinolytic enzymes and low mananolytic levels.

Similarly, during the cultivation of Th. Aurantiacus in wheat bran and beet pulp (1: 1), a strong pectin modifying enzyme system was induced, representing> 56.5-80.0% of the modifying activity profile of total carbohydrates by pectinolytic activities; this enzymatic system also contained ∼22.1-40.1% of xylan modifying enzymes, the rest being mainly β-glucan / cellulase modifying activities. On the contrary, the culture of Th. Aurantiacus in soybean bran induced a strong xylanolytic enzyme system (> 62.1-85.8%), complemented by pectin modifying enzymes -113.5-11.0% being the activities predominantly β-glucan modifiers remaining. Unlike C. thermophi / e, Th. Aurantiacus does not process significant levels of mannan degrading enzymes during culture on one of the substrates.

It has been shown that these systems, by themselves and in combination with each other or with other enzymatic systems (for example, T. emersonii) carry out abundant saccharification (sugar release) of a wide variety of agricultural, food / vegetable, beverage materials , woody / paper and other virgins rich in carbohydrates and of different residues, and can be used for the generation of specialized oligosaccharide products or raw materials rich in sugars for a wide range of biotechnological applications (eg biofuel production).

Example 7: T. emersonii system for the generation of sugar-rich raw materials from food / beverage, papier and oily waste to use in the production of biofuel.

ia) Bioconversion of a food / beverage residue: uapua / apple pomace

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From local fruit suppliers, distributors of food processing and cider / beverage production, apple residues (reduced to pulp), pulp and apple pomace were obtained. T. emersonii was grown in 2-6% pomace / apple pulp (both solid and liquid fermentation) and high levels of a series of carbohydrate modifying enzymes were measured. This system was characterized by high levels of βglucan hydrolases, mainly non-cellulosic β-glucanase (∼27.4% in 120-hour liquid culture filtrates), substantial amounts of key exoglycosidases with especially high levels of α-arabinofuronosidase (13.3% of the total carbohydrase activity) and β-galactosidase (22.6%). Also esterase enzymes, pectin modifiers and additional xylan were detected (> 7.2-33.5%). Therefore using the above analysis, it is possible to design an appropriate enzyme system to degrade apple residues.

Initial studies used an enzyme load containing 2,344 nkat of xylanase, 5,472 nkat of mixed-linked β-glucanase and 8,529 nkat of lichennanase per 3.6 kg of substrate and a reaction temperature of 70 'C was used. The complete pasteurization of the hydrolyzate at 70 'C was achieved, and the hydrolyzate was used to feed up-flow anaerobic reactors mesophiles and thermophiles (UAHR). 100% utilization of sugar raw material has been observed, with joint production of methane (50-70% in the biogas stream). Subsequent optimization studies were performed, which showed that incubation at 80 'C, with gentle agitation (-120 rpm) for a period of 24 hours with approximately 2/3 of the original enzyme dosage, achieved ∼87% saccharification of the carbohydrates present to simple fermentable sugars.

The sugars produced by this enzymatic system can be used as a raw material rich in monosaccharides for the production of biofuel.

ib) �Bioconversion of �pape residues�: �pape� glass and �pape products�

T. emersonii was grown without complement in a variety of paper residues in liquid fermentation (see Example 5). The paper cups were shown to be very effective carbohydrase inducers, producing a strong multicomponent enzyme cocktail with high levels of xylanase enzymatic activities and starch degraders, and cellulase activity levels higher than those presented in conventional growth substrates. The potential of this enzymatic system to effectively release reducing sugars and effect degradation of paper waste was clearly illustrated by biochemical tests and scanning electron microscopy. The effect of thermoenzymatic treatment on the integrity and morphology of the substrate using scanning electron microscopy confirmed the potential of these cocktails as powerful biotechnological tools for converting paper waste. The MEB (scanning electron microscopy) provided clear evidence of extensive degradation of cellulose fibers (complete loss of fibrous structure in certain samples) after treatment of the cellulose-rich substrate with T. emersonii cocktails.

The enzymatic cocktail produced by T. emersonii after 108 hours of growth in paper cups contains an accumulation of cellulose, hemicellulose and starch degrading enzymes and saccharification studies carried out with this multi-component cocktail demonstrate its ability to effectively release glucose and other conventional cellulose reducing sugars and paper waste substrates. It has been discovered that this enzymatic cocktail was active in all the conventional paper and cellulose residue substrates analyzed. Although all substrates were increasingly degraded over time, different biodegradation susceptibilities were shown in response to different substrate compositions.

Before any previous treatment, biodegradable packaging showed the highest susceptibility to enzymatic hydrolysis followed by tissue paper, paper cups and corrugated cardboard. The enzymatic system induced by paper cups, acts optimally, releasing maximum levels of sugar from paper waste, at a temperature of 50 DC, pH 4.5, at an enzymatic dosage of 4 ml / g of substrate and with stirring at 37 rpm. The homogenization of paper cups increased the level of hydrolysis by 2.3 times. Under these experimental conditions (enzymatic dosage of 36 UPF) (filter paper units), a total hydrolysis% of 85% was achieved, representing glucose ∼80% of the released reducing sugars. Glucose and xylose were the main products released (see Figure 3). However, the reduction of the enzymatic dosage to 9 UPF effected an overall hydrolysis of ∼76%. Electron microscopy demonstrated the excellent hydrolytic properties of this cocktail (Figure 4).

The thermal treatment increased the conversion of cardboard by the same enzymatic system, by a factor of 34% (a global carbohydrate hydrolysis based on reducing sugars released from ∼88%), while the combination of both the thermal treatment and homogenization increased the reducing sugars released in 80% producing 1.47 mg / ml glucose (31.7% of the total sugars released). The paper plates were quickly degraded by enzymatic cocktail induced by paper cups representing glucose ∼67% of the total sugars released.

The enzymatic saccharification of paper and food residues in hydrolysis and sequential saccharification (SHF) has been investigated, that is, enzymatic pre-treatment followed by fermentation with yeasts to produce ethanol, and in simultaneous hydrolysis and saccharification (SSF), in which Raw material is continuously generated and fermented immediately by yeast. The reaction temperatures of enzymatic pre-treatment are different in both procedures, that is, higher in SHF since the hydrolyzate is cooled before fermentation and at

19 5

a temperature close to ambient temperatures for yeast growth and fermentation in SSF. Although the enzymes of the strain IMI 393751 of T. emersonii are more efficient and have higher reaction rates, and pasteurization is achieved (and less enzyme is required), the enzymes of T. emersonii still work quite well at 25-37 'C and they compare well with commercial enzyme preparations from other fungal sources. It was found that the raw materials rich in sugars produced were suitable for the production of biofuel (bioethanol and biogas).

ic) Bioconversion of waste of conifers to bioethane production

The remains and woody residues of primary and secondary sources (for example, bark, thinning and processing residues, such as shavings and sawdust) represent an immense resource comprising up to 65-70% dry weight comprising complex carbohydrates such as hemicellulose (∼ 19-28% and mainly xylan and mannan with some other polysaccharides) and cellulose (∼39-46%), which are coated with lignin.

The strain IMI 393751 of T. emersonii was grown in fermentation of liquid or solid state, in woody remains, such as serrin and Sitka spruce and ash shavings to generate enzymatic systems with the appropriate enzyme profile for the conversion of the residues Diana. Different reaction / pretreatment temperatures and enzymatic dosages were investigated. The enzyme systems evaluated included cocktails obtained during the cultivation of T. emersonii in a variety of substrates. Reaction temperatures of 50 'C, 60' C, 70 'C and 80' C were investigated, and several different substrates were used, that is, untreated and pre-treated woody remains. The enzymatic load was also investigated, beginning the initial studies with 60 UPF, subsequently increased to 200 UPF (UPF: filter paper units, a measure of total cellulase activity).

Tabaa 10: `` Salarification of '' hazardous '' expenses by WB / BP1i1: 1) of T. emersonii

Dosage

Temperature Spice Serving Serpentine spike of the moving place in large pieces

% Of Hydrolysis

% Conversion Heiosa �ig)  % Of Hydrolysis % Conversion Heiosa�ig)

60 UPF

50'C 34.3 40.9 0.39 35.2 27.0 0.24

60 UPF

80'C 44.3 50.6 0.49 48.1 39.5 0.38

60 UPF mix *

50'C 48.7 62.3 2.95 41.8 35.1 1.87

60 UPF mix *

80'C 62.4 79.1 3.45 55.9 65.2 3.1

The hexose content is provided as grams released from a 10 g starting batch in a 24-hour reaction period * Mixing = The cocktail-induced hydrolysis of (WB / BP (1: 1) of T was investigated emersonii + coffee residues of C. thermophi / e under buffered and unbuffered conditions, as well as the effect of reaction moisture levels and enzymatic dosing.

Table 11 shows the effects of enzymatic dosing on theoretical ethanol yields.

Table 11: Effect of an enzymatic dosage on% of conversion and performance of aetheric ethanol

Enzyme

IUPF dosage) Reaction Temperature Heiosa iberated ig) % conversion Performance of theoretical ethane / ton of raw material)

Mixture*

60 70 'C 4,5 70.0 325.5

Mixture*

200 70 'C 5.1 76.4 345.4

* Mixture = Cocktail-induced (WB / BP (1: 1) of T. emersonii + coffee residues of C. Thermophi / e; the hexose content is provided as grams released from a starting batch of 10 g in a 24 hour reaction period

Execution �: car Biomass saccharification

Preparation of test substrate. Spruce chips (2-10 mm in diameter) were impregnated with sulfur dioxide (humidity 3% w / v) for 20 minutes at room temperature at an absorption rate of 2.5% w / w moisture. Spruce treated with SO2 was steam treated at 215 'C for 2-5 minutes. The hemicellulose content was almost completely hydrolyzed; The recovery of solids was 60-65% of the starting raw material. MGBG Thermoenzyme Enzymes: MGBG 1, MGBG 2, MGBG 3 and MGBG 4 numbered. Commercial enzymes Celluclast 2 l from T. reesei and Novozym 188 from A. niger (Novo Industri A / S, Bagsvaerd, Denmark) were used.

Evacuation of enzyme hydrolysis

In conventional 300 ml, 1 l and 10 l reaction vessels, with stirring at 30130 rpm, conventional enzymatic hydrolysis was performed at 37 'C, 50' C and 60 'C. The enzymatic dosage of each enzyme was 32 UPF per g of cellulose in a buffered substrate solution (Gilleran, 2004). The pH of the reaction buffer was adjusted to pH 5.0 for the

5 MGBG enzymes and at pH 4.8 for commercial preparations. The samples were removed at temporary intervals and the enzymatic action was finished by boiling each reaction mixture (and controls) for 10 minutes. At the lowest reaction temperatures (37-50 'C), 2 of the enzyme preparations of the invention performed as well as the commercial preparation Celluclast, and (ii) the yield of 3 of the MGBG enzymes is in the same range as that of commercial Celluclast (and Cellulclast / Novozym mixture).

10 Glucose yield using the composition of the invention was similar to that of optimized commercial preparations but produced higher levels of additional fermentable sugars than commercial enzymes.

Enzyme preparations of the invention performed better than commercial enzymes / combinations at higher reaction conditions (60-70 'C), in terms of the overall degree of hydrolysis, product yield and enzymatic stability. A lower enzymatic dosage could be used at higher reaction temperatures to achieve a similar hydrolysis yield (depending on the enzyme preparation, only 62.5-78% of the commercial enzyme load was needed). They are also less affected by inhibitory substances present in the substrate previously treated with steam and higher concentrations of glucose and cellobiose in sugar-rich hydrolysates. They also produced a greater amount of sugar in 24 hours at 60 'C, compared to that achieved by commercial enzymes in 72 hours at 50' C, which is the temperature of

20 optimal performance for commercial enzymes.

Table 12 shows the key results for enzymatic hydrolysis, while Table 13 shows the best reaction temperature / reaction time combinations for the optimal% hydrolysis, for each of the commercial and enzymatic compositions of The invention tested.

Tabaa 12: Summary �de�resutado� comparing�en�im�enzymatic performance / hydroaitic power�of 25 commercial enzymatic preparations and ofMGBG �en�picea�pre · treated

Temp. reaction i'C)

Reaction time ih) Valuation criteria / products ig / a) Ceaauacaast Ceaar Novozym i24: 4) MGBG 1 MGBG 2 MGBG 3 MGBG4

50 'C

24 h Hexose 7.33 13.85 6.8 11.9 3.84 8.64

Pentosa

0.83 0 1.1 1.93 0.59 1.33

Jealous

2.58 0 1.22 2.14 1.28 2.88

% Hydrolysis *

40.2 51.9 34.1 59.8 21.4 48.1

48 h

Hexose 14.1 20.4 14.45 15.75 7.7 15.55

Pentosa

1.59 1.1 2.34 2.55 1.18 2.52

Jealous

4.96 0.6 1.01 0.3 2.57 0.35

% Hydrolysis

77.3 82.7 66.6 69.6 42.9 69.0

72 h

Hexose 12.98 20.8 21.25 21.48 9.16 20.84

Pentosa

2.26 2.34 3.4 3.47 1.40 3.88

Jealous

1.33 0.3 0.93 0.12 3.05 0.16

% Hydrolysis

62.0 87.8 95.8 93.9 51.0 91.3

60 'C

24 h Hexose 4.18 3.74 20.55 22.12 4.65 10.46

Pentosa

0.47 0.31 2.1 1.87 0.71 1.49

Jealous

1.47 0.17 3.69 1.34 1.55 3.61

% Hydrolysis

22.9 15.8 98.6 94.8 25.9 58.3

48 h

Hexose 8.04 11.86 22.71 23.02 9.32 17.38

Pentosa

0.9 0.63 2.47 2.55 1.43 2.67

(continuation)

Reaction temperature i'C)

Reaction time ih) Valuation criteria / products ig / a) Ceaauacaast Ceaar Novozym i24: 4) MGBG 1 MGBG 2 MGBG 3 MGBG 4

Jealous

2.83 0.34 1.31 0.39 3.11 5.80

% Hydrolysis

44.1 48.0 -100.0 97.2 51.9 96.8

72 h

Hexose 12.98 14.80 23.51 23.33 12.56 21.9

Pentosa

2.26 1.33 2.53 2.67 1.92 2.43

Jealous

1.33 0.17 0.85 0.17 5.7 1.34

% Hydrolysis

62.0 61.3 -100.0 98.0 75.6 96.1

70 'C

24 h ** Hexose 1.2 1.07 21.72 24.11 16.8 20.98

Pentosa

0.14 0.09 2.39 2.07 2.58 2.73

Jealous

0.42 0.05 2.43 0.55 5.60 2.44

% Hydrolysis

6.6 4.5 99.4 -100.0 93.5 97.9

*% of hydrolysis: total released sugars expressed as a% of the total available sugars in the substrate; ** subsequent time points were not shown since MGBG enzymes did not achieve complete hydrolysis

Table 13: `` Combination '' of temperature of reaction / reaction time for a minimum% of hydrolysis, minimum, for any of the enzymes, commercial and MBGs investigated

Enzymatic preparation

Optimum reaction temperature i'C) 6th reaction time ih) % of hydrolysis Heiosa g / a

Celluclast

50.0 48 h 77.3 14.1

Cell + Novozym (24: 4)

50.0 48 h 82.7 20.4

50.0

72 h 87.8 20.8

MGBG 1

50.0 48 h 66.6 14.45

50.0

72 h 95.8 21.25

60.0

24 h 98.6 20.55

70.0

24 h 99.4 21.72

MGBG 2

60.0 24 h 94.8 22.12

70.0

24 h -100.0 24.11

MGBG 3

70.0 24 h 93.5 16.8

MGBG 4

70.0 24 h 97.9 20.98

70.0

48 h 96.8 17.38

Simultaneous saccharification and fermentation (SSF)

Discontinuous SSF experiments with spruce hydrolyzate were performed to compare the performance of different enzyme preparations. Fermentation was carried out at a concentration of spruce fibers of 4%, the pretreated material was diluted with sterile water to the desired concentration. The pH was maintained at 5.0 with the addition of

2M NaOH. The fermentation temperature was 37 DC and the stirring speed was 500 rpm. The reactor medium was sprayed with nitrogen (600 ml / minute) and the CO2 content was measured with a gas analyzer. The enzyme preparation was added directly to the fermenter at a load of 25 units of filter paper (UPF) / g cellulose. The fermentation medium is supplemented with nutrients: (NH4) 2HPO4 0.5 g / l, MgSO4.7H2O 0.025 g / l and 1.0 g / l

5 yeast extract. The concentration of yeast cell mass (baker's yeast, Saccharomyces cerevisiae) added was 5 g / l and all SSF experiments were performed at 37 AD for 72 hours. Samples were removed at various time intervals, centrifuged in 1.5 ml microcentrifuge tubes at 14,000 g for 5 minutes (Z 160 M; Hemle Labortechnik, Germany), the supernatant was then prepared for HPLC analysis.

Hydrolysis products generated during degradation of lignocellulosic materials by HPLC were analyzed. Be

10 separated, cellobiose, glucose, xylose, galactose, mannose, HMF and furfural in a polymeric column (Aminex HPX87P) at 85 AD, the mobile phase was millipore water at a flow rate of 0.5 ml / minute. The concentrations of ethanol, glycerol and acetate were determined using an Aminex HPX-87H column at 60 DC, using a Shimadzu HPLC system equipped with a refractory index detector. The mobile phase was a 5 mM aqueous solution of H2SO4 at a flow rate of 0.5 ml / minute. The ethanol produced was also determined using an enzyme-linked assay (r-Biopharm,

15 Germany).

Yeast growth takes place in two phases. Carbon dioxide is an important secondary product of the ethanol fermentation process since anaerobic fermentation of one mole of glucose produces one mole of ethanol and two moles of carbon dioxide. Therefore, measuring the concentration of carbon dioxide in the outlet gas is an indirect measurement of the fermentation rate. In the first phase of growth the

20 glucose is available and ethanol is formed, and the initial rapid response to glucose present is represented by an increase in the evolution of CO2.

The results obtained during the SSF are provided in Table 3. As mentioned above, the total elapsed time for the SSF, for each enzymatic preparation was 72 hours, and the temperature used for the SSF was 37 DC. The efficiency of fermentation was determined by dividing the actual concentration of ethanol.

25 produced (g / l) among the total theoretical ethanol (g / l) that would be produced if all available substrate were converted into fermentable soluble sugar and all sugar was converted to ethanol, and multiplied by 100.

Based on the data obtained, the ethanol yields for each enzyme / SSF combination are provided in liters of ethanol / dry ton, and the corresponding units, liters of ethanol / dry tons.

30 Tabaa 14: �Summary �de�a��iperiments�of�SSF

Enzymatic preparation

Etanoa g / a Efficiency of fermentation i%) Etanoa yield ia / ton) Etanoa�ia yield / ton) Cost of U.S. Enzyme / ton)

Cell + Novo (24: 4) # 1

5.83 54.0 178.78 215.68 Not available

Cell + Novo (24: 4) # 2

9.10 84.3 279.05 334.94 Not available

1 * MGBG

5.02 46.5 153.88 185.6 ∼17 US $

MGBG 1

7.33 67.9 224.62 270.97 ∼21.76 US $

MGBG 2 *

7.24 67.1 221.89 267.68 ∼16.8 US $

MGBG 2

7.89 73.1 241.79 291.68 ∼23 US $

MGBG 3 **

8.62 79.9 264.46 319.01 ∼17.4 US $

MGBG 4 **

9.67 89.6 296.37 357.54 ∼15.4 US $

* An enzymatic load of 21.3 UPF was used instead of 32.0 UPF ** More optimized mixtures of MGBG 3 and MGBG 4.

Example 9: Comparison of commercial enzymes and MGBG in a strategy of hydrolysis and sequential fermentation isSHF) for production of bioethane.

The bioethanol yields obtained by sequential hydrolysis and fermentation were investigated for the commercial and enzymatic preparations of the invention. An advantage of the SSF is that the procedure consists of an initial rapid fermentation and metabolism of monomeric sugars resulting from the pretreatment stage. A glucose is released from the substrate by the action of the hydrolytic enzymes that have been added, the fermentation is rapid, which means that, in the SSF, the concentration of free sugars always remains low. In the SSF, the fermentation rate decreases over time as a result of a decrease in the rate of substrate conversion by enzymes, or inhibition of yeast metabolism, whichever

10 speed limiter. The data obtained in these experiments are summarized in Table 15.

Tabaa 15: �Summary � of�SHF

Enzymatic preparation

Hydr6ysis temperature and time Etanoa g / a Fermentation efficiency i%) Etanoa yield ia / ton) Ethane yield ia / ton) Cost of aa enzyme (U.S.) / tonne))

Celluclast

50 DC for 48 h 7.21 66.8 220.94 269.55 Not available

Cell + Novo (24: 4) # 1

50 DC for 48 h 9.35 86.7 286.7 345.89 Not available

Cell + Novo (24: 4) # 2

50 DC for 48 h 10.42 96.6 319.53 385.45 Not available

MGBG 1

60 DC for 24 h 10.50 93.37 321.98 388.40 ∼21.76 US $

1 * MGBG

70 DC for 24 h 11.10 -100.0 340.38 410.62 ∼21.76 US $

MGBG 2

60 DC for 24 h 11.30 -100.0 346.51 418.05 ∼21.8 US $

MGBG 2 *

70 DC for 24 h 12.32 > 100.0 ** 377.79 455.91 ∼21.8 US $

MGBG3%

70 DC for 24 h 8.59 79.6 263.26 317.40 $ 21.4

MGBG 4

70 DC for 24 h 10.72 99.4 328.73 396.55 ∼21.4 US $

* Enzymatic preparation used at higher reaction temperature. % This enzyme cocktail is particularly effective in releasing fermentable sugars from hemicellulose-rich substrates and would not be expected to be as effective in a substrate that has a significant part of the hemicellulose fraction removed (pre-treatment stage).

 The HPLC analysis confirmed that the main products formed are monosaccharides (simple sugars) with very small amounts of larger oligomers formed (being cellobiose, which is a disaccharide, the main one,

or unique, major chain sugar present in hydrolysates).

15 Execution 10�Apps �for�thinking� animals:

Each enzymatic composition was evaluated in individual target applications, carrying out laboratory scale model studies with 5-25 grams of the substrate (cereal, cereal flour or other vegetable residue) in final reaction volumes of 50-250 ml, at pH 2, 5-7.0 and 37-85 AD, with or without agitation. Enzymatic performance was evaluated with and without substrate pretreatment, i.e. pre-treatment of mild steam (105 DC, 55.16 kPa (8 psi) for 5 minutes), 20 grinding using a mortar, homogenization in a Parvalux or Ultraturrax homogenizer . For tissues / remains of soft fruits and vegetables, the substrate is macerated without much precision, mixing, and incubated with enzyme, without pretreatment. Substrate hydrolysis was monitored by (i) measurement of released reducing sugars and tests to detect and quantify individual sugars, (ii) TLC and HPLC analysis of confirmation of sugar hydrolysis products, (iii) weight reduction analysis / volume of the rest, (iv) comparison of contents of

Cellulose, hemicellulose, starch and pectin before and after enzymatic treatment, and (v) physical analysis of substrate degradation by scanning electron microscopy (SEM) for fibrous substrates such as paper and woody residues.

Tabaa 16

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimal treatment temperature�i�C) products

MGBG15

CBH I (25-30%) CBH II (1-5%) β (1,3) 4-glucanase (10-15%) β-glucosidase (2-10%) Xylanase (44-48%) β-Xylosidase 0.1-1.5%, α-Glucuronidase 0.1-2.0% αL-arabinofuranosidase 0.11.5%; peptinolytic enzymes and esterases 2-5% protease 7-15% Enhanced digestibility Wheat, oats > 60 DC (up to 75 DC) Mainly glucooligosaccharides, xylooligosaccharides and some other minor amounts of various sugars

MGBG16

CBH I (10-15%) CBH II (10-15%) β (1,3) 4glucanase (25-30%) β-glucosidase (2-8%) Xylanase (25-30%) β-Xylosidase 1- 2.0%, α-Glucuronidase 1-2.0% α-Larabinofuranosidase 0.12.0%; 5-10% peptinolytic enzymes, ∼5% starch modifying activity; oxidoreductase / oxidase and esterases 5-10% protease 10-15% Enhanced digestibility Feed enriched in Galacto-yGlucooligosacaridos Antioxidant release Wheat; oats; rye > 60 DC (up to 80-85 C) Galactooligosaccharides, glycooligosaccharides and xylooligosaccharides

25

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimum treatment temperature�i�C) products

MGBG17

CBH I (5-10%) CBH II (5-10%) β (1,3) 4-glucanase (20-30%) β-glucosidase (2-8%) Xylanase (30-35%) - Xylosidase 1 -2.0%, α-Glucuronidase 1-3.0% α-L-arabinofuranosidase 1.54.0%; 5-10% peptinolytic enzymes, 5-8% dealmidon modifying activity; 1-4% oxidoreductase / oxidase and esterases, 18-25% protease Enhanced digestibility Wheat; oats; rye; corn, barley; rape > 60DC (up to 80DC) Glucooligosaccharides and xylooligosaccharides

MGBG13

CBH I (5-10%) CBH II (5-10%) �� β (1,3) 4-glucanase (25-40%) β-glucosidase (∼5%) Xylanase (18-25%) β- Xylosidase 1-2.0%, Exoxylanase 1-3.0%, α-Glucuronidase 8-10%, α-L-arabinofuranosidase 1.55.0%; peptinolytic enzymes 1015%, modmidon modifying activity 5-7%; other hemicellulases 5-10%, oxidoreductase / oxidase and esterases 1-4%, protease 8-12% Enhanced digestibility Antioxidant release Wheat; oats; rye; corn, barley; rape > 60DC (up to 85 / 90DC) Glycooligosaccharides and xylooligosaccharides, some monosaccharides and disaccharides from pectin polymers (e.g., arabinooligosaccharides, galacturonic acid, rhamnose, etc.)

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimum treatment temperature�i�C) products

MGBG19

CBH I (10-15%) CBH II (10-15%) β (1,3) 4-glucanase (25-30%) β-glucosidase (∼10-15%) Xylanase (20-27%) β- Xylosidase 4.0-7.0%, Exoxylanase 5-10%, α-Glucuronidase 1-4% α-L-arabinofuranosidase 2.05.0%; 5% peptinolytic enzymes, mid5% dealmidon modifying activity, other hemicellulases ∼ 5%, oxidoreductase / oxidase and esterases 1-4%, protease ∼25% Enhanced digestibility; Flexibility for different feeds Sorghum; rape; also other cereals, for example wheat; oats; rye; corn; barley > 60DC (up to 80 / 85DC) Glucooligosaccharides and xylooligosaccharides, some monosaccharides or some other minor amounts of sugars from peptide polymers

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimum treatment temperature�i�C) products

MGBG20

CBHI (5-10%) CBH II (5-10%) β (1,3) 4-glucanase (20-32%) β-glucosidase (∼2-9.5%) Xylanase (15-25%) β -Xylosidase 15-20.0%, Exoxilanase 10-15%, α-Glucuronidase 2-5%, α-L-Arabinofuranosidase 1.55%; peptinolytic enzymes 1015%, modifier activity dealmidon ∼10%; other hemicellulases ∼5% , oxidoreductase / oxidase and esterases 2-5%, protease�∼7-10% Enhanced digestibility; Wheat, barley ycenteno > 60DC (up to 80 / 85DC) Glucooligosaccharides and xylooligosaccharides or some other minor amounts of sugars from peptide polymers

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimum treatment temperature�i�C) products

MGBG21

CBH I (5-10%) CBH II (5-10%) β (1,3) 4-glucanase (15%) β-glucosidase (∼2-10%) Xylanase (50-55%) Xylosidase 1-5 %; Exoxylanase 5-10%, α-Glucuronidase 1-4%, α-L-Arabinofuranosidase 25%; peptinolytic enzymes 2025%, modmidon modifying activity 4-6%; other hemicellulases 7-10%, oxidoreductase / oxidase and esterases 2- 4%, protease ∼10% Enhanced digestibility; Fructooligosaccharide release Wheat, barley and corn; also beet pulp > 60DC (up to 80 / 85DC) Glycooligosaccharides, xylooligosaccharides, some monosaccharides or some other minor amounts of sugars from peptide polymers and fructooligosaccharides

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimum treatment temperature�i�C) products

MGBG22

CBH I (∼5%) CBH II (∼5%) β (1,3) 4-glucanase (27-45%) β-glucosidase (∼1-5%) Xylanase (16-25%) Xylosidase 0.5 -4%, Exoxylanase ∼10%, α-Glucuronidase 0.5-4%, α-L-Arabinofuranosidase 15%; peptinolytic enzymes 510%, dealmidon modifying activity 4-8%; other hemicellulases 8-10%, oxidoreductase / oxidase yesterases 1-4% protease ∼5-6% Enhanced digestibility; Formation deglucooligosacaridos Wheat, barley, rye > 60DC (up to 85 / 90DC) Glucooligosaccharides, xylooligosaccharides, some monosaccharides or some other minor amounts of sugars, etc.

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum Optimum treatment temperature�i�C) products

MGBG23

CBH I (∼3-6%) CBH II (∼3-6%) β (1,3) 4-glucanase (35-45%) β-glucosidase (∼3-6%) Xylanase (∼30%) Xylosidase 0.5-2.0%, Exoxylanase -52-5%, α-Glucuronidase 0.5-2%, α-L-Arabinofuranosidase 15%; peptinolytic enzymes 510%, modifying activity ofmidmidon ∼2-4%; other hemicellulases ∼10%, oxidoreductase / oxidase and esterases 6-10% protease ∼8-10% Enhanced digestibility; Glucooligosaccharide, xylooligosaccharides High potential for antioxidant release Wheat, barley, rye > 60DC (up to 85 / 90DC) Low DP glycooligosaccharides, xylooligosaccharides, with significant monosaccharides or some other minor amounts of sugars, etc.

For feeds enriched in key oligosaccharides, for example Galactooligosaccharides - the MGBG 16 Glucooligosaccharide enzyme cocktail is better - the MGBG 16 and 22 Fructooligosaccharides are better - the MGBG cocktail 21 is better

For feed preparations enriched with antioxidants, the best cocktails to use for treatment are those prepared in: MGBG 13, 16 and 23.

Example 11� Feed �Applications� for �monogastric animations:

Studies were conducted at temperatures in the range 50-85 AD (with stirring at 140 rpm), with fractions of raw cereals (batches of 1-5 g). The hydrolysis of carbohydrates in the substrate was monitored, for each enzymatic preparation (maximum dosage 0.5 IU per g of substrate), for 24 hours quantifying the released reducing sugars, and products formed in samples removed from the reaction mixture at periodic intervals .

Tabaa 17

C6ctea

Composition Daily Appearance Substratum Temperature products

c6ctea�i%)

6th optimal treatment)

MGBG

CBH I (1-5%) Digestibility Barley, > 60DC (up to Mainly

24

CBH II (1-5%) β (1,3) 4-glucanase (27-43%) β-glucosidase (210%) Xylanase (44-48%) -Xylosidase 0.10.4%, α-Glucuronidase 0 , 1-3.0% α-L-Arabinofuranosidase 0.1-0.4%; peptinolytic enzymes and esterases 5-10% protease 20-25% boosted Feed production based on easily digested cereals low in pentose for poultry and pigs, in which the water binding properties of β1,3 have been reduced; 1,4-viscous glucans by enzymatic fragmentation Millet, wheat, oats. Barley soy diets. Corn-wheat diets. Wheat-orange diets 85DC) glucooligosaccharides, xylooligosaccharides and some other minor amounts of various sugars. Some of the oligosaccharide products (and peptides) would have health-enhancing properties

MGBG 17

Like before

MGBG 25

CBH I (0.5-2.5%) CBH II (0.5-2.5%) β (1,3) 4-glucanase (15-20%) β-glucosidase (410%) Xylanase (70- 88%) -Xylosidase 0.10.4%, α-Glucuronidase 0.1-2.0% α-L-Arabinofuranosidase 0.1-1.0%; peptinolytic enzymes and esterases 1-6% protease 8-10% Enhanced digestibility Feed production based on easily digested cereals low in pentose for poultry and pigs. More than 30% hydrolysis of non-cellulosic βglucan present in rye, which is accompanied by a notable reduction in viscosity Barley, oats, rye > 60DC (up to 85DC) Mainly glucooligosaccharides, xylooligosaccharides (DP 26)

Significant hydrolysis of xylan and non-cellulosic β-glucan fraction were observed, which resulted in the formation of medium-to-long chain oligosaccharide hydrolysis products, which would also be suitable fermentation substrates for probiotic microorganisms.

Biogas Production Execution

5 Two anaerobic, 10 l higher flow hybrid reactors (UAHR; Reynolds, 1986) were used, one maintained at 37 DC and the other at 55 DC for biogas production by individual mixed populations of mesophilic and thermophilic bacteria, respectively. The sugar-rich hydrolyzate was pumped through the mud bed and degraded by the communities of microorganisms present. A separating device was used on the top of the reactor to separate the gas produced from any sludge particles that could have broken up during digestion.

10 anaerobic Both reactors were continuously evaluated during a 650-day operating period, monitoring the effectiveness of COD withdrawal (APHA, 1992), total carbohydrate reduction (Dubois procedure) and methane production. The production of fatty acids, an indicator of sugar metabolism, and the production of methane by gas chromatography (GC) were monitored.

Biofuel can be produced from various raw materials. Many of these require the use of different

15 enzymatic cocktails. MGBG 16 is the best cocktail for the production of raw materials from the food and vegetable waste listed below for the production of biogas by anaerobic digestion.

Tabaa 1�

Treatment C6

Remains of used waste Main sugar free % Carbon Reduction by Anaerobic Messenger Digestion % Carbon Reduction by Anaerobic Termological Relief % of methane in biogas by Anaerobic Messenger Digestion % of methane in biogas by Anaerobic Termiophy Digestion

MGBG 16

Carob Xylose, mannose glucose, cellobiose 99-100 94.0-96.6 58.0 49.0

Bread

Xylose, xylobiose, Glucose, cellobiose 96.1-99.7 96.0-99.3 71.9 69.0

Apple Pomace

Glucose, Galacturonic acid, Fructose, Arabinose, Galactose, Xylose 96.0-99.3 95.4-99.5 58.0 52.0

Paperboard

Xylose, Glucose (older trace oligos) 95.4-96.9 94.0-96.6 63.0 49.0

MGBG 18

Mixed vegetable / fruit waste (restaurant) Xylose, Glucose, Galacturonic Acid, Fructose; Arabinose, Galactose, Ramnosa

(*% of methane in biogas is normally 50-65% maximum);

 The data in Table 18 were obtained at retention times of only 3 days, unlike the literature reports, in which retention times are normally between 9 and 30 days. Retention times 20 indicate the period it takes (or latency period) the metabolism of carbohydrate raw materials and the

biogas production.

Execution 12 Bioethane Production

Conventional enzymatic hydrolysis was performed at 37 DC, 50 DC and 60 DC in 300 ml and 1 L containers with stirring at ∼ 130 rpm. The enzymatic dosage was 32 UPF of each enzyme preparation per gram of cellulose in a buffered substrate solution (as described by Gilleran, (NUI, Galway, Ph.D. Thesis, 2004) total processed weight =

5 to 100 g in laboratory scale studies). The pH of the reaction buffer was adjusted to pH 5.0. The samples were removed at timed intervals and the enzymatic action was boiling for 10 minutes. The following was measured during a period of 0-72 hours:

• Release of reducing sugars and detection and quantification of individual sugars (expressed as g / l)

• HPLC analysis of the sugars produced by hydrolysis (quantities of product expressed in g / l) 10 • Reduction of weight / volume of the rest

• Cellulose fiber content before and after enzymatic treatment

Scanning electron microscopy (SEM) of substrate integrity, before and after production, by enzymatic treatment, of key fatty acids during sugar metabolism by anaerobic bacteria, and methane production, were monitored by chromatography of gases (CG). The bioethanol produced by

15 yeast fermentation using two strategies, an enzyme-linked test kit, for the quantification of ethanol (r-Biopharm, Germany) and also by high performance liquid chromatography (HPLC).

Residues of sitka spruce hydrolyzate, paper and woody debris (mixture of coniferous debris, mainly sitka spruce) were tested in laboratory scale studies. Two formats of ethanol production, SHF (Sequential Hydrolysis and Fermentation, Secuentia / Hydro / isis and Fermentation) and SSF (Saccharification) were investigated

20 and Simultaneous Fermentation, Simu / taneous Saccharification and Fermentation).

Tabaa 19: Enzymatic composition of the enzyme for bioethane production

Enzyme

MGBG�1a% MGBG2b% MGBG�3c% MGBG4d%

CBH I CBH II β- (1,3) 4-glucanase

20-28 15-20 20-25 15-20 20-28 20-26 15-17 22-26 25 12-15 24-30 20-22

β-glucosidase Xylanase β-Xylosidase, α-Glucuronidase α-L-Arabinofuranosidase (Other hydrolases, including Pectinolytic enzymes, Phenolic acid and acetyl (xylan) esterases, Protease; Lignin modifying oxidase activities)

10-12 20-25 5-10 5-8 0.5-2.0 8-15 10-11 18-30 8-10 8-10 0.5-2.0 6-17 11-15 24-27 10-12 6-8 2-4.0 12-15 ∼10.0 20-30 5-8 8-10 1.5-3.0 10-15

Study data at 1 l laboratory scale in SHF and SSF; Hydrolysis values are based on available cellulose and any residual hemicellulose in substrate

34 35

Tabaa 20: Comparison of the production of hydroethane bioethane generated by spikes generated by inventions and commercial enzymes.

Dynamic Preparation

Reaction Time Optimum) % �de�Hidr6aisis * Heiosa�g / a Etanoa�SSF�g / a�i37�C) Efficacy of `` Fermentation of SSF '' 37�C) Reaction Temperature Optimum�i�C) �for�SHF Etanoa�de�SHF�g / a Efficacy of Fermentation of HSF

Celluclast

48 h 74.5 13.59 4.69 43.44 50.0 6.93 64.21

Cell + Novozym (24: 4)

48 h 78.9 19.46 nd nd 50.0 8.97 83.18

72 h

82.3 19.50 5.70 52.80 50.0 nd nd

MGBG 1

48 h 57.9 12.56 4.83 44.74 50.0 nd nd

72 h

87.2 19.34 nd nd 50.0 nd nd

24 h

88.1 19.14 nd nd 60.0 10.29 91.50

24 h

90.4 19.75 6.89 63.82 70.0 10.51 93.46

MGBG 2

24 h 89.3 20.84 7.04 65.25 60.0 10.39 92.39

24 h

91.2 22.32 8.01 74.21 70.0 10.53 93.64

MGBG 3

24 h 87.3 15.69 8.58 79.53 70.0 8.71 80.71

MGBG 4

24 h 92.2 19.76 9.55 88.49 70.0 10.65 98.75

48 h

89.9 16.14 nd nd 70.0 nd nd

Tabaa 21: Production of `` bioethane '' of `` waste of paper and waste '' of conifers generated by `` those '' militias is of `` enzymes '' were of T. emersonii in comparison with `` traded '' business enzymes. boaaboratory�de�1a �en�SHF and�SSF scale; `` Several '' values of hydrisisasis� are�basan�aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa and

Dynamic Preparation

Optimum Temperature Reaction�i�C) Reaction Time Optimum) % �de�Hidr6aisis * Heiosa�g / a�i37�C) Etanoa�deSSF�g / a�i37�C) Efficacy of the SSF Fermentation Etanoa�deSHF�g / a�i37�C) Efficacy of Fermentation of HSF 37)

Paper waste

MGBG 1

70.0 24 h 76.5 13.59 8.36 74.3 7.18 66.5

MGBG 3

70.0 24 h 79.2 17.57 7.89 70.2 8.08 74.6

MGBG 4

70.0 24 h 83.4 19.46 7.54 69.9 8.22 76.2

Coniferas waste

MGBG 1

70.0 24 h 63.8 11.21 7.57 67.3 7.03 65.1

MGBG 3

70.0 24 h 74.9 13.09 7.92 70.4 7.53 69.8

MGBG 4

70.0 24 h 77.9 13.57 7.74 71.8 7.82 72.5

Tabaa 22: Other cocktails are for the production of hydrogenated products in sugars, which were to be distributed from residues of vegetables / fruits

C6ctea

Composition of dairy%) Daily Appearance Substratum 6th optimal treatment temperature Sugar from years products

MGBG 18

CBH I (5-10%) CBH II (5-10%) β (1,3) 4-glucanase (2540%) βglucosidase (∼5%) Xylanase (18-25%) + [-Xylosidase 1-2, 0%, Exoxylanase 1-3.0%, α-Glucuronidase 8-10%, α-L-Arabinofuranosidase 1.5-5.0%; 10-15% peptinolytic enzymes, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 5-7% starch modifying activity; other 510% hemicellulases including βgalactosidase, oxidoreductase / oxidase and esterases 1-4% + protease 8-12%] Production of fermentable sugars for the manufacture of biofuel and / or other products of high value, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, etc. Mixed vegetable / fruit waste (restaurant) > 60DC (up to 80DC) Xylose, Glucose, Galacturonic acid, Fructose, Arabinose, Galactose, Ramnosa

MGBG 32

CBH I (1-5%) CBH II (1-5%) β (1,3) 4-glucanase (2025%) βglucosidase (8-12%) Xylanase (30-35%) + [-Xylosidase 0.51 , 5%, α-L-Arabinofuranosidase 12.5%; 18-25% peptinolytic enzymes, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, -105-10% starch modifying activity; oxidoreductase / oxidase and esterases 8-15% + protease 10-15%] Production of fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, aroma and flavor precursors, etc. They can also be used for extracting boosted juice and producing "healthy drinks" Mixed vegetable / fruit waste (restaurant) Also mixed soft fruits > 60DC (up to 80DC) Xylose, Glucose, Galacturonic acid, Fructose, Arabinose, Galactose, Ramnosa

(continuation)

C6ctea

Composition of the dairy%) Daily Appearance Substratum Treatment temperature 6th optimal�i�C) Sugar �de�aos products

MGBG 6

CBH I (1-5%) CBH II Production of Waste > 60DC (up to Xilosa,

(4%

(1-5%) sugars mixed of 80DC) Glucose,

CarrotBP;

fermentable for vegetables / fruits

1: 1)

β (1,3) 4-glucanase (2228%) βglucosidase (10-15%) Xylanase (25-30%) + [-Xylosidase 0.7-2.1%, α-L-Arabinofuranosidase 24%; 20-25% peptinolytic enzymes, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, ∼5-8% starch modifying activity; oxidoreductase / oxidase and esterases 12-15% + protease 8-12%] the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, aroma and flavor precursors, etc. They can also be used for extracting boosted juice and producing "healthy drinks" (restaurant) Also mixed soft fruits Galacturonic acid, Fructose, Arabinose, Galactose, Ramnosa

Tabaa 23: `` Cocktails '' for `` production '' of hydrofoils `` in sugars '' was fermenting and distributing `` fluxes '' of `` Arabian '' wastes.

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum 6th optimal treatment temperature Sugar �de�aos products

MGBG

CBH I (20-28%) Production of Flow of 65DC (up to Xylose, Glucose,

one

CBH II (15-20%) β (1,3) 4glucanase (20-25%) β-glucosidase (10-12%) Xylanase (20-25%) -Xylosidase (5-10%) α-Glucuronidase (5 -8%) α-L-Arabinofuranosidase (0.5-2.0%) (8-15%: other hydrolases, including Peptinolytic enzymes, phenolic acid and acetyl (xilan) esterases, Protease; Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) as the main sugars, with some galactose

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum 6th optimal treatment temperature Sugar �de�aos products

MGBG

CBH I (15-20%) Production of Flow of 60DC (up to Glucose and

2

CBH II (20-28%) β (1,3) 4glucanase (20-26%) fermentable sugars for manufacturing hospital waste rich in cellulose 85DC) Xylose, like the main sugars, with

β-glucosidase (10-11%)

biofuel sterilized some mannose

Xylanase (18-30%) -

and galactose

Xylosidase (8-10%) α-

Glucuronidase (8-10%)

α-L-Arabinofuranosidase

(0.5-2.0%) (6-17%:

other hydrolases, including

Peptinolytic enzymes, acid

phenolic and acetyl (xilan)

esterases, protease;

oxidase activities

Lignin modifiers)

MGBG

CBH I (12-15%) Production of Flow of 60DC (up to Glucose and

4

CBH II (24-30%) β (1,3) 4glucanase (20-22%) fermentable sugars for manufacturing hospital waste rich in cellulose 85DC) Xylose, like the main sugars, with

β-glucosidase (∼10%) Xylanase (20-30%) -

biofuel sterilized some mannose and galactose

Xylosidase (5-8%) α-

Glucuronidase (8-10%)

α-L-Arabinofuranosidase

(1.5-3.0%) (10-15%:

other hydrolases, including

Peptinolytic enzymes, acid

phenolic and acetyl (xilan)

esterases, protease;

oxidase activities

Lignin modifiers)

MGBG

CBH I (50-55%) Production of Flow of 60DC (up to Mainly

27

CBH II (20-25%) β (1,3) 4glucanase (12-20%) fermentable sugars for manufacturing hospital waste rich in cellulose 85DC) glucose; smaller amount of xylose

β-glucosidase (5-8%)

biofuel sterilized

Xylanase (∼5%) -

Xylosidase (0.1-0.5%) α-L-

Arabinofuranosidase (0.5

2.0%) (5-8%: others

hydrolases, including

Peptinolytic enzymes

selected esterases;

0.5-1.5% protease;

0.5 oxidase activities

1.5%)

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum 6th optimal treatment temperature Sugar �de�aos products

MGBG 31

CBH I (5-10%) CBH II (∼15%) β (1,3) 4-glucanase (28-32%) β-glucosidase (8-12%) Xylanase (22-30%) -Xylosidase 10- 15% α-Glucuronidase (2-5%) α-L-Arabinofuranosidase (13.0%) (20-25%: other hydrolases, including Peptinolytic enzymes, Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes , Lignin modifying oxidase activities) Production of fermentable sugars for biofuel manufacturing Flow of sterile cellulose-rich hospital waste 60DC (up to 85DC) Glucose and xylose, like the main sugars, with some mannose, galactose and galacturonic acid

MGBG

CBH I (20-25%) Production of Flow of 60DC (up to Glucose and

3. 4

CBH II (10-15%) β (1,3) 4glucanase (24-28%) β-glucosidase (10-15%) Xylanase (25-30%) -Xylosidase (5-8%) α-Glucuronidase (1 -3%) α-L-Arabinofuranosidase (23.0%) (5-10%: other hydrolases, including Peptinolytic enzymes, Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) Xylose, like the main sugars, with some mannose, galactose and galacturonic acid

MGBG

CBH I (10-12%) CBH II Production of Flow of 60DC (up to Glucose and

35

(∼15%) β (1,3) 4-glucanase (30-40%) βglucosidase (5-10%) Xylanase (15-28%) -Xylosidase (2-5%) α-Glucuronidase (1-3% ) α-L-Arabinofuranosidase (13.0%) (∼15%: other hydrolases, including Peptinolytic enzymes, Phenolic acid and acetyl (xylan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) Xylose, like the main sugars, with some mannose, galactose and galacturonic acid

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum 6th optimal treatment temperature Sugar �de�aos products

MGBG

CBH I (10-12%) CBH II Production of Flow of 60DC (up to Glucose and

36

(∼15%) β (1,3) 4-glucanase (30-40%) βglucosidase (5-10%) Xylanase (15-28%) -Xylosidase (2-5%) α-Glucuronidase (1-3% ) α-L-Arabinofuranosidase (13.0%) (∼15%: other hydrolases, including Peptinolytic enzymes, such as β-galactosidase, ramnogalacturonase, polygalacturonase, exo-galacturonase, mannan degrading enzymes such as αgalactosidase; Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) Xylose, like the main sugars, with some mannose, galactose and galacturonic acid

MGBG

CBH I (15-20%) CBH II Production of Flow of 60DC (up to Glucose and

37

(35-40%) β (1,3) 4-glucanase (20-25%) βglucosidase (5-10%) Xylanase (15-20%) -Xylosidase (1-3%) α-Glucuronidase (0.5 -2%) α-L-Arabinofuranosidase (13.0%) (12-15%: other hydrolases, including Peptinolytic enzymes, such as β-galactosidase, ramnogalacturonase, polygalacturonase, exo-galacturonase, mannan degrading enzymes such as αgalactosidase; Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) Xylose, like the main sugars, with some mannose, galactose and galacturonic acid

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum 6th optimal treatment temperature Sugar �de�aos products

MGBG

CBH I (25-30%) Production of Rich textiles 60DC (up to Glucose, something

38

CBH II (15-20%) β (1,3) 4glucanase (15-20%) β-glucosidase (2-8%) Xylanase (25-30%) -Xylosidase (1-3%) α-Glucuronidase (0 , 5-2%) α-L-Arabinofuranosidase (13.0%) (15-20%: other hydrolases, including Peptinolytic enzymes, such as β-galactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase, mannan degrading enzymes such as α- galactosidase; Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for the manufacture of biofuel and / or other high-value products, including antibiotics, carotenoids, aroma compounds and food saporifers, chemical raw materials etc. in cotton, especially in highly purified cotton forms 85DC) of mannose, galactose and galacturonic acid. Lesser amounts of xylose

MGBG

CBH I (20-25%) Production of Rich textiles 60DC (up to Mainly

39

CBH II (20-25%) β (1,3) 4glucanase (20-25%) β-glucosidase (8-12%) Xylanase (20-25%) -Xylosidase (2-5%) α-Glucuronidase (1 -3%) α-L-Arabinofuranosidase (13.0%) (8-12%: other hydrolases, including Peptinolytic enzymes, such as β-galactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase, mannan degrading enzymes such as α-galactosidase; Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for the manufacture of biofuel and / or other high-value products, including antibiotics, carotenoids, aroma compounds and food saporifers, chemical raw materials etc. in cotton, including more mixed fibers 85DC) glucose, with some xylose and mannose

(continuation)

C6ctea

Composition�dea�c6ctea�i%) Daily Appearance Substratum 6th optimal treatment temperature Sugar �de�aos products

MGBG

CBH I (18-25%) Production of Flow of 60DC (up to Glucose and

40

CBH II (15-20%) β (1,3) 4glucanase (25-30%) β-glucosidase (5-10%) Xylanase (20-25%) -Xylosidase (0.5-2%) α-Glucuronidase (0.5-2%) α-L-Arabinofuranosidase (1.5-4.0%) (20-25%: other hydrolases, including Peptinolytic enzymes, such as β-galactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase, degrading enzymes of mannan such as α-galactosidase; Phenolic acid and acetyl (xylan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) Xylose, like the main sugars, with some mannose and galactose

MGBG

CBH I (10-15%) Production of Flow of 60DC (up to Glucose and

43

CBH II (20-25%) β (1,3) 4glucanase (25-30%) β-glucosidase (5-10%) Xylanase (25-30%) -Xylosidase (5-10%) α-Glucuronidase (1 -3%) α-L-Arabinofuranosidase (13.0%) (10-14%: other hydrolases, including Peptinolytic enzymes, Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for biofuel manufacturing sterile cellulose-rich hospital waste 85DC) Xylose, like the main sugars, with some mannose

Tabaa 24: C6cteaes for the production of hydrofoils rich in sugars fermented from faujos de teitiaes waste.

C6ctea

Composition of the dairy i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Sugars of products

MGBG

CBH I (20-25%) Production of Rich textiles 60 DC (up to Glucose

3. 4

CBH II (10-15%) � (1,3) 4-glucanase (24-28%) �-glucosidase (10-15%) Xylanase (25-30%) �-Xylosidase (5-8%) a- Glucuronidase (1-3%) aL-Arabinofuranosidase (23.0%) (5-10%: other hydrolases, including pectinolytic enzymes, phenolic acid and acetyl (xylan) esterases, protease; starch modifying enzymes, oxidase activities or modifiers of Lignin) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, flavoring compounds and food flavorings, chemical raw materials, etc. in cotton 85 AD) mainly with some xylose, galactose and galacturonic acid.

MGBG

CBH I (15-20%) Production of Rich textiles 65 DC (up to Mainly

37

CBH II (35-40%) � (1,3) 4-glucanase (20-25%) �-glucosidase (5-10%) Xylanase (15-20%) �-Xylosidase (1-3%) a- Glucuronidase (0.5-2%) aL-Arabinofuranosidase (0.13.0%) (12-15%: Other hydrolases, including pectinolytic enzymes such as �-galactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase, mannan degrading enzymes such as a-galactosidase, phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, flavoring compounds and food flavorings, chemical raw materials, etc. in cotton, including more mixed fibers and more processed cotton textiles. 85 AD) glucose, some minor amounts of xylose and mannose.

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Products sugars

MGBG

CBH I (25-30%) Production of Rich textiles 60 DC (up to Glucose, something

38

CBH II (15-20%) � (1,3) 4-glucanase (15-20%) -glucosidase (2-8%) Xylanase (25-30%) -Xylosidase (1-3%) a-Glucuronidase ( 0.5-2%) aL-Arabinofuranosidase (13.0%) (15-20%: Other hydrolases, including pectinolytic enzymes such as, -galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase, mannan degrading enzymes such as a-galactosidase, Phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, flavoring compounds and food flavorings, chemical raw materials, etc. in cotton, especially in highly purified cotton forms 85 AD) of mannose, galactose and galacturonic acid. Lesser amounts of xylose.

MGBG

CBH I (20-25%) Production of Rich textiles 65 DC (up to Mainly

39

CBH II (20-25%) � (1,3) 4-glucanase (20-25%) -glucosidase (8-12%) Xylanase (20-25%) -Xylosidase (2-5%) a-Glucuronidase ( 1-3%) aL-Arabinofuranosidase (13.0%) (8-12%: Other hydrolases, including pectinolytic enzymes such as, -galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase, mannan degrading enzymes such as a-galactosidase, phenolic acid and acetyl (xilan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, flavoring compounds and food flavorings, chemical raw materials, etc. in cotton, including more mixed fibers. 85 AD) glucose, with some xylose and mannose.

Tabaa 25: `` Cocktails '' for `` production of hydrogenated products '' in sugars `` fermented '' of areas, seizing `` combatants '' equipment, grains, `` processing '' waste and `` waste '' of manufacturing Beer.

C6ctea

Composition of the dairy i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Sugars of products

MGBG

CBH I (5-10%) Production of Cereals, > 60 DC. Xilosa,

5

CBH II (8-15%) � (1,3) 4-glucanase (18-22%) -glucosidase (6-10%) Xylanase (30-35%) + [�-Xylosidase 5-12% Exoxylanase 10- fifteen %; a-Glucuronidase (3-5%), a-L-Arabinofuranosidase (1.5-5.0%); Pectinolytic enzymes 8-10%, including -galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 57% starch modifying activity; other hemicellulases including agalactosidase 4-7%, oxidoreductase / oxidase and esterases 1-4%, + protease 2-6%] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. including whole crops, grains, processing debris and beer manufacturing residues, especially barley, wheat, rye, corn, malt / dough residues (including distillation grains). (up to 80 AD) arabinose, glucose, cellobiose, xelobiosa, galactose, glucuronic acid, some phenolic acids, small amounts of glucuronic acids, galacturonic acid and Ramnosa and higher oligosaccharides.

MGBG

CBH I (1-5%) Production of Cereals, > 60 DC. Xilosa,

6

CBH II (1-5%) (1,3) 4-glucanase (22-28%) -glucosidase (10-15%) Xylanase (25-30%) + [�-Xylosidase 0.7-2.1% , aL-Arabinofuranosidase 2-4%; Pectinolytic enzymes 20-25%, including -galactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, starch modifying activity ∼ 5-8%; oxidoreductase / oxidase and esterases 12-15%, + 8-12% protease] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, precursors of flavoring and flavoring, natural sweeteners, etc. including whole crops, grains, processing debris and manufacturing waste from beer especially barley, wheat and oats. (up to 85 AD) arabinose, mainly glucose, small amounts of glucuronic acids, galactose, galacturonic acid and Ramnosa and disacarids (cellobiose and xylobiose).

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Products sugars

MGBG

CBH I (5-10%) Production of Remains of > 60 DC. Mainly

7

CBH II (8-12%) (1,3) 4-glucanase (25-30%) -glucosidase (5-10%) Xylanase (40-45%) + [�-Xylosidase 0.5-2.0% , α-glucuronidase, aL-Arabinofuranosidase 0.52.0%; Pectinolytic enzymes 8-15%, mainly �-galactosidase and exogalacturonase, esterases 2-4%, + 10-15% protease] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example, (bio) chemical products / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. barley, millet, wheat, oats. (up to 85 AD) xylose, arabinose, glucose, cellobiose, xylobiose, glucuronic acid, some galactose, phenolic acids, galacturonic acid.

MGBG

CBH I (5-10%) Production of Cereals, > 60 DC. Xilosa,

8

CBH II (5-10%) (1,3) 4-glucanase (∼20%) -glucosidase (4-8%) Xylanase (25-30%), including Exoxylanase (5-10%) + [β-Xylosidase 12.0%, a-Glucuronidase 58%, aL-Arabinofuranosidase 0.53.0%; Pectinolytic enzymes 8-12%, including -galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, starch modifying activity 8-12%; other hemicellulases 5-10%, including agalactosidase, oxidoreductase / oxidase and esterases 1-3%, + 10-12% protease] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. including whole crops, grains, processing residues and beer manufacturing residues, especially residues of wheat, oats, barley, malt / dough (including distillation grains). (up to 80 AD) arabinose, glucose, cellobiose, xylobiose; smaller amounts of glucuronic acid, phenolic acids and galactose.

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Products sugars

MGBG

CBH I (5-10%) Production of Cereals, > 60 DC. Xilosa,

9

CBH II (5-10%) (1,3) 4-glucanase (20-25%, including higher lichenase) -glucosidase (8-10%) Xylanase (25-30%), including Exoxylanase at -12 8-12% ) + [β-xylosidase 25.0%, a-Glucuronidase 14%, aL-Arabinofuranosidase (25.0%); Pectinolytic enzymes 5-10%, including α-galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 5-10% starch modifying activity; other 3-6% hemicellulases, including βgalactosidase, oxidoreductase / oxidase and esterases 2-4%, + 5-10% protease] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. including whole crops, grains, processing residues and beer manufacturing residues, especially wheat, oats, barley, sorghum and more new adjuvant residues, malt / dough residues (including distillation grains). (up to 80 AD) arabinose, glucose, cellobiose, xylobiose; smaller amounts of glucuronic acid, phenolic acids and galactose.

MGBG

CBH I (3-6%) Production of Cereals, > 60 DC. Xilosa,

10

CBH II (5-10%) (1,3) 4-glucanase (25-40%, including higher lichenase) -glucosidase (2-5%) Xylanase ∼ (25-30%), including o10-15 Exoxylanase %) + [β-xylosidase 5-12%, a-Glucuronidase 2-5%, aL-Arabinofuranosidase (25.0%); Pectinolytic enzymes ∼ 5-8%, including β-galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 2-5% starch modifying activity; other 2-5% hemicellulases, including βgalactosidase, oxidoreductase / oxidase and esterases 1-3%, + 3-6% protease] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. including whole crops, grains, processing residues and beer manufacturing residues, especially residues of wheat, oats, barley, corn, rye, malt / dough (including distillation grains). (up to 80 AD) arabinose, glucose, cellobiose, xylobiose; smaller amounts of glucuronic acid, phenolic acids and galactose. Some higher oligosaccharides.

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Products sugars

MGBG

CBH I (1-5%) Production of Cereals, > 60 DC. Xilosa,

eleven

CBH II (5-8%) � (1,3) 4-glucanase (20-25%, including high activity against higher and middle DP mixed bond glucans) -glucosidase (20-30%) Xylanase (∼ 15- 20%), including Exoxylanase ∼ 3-6%) + β-xylosidase 2-6%, a-Glucuronidase 13%, aL-Arabinofuranosidase 1.5-3.0%; Pectinolytic enzymes ol8-12%, including β-galctosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 5-10% starch modifying activity; other 6-11% hemicellulases, including αgalactosidase, oxidoreductase / oxidase and esterases 3-7%, + 10-15% protease] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. including whole crops, grains, processing residues and beer manufacturing residues, especially remains of wheat, corn, oats, barley, malt / rye dough (including distillation grains). (up to 80 AD) arabinose, glucose, minor amounts of glucuronic acid, phenolic acids galactose and Ramnosa. Some higher oligosaccharides.

MGBG

CBH I (5-10%) Production of Cereals, > 60 DC Xilosa,

13

CBH II (5-10%) (1,3) 4-glucanase (25-40%) -glucosidase (∼5%) Xylanase (18-25%), + [β-xylosidase 1-2.0%, exoxylanase 1-3.0%, a-Glucuronidase 8-10%, aL-Arabinofuranosidase 1.5-5.0%; Pectinolitic enzymes 10-15%, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 5-7% starch modifying activity; other hemicellulases 5-10%, including αgalactosidase, oxidoreductase / oxidase and esterases 1-4%, + 8-12% protease] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. including whole crops, grains, processing debris and beer manufacturing residues, especially oats, barley, wheat, malt / dough (including distillation grains). (up to 80 AD) arabinose, glucose, cellobiose, xylobiose, galactose, glucuronic acid, some phenolic acids, galacturonic acid and Ramnosa.

Tabaa 26: `` Cocktails '' for `` production '' of hydrofoils `` in sugars '' was fermenting and distributing vegetable residues based on soil or cereaaes, seizing herbs and testing), leaves, `` Trimming '' of `` pruning '' and `` waste flows '' of faculty.

C6ctea

Composition of the dairy i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Sugars of products

MGBG

CBH I (15-20%) Production of  Waste 60 DC (until Glucose and

2

CBH II (20-28%) � (1,3) 4-glucanase (20-26%) -glucosidase (10-11%) Xylanase (18-30%) β-xylosidase (8-10%) a-Glucuronidase (8-10%), aL-Arabinofuranosidase (0.5-2.0%) (6-17%: other hydrolases, including pectinolytic enzymes, phenolic acid and acetyl (xilan) esterases, protease; Lignin modifying oxidase activities) . fermentable sugars for the manufacture of biofuel and / or other high-value products, for example, (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, antioxidant molecules, etc. vegetables, including herbs, leaves, pruning clippings and mixed florist waste streams; Especially good for more woody materials. 85 AD) Xylose as the main sugars, with some arabinose, mannose and galactose, glucuronic and galacturonic acids, some phenolic acids and rhamnose, as well as some higher DP oligosaccharides.

MGBG

CBH I (5-10%) Production of Waste > 60 DC. Xilosa,

13

CBH II (5-10%) (1,3) 4-glucanase (25-40%) -glucosidase (∼5%) Xylanase (18-25%), + [β-xylosidase 1-2.0%, 1-3 , 0; exoxylanase 8-10, a-Glucuronidase 1.5-5.0%), a-L-Arabinofuranosidase; 10-15%; pectinolytic enzymes including α galactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactose, starch modifying activity 5-7%; other hemicellulases 5-10%, including αgalactosidase, 1-4%, oxide reductase / oxidase and esterases + protease 812%] fermentable sugars for the manufacture of biofuel and / or other high-value products, for example, (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, antioxidant molecules, etc. vegetables, including herbs, leaves, pruning clippings and mixed florist waste streams; Especially good herbs and silage. (up to 80 AD) arabinose, glucose, cellobiose, xylobiose, galactose, mannose, glucuronic acid, some phenolic acids, galacturonic acid and Ramnosa. Some higher oligosaccharides.

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature �C) Products sugars

MGBG

CBH I (5-10%) Production of Waste > 60 DC. Xylose, glucose,

twenty-one

CBH II (5-10%) (1,3) 4-glucanase (15%) -glucosidase (∼2-10%) Xylanase (50-55%), + [xylosidase 1-5%); 5-10% exoxylanase, a-Glucuronidase (1-4%), a-L-Arabinofuranosidase (25%); 20-25% pectinolytic enzymes, 4-6% starch modifying activity; other 7-10% hemicellulases, oxidoreductase / oxidase and 2-4% esterases + ∼10% protease]. fermentable sugars for the manufacture of biofuels and / or other high-value products, for example, (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, aroma and flavor precursors, antioxidant molecules, etc. vegetables, including herbs, leaves, pruning clippings and mixed florist waste streams; also good for crust. (up to 80/85 AD) arabinose, galactose, mannose, galacturonic acid, some fructose, Ramnosa; some phenolic acids and sucrose; some higher oligosaccharides.

MGBG

CBH I (1-5%) Production of Waste > 60 DC. Xylose, glucose,

32

CBH II (1-5%) � (1,3) 4-glucanase (20-25%) -glucosidase (8-12%) Xylanase (30-35%) + [β-xylosidase (0.5-1.5% ), aL-Arabinofuranosidase (12.5%); 18-25% pectinolytic enzymes, including αgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, -105-10% starch modifying activity; Oxide reductase / oxidase and esterases 8-15% + 10-15% protease]. fermentable sugars for the manufacture of biofuel and / or other high-value products, for example, (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, antioxidant molecules, etc. Vegetables, including herbs, leaves, pruning cuttings and mixed florist waste streams. (up to 80 AD) arabinose, galactose, galacturonic acid, some fructose, Ramnosa; Some phenolic acids.

MGBG

CBH I (20-25%) Production of Waste 60 DC (until Glucose,

3. 4

CBH II (10-15%) sugars vegetables, 85 AD) arabinosa and

CBH

� (1,3) 4-glucanase (24-28%) -glucosidase (10-15%) Xylanase (25-30%), + β-xylosidase (5-8%), a-Glucuronidase (1-3%), aL-Arabinofuranosidase (23.0%) (5-10%: other hydrolases, including pectinolytic enzymes, phenolic acid and acetyl (xylan) esterases, protease; starch modifying enzymes, Lignin modifying oxidase activities). fermentable for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, aromatic compounds and food flavorings, chemical raw materials, etc. including herbs, leaves, pruning clippings and mixed florist waste streams. Xylose mainly with some galactose and galacturonic acid; phenolic acids, Ramnosa and some oligosaccharides.

Tabaa 27: `` Cocktails '' for `` production of hydrogenated products '' in sugars - fermented as part of `` waste streams '' of papal

Paper waste includes: black and white newspaper paper, color newspaper paper, glossy magazines, paper cups, paper plates, paper handkerchiefs / wipes, brown paper, corrugated cardboard, white office paper, cellophane, kitchen paper, KleenexTM and domestic cotton bandages

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars�de�aos products

MGBG

CBH I (5-10%) Production of Glasses of 60 DC (up to Glucose,

26

CBH II (45-50%) β (1,3) 4-glucanase (10-15%) -glucosidase (0.5-2.0%) Xylanase (15-20%) β-Xylosidase (0.2- 1.0%) α-Glucuronidase (0.5-2.0%) aL-Arabinofuranosidase (0.5-1.5%) Other hydrolases, including 3-6% pectinolytic enzymes, Phenolic acid and acetyl (xilan) esterases 0.2-1.0, Protease 1-5%; starch modifying enzymes 5-10%, oxidoreductase / oxidase activities 25 fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food and aromatic compounds, chemical raw materials, etc. paper, brown paper, KleenexTM and related paper waste products 85 AD) arabinose and xylose, some galactose and galacturonic acid; some oligosaccharides

MGBG

CBH I (50-55%) Production of White paper 60 DC (up to Glucose,

27

CBH II (20-25%) β (1,3) 4-glucanase (12-20%) -glucosidase (5-8%) Xylanase (∼5%) β-Xylosidase (0.1-0.5%) aL-Arabinofuranosidase (0.5-2.0%) (5-8%: other hydrolases, including selected pectinolytic enzymes, esterases; 0.5-1.5% protease; 0.51.5% oxidase activities) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food and aromatic compounds, chemical raw materials, etc. of office, paper plates and related products 85 AD) smaller amounts of arabinose and xylose, galactose and galacturonic acid; some oligosaccharides, but mainly monosaccharides

MGBG

CBH I (10-15%) Production of White paper 60 DC (up to Glucose,

28

CBH II (30-35%) β (1,3) 4-glucanase (15-20%) -glucosidase (2-5%) Xylanase (20-25%) β-Xylosidase (0.5-2.0% ) aL-Arabinofuranosidase (0.5-2.0%) (17-26%: other hydrolases, including selected pectinolytic enzymes and starch modifiers, 25% esterases; 8-10% protease; 2-5% oxidase activities) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food and aromatic compounds, chemical raw materials, etc. of office, paper plates and related products 85 AD) smaller amounts of arabinose and xylose, galactose and galacturonic acid; some oligosaccharides, but mainly monosaccharides

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of years products

MGBG

CBH I (8-10%) Production of Wipes, 60 DC (up to Glucose,

29

CBH II (8-10%) β (1,3) 4-glucanase (20-30%) -glucosidase (25-30%) Xylanase (20-30%) β-Xylosidase (0.5-2.0% ) β-L-Arabinofuranosidase (0.5-2.0%) Hydrolases, including 510% pectinolytic enzymes and 5-8% starch modifiers, 13% esterases; 5-10% protease; 2-5% oxidases fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food and aromatic compounds, chemical raw materials, etc. cotton, cardboard and cellophane bandages 85 AD) smaller amounts of arabinose and xylose, galactose and galacturonic acid; some oligosaccharides, but mainly monosaccharides

Tabaa 2�: Cocktails are for the production of rich sugars in sugars and were distributed from residues / residues of packaging or biodegradation.

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Products sugars

MGBG

CBH I (20-25%) Production of Waste / Remains 60 DC (up to Xylose, glucose,

30

CBH II (20-25%) β (1,3) 4-glucanase (1520%) -glucosidase (1-5%) Xylanase (25-30%) β-Xylosidase (5-10%) α-L-Arabinofuranosidase (0.6-2.0%) (other hydrolases, including selected 812% pectinolytic enzymes, 4-8% starch modifying enzymes, 13% mannan degrading enzymes, 1-5% esterases, 3-6.0% protease; ; oxidase activities 2-5%) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food and aromatic compounds, chemical raw materials, etc. biodegradable packaging 85 AD) galactose, small amounts of mannose, galacturonic acid; and some oligosaccharides, but mainly monosaccharides

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of products

MGBG

CBH I (10-12%) Production of Waste / Remains 60 DC (up to Glucose and Xylose

35

CBH II (∼15%) β (1,3) 4-glucanase (3040%) -glucosidase (5-10%) Xylanase (15-28%) β-Xylosidase (2-5%) α-Glucuronidase (1- 3%) α-L-Arabinofuranosidase (1-3.0%) (∼15%: other hydrolases, including pectinolytic enzymes fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food saporifers and biodegradable packaging 85 AD) as the main sugars, with some mannose, galactose and galacturonic acid

such as αgalactosidase, ramnogalacturonase, polygalacturonase, exo

aromatic compounds, chemical raw materials, etc.

galacturonase, mannan degrading enzymes such as βgalactosidase, phenolic acid and acetyl (xylan) esterases, Protease; starch modifying enzymes, Lignin modifying oxidase activities)

MGBG

CBH I (8-10%) Production of Waste / Remains 60 DC (up to Glucose,

29

CBH II (8-10%) β (1,3) 4-glucanase (2030%) -glucosidase (25-30%) Xylanase (20-30%) β-Xylosidase (0.5-2.0%) α -L-Arabinofuranosidase (0.5-2.0%) (Hydrolases, including selected 5-10% pectinolytic enzymes and 5-8% starch modifiers, 1-3% esterases; 5-10% protease; 2- oxidase activities 5%) fermentable sugars for the manufacture of biofuels and other high-value products, including antibiotics, carotenoids, food and aromatic compounds, chemical raw materials, etc. biodegradable packaging 85 AD) smaller amounts of arabinose and xylose, galactose and galacturonic acid; mainly monosaccharides

Tabaa 29: C6ctea is `` for production '' of hydrogenated products in sugars - it was being distributed to distribute fungal biomass by undermining biomass and wasting water from biomass. Marine

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Products sugars

MGBG

CBH I (5-10%) Sugar production Biomass of > 60 DC (up to Glucose,

9

CBH II (5-10%) β (1,3) 4-glucanase (2025%, including higher lichenase) -glucosidase (8-10%) Xylanase (25-30%, including Exoxylanase -128-12%) + [ β-Xylosidase 2-5.0% α-Glucuronidase 1-4%, α-L-Arabinofuranosidase 2-5.0%; 5-10% pectinolytic enzymes, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 5-10% starch modifying activity; other 3-6% hemicellulases, including αgalactosidase, oxidoreductase / oxidase and esterases 2-4% + protease 5-10%] fermentable for the manufacture of biofuels and / or other products of high value, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

MGBG

CBH I (5-10%) Sugar production Biomass of > 60 DC (up to Glucose,

13

CBH II (5-10%) β (1,3) 4-glucanase (2540%) -glucosidase (∼5%) Xylanase (18-25%) + [β-Xylosidase 1-2.0%, Exoxylanase 1- 3.0%, α-Glucuronidase 8-10%, α-L-Arabinofuranosidase 1.5-5.0%; 10-15% pectinolytic enzymes, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, 5-7% starch modifying activity; other hemicellulases 5-10%, including αgalactosidase, oxidoreductase / oxidase and esterases 1-4% + protease 8-12%] fermentable for the manufacture of biofuels and / or other products of high value, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of products

MGBG

CBH I (2-5%) Production of Biomass of > 60 DC (up to Glucose,

44

CBH II (2-5%) β (1,3) 4-glucanase (2025%) (1,3) 6-glucanase (2025%) N-Acetylglucosaminidase and / or chitinase 2-5% -glucosidase (5-10 %) Xylanase (10-15%) + � [β-Xylosidase 5-10%, α-Glucuronidase 1-5%, α-L-Arabinofuranosidase 15.0%; 2-5% pectinolytic enzymes, including αgalactosidase and galactanase, 2-5% starch modifying activity; other 3-5% hemicellulases, including mannanase, 2-5% oxidoreductase / oxidase and 2-4% esterases + 25-30% protease] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

MGBG

CBH I (5-10%) Production of Biomass of > 60 DC (up to Glucose,

Four. Five

CBH II (5-10%) β (1,3) 4-glucanase (1520%) (1,3) 6-glucanase (2530%) N-Acetylglucosaminidase and / or chitinase 5-10% -glucosidase (5-10 %) Xylanase (1-4%) + � [β-Xylosidase 2-5%, pectinolytic enzymes 5-10%, including βgalactosidase and galactanase, 12-15% starch modifying activity; other 2-5% hemicellulases, including mannanase, 2-5% oxidoreductase / oxidase and 2-4% esterases + 10-15% protease] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, natural sweeteners, etc. yeast and filamentous fungi or algae biomass and processing debris 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of products

MGBG

CBH I (5-10%) Production of Biomass of > 60 DC (up to Glucose,

46

CBH II (5-10%) β (1,3) 4-glucanase (2530%) -glucosidase (5-10%) Xylanase (20-25%) + � [β-Xylosidase 2-5%, α-Glucuronidase 1-4%, α-L-Arabinofuranosidase 25%; 5-8% pectinolytic enzymes, including βgalactosidase and galactanase, 5-10% starch modifying activity; other 1-2% hemicellulases, including mannanase, 2-5% oxidoreductase / oxidase and 2-5% esterases + 30-35% protease] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

MGBG

CBH I (2-5%) Production of Biomass of > 60 DC (up to Glucose,

47

CBH II (2-5%) β (1,3) 4-glucanase (1015%) (1,3) 6-glucanase (4045%) -glucosidase (8-10%) Xylanase (2-5%) + � [β-Xylosidase 1-3%, αglucuronidase 1-2%, α-L-Arabinofuranosidase 25%; 2-5% pectinolytic enzymes, including βgalactosidase and galactanase, 2-5% starch modifying activity; other mic5% hemicellulases, including mannanase, oxidoreductase / oxidase 2-5% and esterases 1-3%] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of products

MGBG

CBH I (5-10%) Production of Biomass of > 60 DC (up to Glucose,

48

CBH II (5-10%) β (1,3) 4-glucanase (4045%) (1,3) 6-glucanase (1015%) -glucosidase (8-12%) Xylanase (10-15%) + � [β-Xylosidase 5-8%, α-glucuronidase 0.5-1.5%, α-L-Arabinofuranosidase 0.5-1.5%; 2-5% pectinolytic enzymes, including βgalactosidase and galactanase, 2-5% starch modifying activity; other mic5% hemicellulases, including mannanase, oxidoreductase / oxidase 1-3% and esterases 1-3%] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

MGBG

CBH I (0.4-2%) Production of Biomass of > 60 DC (up to Glucose,

49

CBH II (0.4-1.0%) β (1.3) 4-glucanase (7.015.0%) (1.3) 6-glucanase (1215%) -glucosidase (3.0-5.0% ) Xylanase (75.0-78.0%) + [β-Xylosidase 0.4-2.0%, α-glucuronidase 0.51.5%, α-L-Arabinofuranosidase 0.21.5%; 2-5% pectinolytic enzymes, including βgalactosidase and galactanase, 2-5% starch modifying activity; other hemicellulases ,04.0-7.0%, including mannanase, oxidoreductase / oxidase 1-3% and esterases 1-3%] fermentable sugars for the manufacture of biofuels and / or other high-value products, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, natural sweeteners, etc. yeast and filamentous fungi 80 AD) cellobiose, mannose, galactose and some oligosaccharides, mainly

Tabaa 30: `` Cocktails '' for production of `` hydrogenated '' foods in sugars was used as a basis for distribution of sugar beet sugar, sugarcane, and the rest of the same.

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of the products

MGBG

CBH I (1-5%) Production of Beet > 60 DC (up to Xilosa,

6

CBH II (1-5%) β (1,3) 4-glucanase (2228%) -glucosidase (10-15%) Xylanase (25-30%) + [β-Xylosidase 0.7-2.1%, α-L-Arabinofuranosidase 24%; 20-25% pectinolytic enzymes, including βgalactosidase, ramnogalacturonase, polygalacturonase, exogalacturonase and galactanase, ∼5-8% starch modifying activity; oxidoreductase / oxidase and esterases 12-15% + protease 8-12%] fermentable sugars for the manufacture of biofuels and / or other products of high value, for example (bio) chemical compounds / chemical raw materials, antibiotic carotenoids, probiotics, aroma and flavor precursors, etc. sugar bowl, including top parts, beet pulp and whole plant, as well as sugar cane and processing waste 85 AD) Glucose, Galacturonic acid, Fructose, Arabinose, Galactose, Ramnose, Some oligosaccharides; phenolic acid released

MGBG

CBH I (5-10%) Production of Beet > 60 DC (up to Xilosa,

twenty

CBH II (5-10%) β (1,3) 4-glucanase (2032%) -glucosidase (-102-10%) Xylanase (15-25%) + [β-Xylosidase 15-20.0% Exoxylanase 10 -15%, α-Glucuronidase 2-5%, α-L-Arabinofuranosidase 1.5-5%; 10-15% pectinolytic enzymes, modifying activity of fermentable sugars for the manufacture of biofuels and / or other high value products, for example (bio) chemical compounds / chemical raw materials, carotenoids, antibiotics, probiotics, etc. sugar bowl, including top parts, beet pulp and whole plant, as well as sugar cane and processing waste 80/85 AD) Glucose, Galacturonic acid, Fructose, Sucrose, Arabinose, Galactose, Ramnose, Some oligosaccharides; phenolic acid released

∼10% starch; other hemicellulases ∼5%, oxidoreductase / oxidase and esterases 2-5% + protease ∼7-10%]

(continuation)

C6ctea

Composition cdeactea i%) Daily Appearance Substratum 6th optimal treatment temperature Sugars of years products

MGBG

CBH I (5-10%) Production of Beet > 60 DC Xilosa,

18

fermentable sugars sugar bowl, (up to 80 AD)

CBH II (5-10%)

for the manufacture of including the Glucose,

β (1,3) 4-glucanase (25

biofuels and / or parts acid

40%)

other high products superior, Galacturonic,

-glucosidase (∼5%)

value for example pulp of

Xylanase (18-25%) + [β

compounds beet and Fructose,

Xylosidase 1-2.0%,

(bio) chemicals / materials plant Saccharose,

Exoxylanase 1-3.0%, α

chemical premiums, complete as well Arabinosa,

Glucuronidase 8-10%, αL-Arabinofuranosidase 1.5-5.0%; 10-15% pectinolytic enzymes, including βgalactosidase, ramnogalacturonase,

carotenoids, antibiotics, probiotics, etc. as sugarcane and processing waste Galactose, Ramnosa Some oligosaccharides; phenolic acid released

polygalacturonase,

exogalacturonase and

galactanase activity

starch modifier

5-7%; other hemicellulases

5-10%, including α

galactosidase,

oxidoreductase / oxidase and

esterases 1-4% +

8-12% protease]

Several of the cocktails listed above have applications in a wide range of other applications. In these applications, the key differences in the use of cocktails correspond to (a) enzyme dosage, or amount used in each treatment, and (b) the duration of the incubation. For example, MGBG 18 is very well adapted for the treatment of certain waste streams, as well as in nutraceutical applications. In the "waste" saccharification / treatment stages, a larger enzyme dosage is used and the reaction time is ∼18-24 hours (∼25-32 filter paper units). The ultimate goal is to achieve thorough degradation of the target residue in fermentable monosaccharides. On the contrary, when production of bioactive oligosaccharides (glucooligosaccharides and xylooligosaccharides) is required, and / or a change in texture for breads, a

10 less enzymatic concentration and the modification time (or reaction) may take no more than 1 (maximum 2) hours to achieve the desired end point.

When the target substrate is primarily rich in cellulose, enzyme concentrations have been based on "filter paper units or UPF". When a bioactive oligosaccharide (for example non-cellulosic β-glucooligosaccharide) is produced, enzyme concentrations are based on the main activity required to ferment the substrate.

15 target (for example β-1,3; 1,4-glucans or β-1,3; 1,6-non-cellulosic, mixed-link glucans from fungal sources or algae).

Example 13 Enzyme production by strains of T. emersonii during the aforementioned fermentation

The Ta / aromyces emersonii strains were examined:

IMI (Imperial Mycological Institute (CABI Bioscience)) 393751 (Patent strain), IMI 393753 (CBS (Centraal

20 Bureau voor Schimmelcultures) 180.68), IMI 393755 (CBS 355.92), IMI 393756 (CBS 393.64), IMI 393757 (CBS 394.64), IMI 393758 (CBS 395.64), IMI 393759 (CBS 397.64), IMI 393760 (CBS 39372) IMI 393752 (CBS 549.92), IMI 393761 (CBS 759.71).

Liquid fermentation; Repeated liquid cultures of individual T. emersonii were grown at 45 AD, in the medium described by Moloney et al. (1983) and Tuohy and Coughlan (1992), under conditions with uncontrolled pH. The four 25 selected carbon sources were: glucose (monosaccharide), oatmeal husk xylan (arabinoglucuronoxylan), locust bean powder and a mixture of tea leaves / paper plates 1: 1 (fragmented in a mixer for ∼15-20 second, culture supernatants (Tuohy and Coughlan, 1992; Murray et al., 2002) were recovered and used to analyze extracellular enzyme production. Enzyme assays; activity

60 5

Enzyme was expressed in International Enzyme Units (IU) per gram of inductive carbon source. A UI unit releases 1 micromol of product (reducing sugar, 4-nitrophenol, etc.) per minute. All exoglycosidase and endohydrolase enzyme assays were performed as previously described (Tuohy and Coughlan, 1992; Tuohy et al., 1994, 2002; Murray et al., 2002; Gilleran, 2004). Unless indicated otherwise, all initial activity measurements were performed at 50 DC and pH 5.0. Exoglycosidase activities included: β-Glucosidase, α-Glucosidase, β-Xilosidase, β-Galactosidase, β-Mannosidase, β-Fucosidase, α-Arabinofuranosidase, N-Acetylglucosaminidase, α-Ramnopyranosidase, α-Galactosidase, α-Fucosidase, α -Arabinopyranosidase, α-Mannosidase and α-Xylosidase. The α-Glucuronidase activity was assayed by a process of reducing sugars using a mixture of reduced aldouronic acids as a substrate (Megazyme International Ltd). This substrate contained aldotriouronic, aldotetrauronic and aldopentauronic acids reduced in a ratio of approximately 40:40:20. The activity was measured at pH 5.0 with a reserve of 5 mg / ml of this mixture. Reducing groups released during a 30 minute incubation period were detected by the DNS procedure. Since some of the enzyme samples contained appreciable β-xylosidase activity that could release xylose residues from aldouronic acids, the assay was repeated and xylose was included in the reaction mixture to inhibit β-xylosidase activity.

Additional external-acting xylanolytic enzymes such as: α-arabinoxylan arabinofurannohydrolase (release of wheat straw arabinoxylan arabinose using an enzyme-linked assay), acetyl esterase (using 4-nitrophenyl and 4-methylumbelliferyl acetate substrates) were also measured, Acetyl xylan esterase activity (monitoring the release of acetylated beech xylan acetate), ferulic acid esterase (spectrophotometric and HPLC test procedures).

Endohydrolase activities included: β-D- (1,3; 1,4) -glucanase (barley β-glucan (BBG) or lichenan as test substrates), xyloglucanase (tamarind xyloglucan), laminarinase (Laminaria digitata lamellar ), endo-1,4-β-glucanase, (called CMCase), based on the activity against a commercial substrate carboxymethyl cellulose, β-mannanase (carob galactomannan) pectinase and polygalacturonase, ramnogalacturonase (soybean ramnogalacturonan), galactanose (pect galactanos of potato and lupine as substrates), arabinanase (sugar beet arabinan), amylase, glucoamylase and dextrinase.

Optimum temperature / pH and stability; the optimum temperature for the activity was determined by carrying out the appropriate conventional tests at temperature increases in the range 30-100 DC, in normal test buffer (100 mM NaOAc, 5.0). The variation in pH with temperature was considered. Optimum pH was determined using the following pH 2.2-7.6 buffers: McIlvaine constant ionic strength citrate phosphate buffer; pH 7-pH 10 Tris-HCl buffer. All buffers regardless of pH were adjusted to the same ionic strength with KCl.

Temperature and pH stabilities were determined as previously described (Tuohy et al., 1993; Gilleran, 2004; Braet, 2005).

Protein determination; Protein concentration in enzyme samples (raw culture samples) was estimated by modification of Bensadoun and Weinstein of the Lowry procedure (Bensadoun and Weinstein, 1976; Lowry et al., 1951) using fraction V of BSA as a standard (Murray et al., 2001). Electrophoresis and zymography; To determine the protein profile present in the culture filtrates, a known volume of each sample was concentrated by lyophilization and analyzed by native SDS-PAGE and / or renaturalizing or isoelectric focusing (IEF; Tuohy and Coughlan, 1992). The active bands for endoglycanase in the renatured SDS-PAGE gels and IEF gels were identified using a modification of the overlay technique of MacKenzie and Williams (1984) gels (Tuohy and Coughlan, 1992). To detect exoglycosidase activity, the gels were immediately incubated in 50-100 pM solution of the appropriate derivative of 4-methylumbelliferyl glycoside (reaction period of 2-30 minutes). The active enzyme band or bands were visualized with ultraviolet light using a Fluor-S 'Multimager (Bio-Rad).

Results:

The liquid culture of the strains was repeated at least 3 times, in independent experiments carried out in different periods of time. The culture filtrates collected (120 hours) from repeating flasks (for each combination of carbon source / strain) were tested for enzymatic activity; Multiple repetitions were tested in a range of enzyme dilutions. In addition, the results were validated by measurements within the test and between the tests, and volume independence tests. There were clear differences between crops with respect to crop appearance and growth pattern. For example, strain IMI393751 rapidly produced a fairly dense, filamentous glucose culture, while several of the other strains (for example, CBS180.68, CBS355.92, CBS393.64, CBS395.64, CBS397.64, CBS 549.92 and CBS 759.71) presented limited growth and atypical morphology, that is, absence of normal filamentous growth and formation of a limited viscous culture mass. The strain CBS394.64 produced a lower mycelium biomass, but grew as a filamentous culture, while CBS472.92 adopted granule morphology under identical growth conditions. A 120-hour time growth point was selected, since previous studies have shown that extracellular exoglycosidase and endoglycanase activities are present in significant amounts during the growth of T. emersonii in most carbon sources (under pH conditions not controlled, they have

observed "peaks and valleys" of key activities. However, maximum activity is usually detected towards the end of the fermentation cycle).

The glucose utilization was only about 15-25% at 120 hours for many of the strains. The CBS 394.64 strain used ∼ 50-55%, while CBS472.92 (which presented granule morphology) used ∼ 20-25% of the glucose in the medium. In contrast, ∼95% of the glucose in the culture medium was used by the strain of the invention (IMI393751) at 72 hours and no glucose was detected in the culture medium at the 120 hour collection time point.

Tables 31A-D show the production of exoglycosidases selected by the strains. As the results reveal, clear distinctions can be seen between the strain of the invention and other strains of T. emersonii with respect to exoglycosidase production.

Glucose does not completely suppress the production of exoglycosidase by strains of T. emersonii (Table 31A). Strain 393751 produces significantly higher levels of β-glucosidase (BGase) than the other strains and the second highest levels of N-acetylglucosaminidase (NAGase) during glucose growth. The production pattern obtained for strain 393751 contracts significantly with that of strain CBS549.92 (previously CBS814.70). The production of various exoglycosidase activities by the last strain seems to be repressed by glucose. It should be noted that the levels of exoglycosidase activity were measured in non-dialyzed and dialyzed culture filtrates in case the residual glucose in the medium was inhibiting BGase and / or NAGase present (similar patterns were observed in the dialyzed samples).

Carob induces differential production of extracellular glycosidases by strains (Table 31B). CBS strain

394.64 produces relatively no exoglycosidase activity other than α-arabinofuronasidase. Low levels of all exoglycosidases were produced by strain CBS 393.64, CBS 395.64 and CBS 549.92. Strain 393751 produced significant levels of a wide range of exoglycosidases (higher levels of certain activities, for example, pectin modifying β-glucosidase exoglycosidase).

Production of inbreeding by strains of T. emersonii

A. Xylanase production: Two of the main types of endohydrolase activities required for the conversion of plant biomass and residual residues rich in non-starch polysaccharides are glucanase and xylanase. Of all the degrading activities of polymeric glycan tested, glucanase and xylanase were the predominant glycanase activities present.

Tables 32A-D present values for xylanase production by all strains in the same carbon sources. Previous studies have shown that the wild type (CBS814.70) and other mutant strains produce a complex xylanolytic enzyme system (Tuohy et al., 1993; 1994), with multiple endoxylanases. Several of the isolated xylanases have selective specificity for different types of xylanes, for example, arabinoxylans, arabinoglucuronoxylanes, glucuronoxylanes, plus substituted xylanes against unsubstituted xylanes (from previous results and continued results with the enzymes of the strain of the invention) . As the results show, glucose is a strong repressor of xylanase expression in all strains of T. emersonii. Significant levels of active xylanase activity are expressed against oat husk arabinoxylan (OSX) by strain 393751. This component is not active against rye or wheat arabinoxylanes.

Carob, which is mainly rich in galactomannans (contains some xylan), is a potent inducer of very high levels of xylanase activity against all xylan substrates by strain 393751. The role of carob as an inducer of potent xylanase activity would not be expected based on the knowledge of its composition. The appearance of the cultures obtained for several of the CBS strains (after 120 hours) was significantly different and clear morphological differences could be observed between strains 393751 and CBS549.92, that is, dense mycelium growth (filamentous) for IMI 393751 and formation of a limited, viscous (non-filamentous) culture mass for strain CBS549.92. As shown in Table 31B, xylanase levels are significantly higher for strain 393751 than any other. Unlike strain 393751, carob is a very low xylanase inducer in strains CBS394.64, CBS395.64 and CBS549.92. Another notable difference can be seen in the type of carob-induced xylanase activity. In strain 393751 potent activity occurs against all xylanes. In the other strains, in general, there is little or no activity against rye and wheat arabinoxylans. Only two different strains of strain 393751 produce appreciable levels of activity against both of these xylanes, that is, CBS472.92 and CBS759.71. The zymogram analysis of strain culture filtrate 393751 revealed high levels of multiple active bands for xylanase, including a new bifunctional xylanase that has been isolated.

The mixture of tea leaves / paper plates (TL / PPL) also proved to be a potent inducer of xylanase activity in strain 393751 (Table 32C), and although this mixture induced xylanase expression in the other strains, the levels were significantly minors As observed with the carob, potent activity was produced by strain 393751 against all xylanes, obtaining an activity almost 1: 8 times higher against rye arabinoxylan (AX rye) and lower activity against OSX and birch xylan. This suggests differential expression of individual xylanases by the TL / PPL and carob inductors, which was subsequently supported by zymogram analysis.

TL / PPL also induces a multicomponent xylanolytic enzyme system, and complementary esterase and oxidase / peroxidase activities in strain 393751 and not in the other strains. These complementary activities enhance the effectiveness of polysaccharide hydrolases in enzyme cocktails optimized for key biomass degradation applications (eg cereals, plant residues, woody residues, paper products). Compared to carob, TL / PPL induces major xylanase production by all strains (especially activity against arabinoxylanes), but the levels are much lower than for strain 393751. Although OSX is a known fungal xylanase inducer and induced Enzyme production by all strains, the most pronounced induction was with strain 393751. However, unlike strain 393751, only OSX induced high xylanase activity and TL / PPL was a low inducer of xylanase production by strain 472.92 . The pattern of enzyme production in OSX is different from that obtained with carob and TL / PPL. In general, the results for xylanase production in OSX suggest that strain 393751 can metabolize raw substrates very quickly and efficiently to generate soluble xylanase inducers. Hemicellulose in more complex raw substrates is more accessible to this strain. The results also suggest that enzyme cocktails produced by strain 393751 in such complex substrates would be more suitable for hydrolysis of complex raw plant materials and residues. The model studies and applications investigated to date (for example, conversion of woody biomass, saccharification of vegetable residues and carbohydrate-rich foods and OFMSW and cereals) have confirmed the potential of these cocktails.

Finally, Figures 32A-D compare and contrast the production of active xylanase against the different test substrates (i.e., OSX, Rye AX, etc.) by strain 393751 and parental strain CBS549.92 (also CBS814. 70) in the four carbon sources.

B. Production of glucanase and mannanase: previous studies have shown that the wild-type strain (CBS814.70) and other mutant strains produce a complex glucanolytic enzyme system (Murray et al. 2001, 2004; Tuohy et al., 2002; McCarthy et al., 2003, 2005), which includes cellulases and a series of non-cellulolytic βglucan modifying activities. As observed for the xylanolytic system, multiple endoglucanases are produced, depending on the carbon source. Several of the isolated -glucanases have selective specificity for different types of β-glucans.

Tables 7A-D show activity against modified commercial β-1,4-glucan (CMC) (Sigma Aldrich), β-1,3; 1,4 barley glucans (BBG; Megazyme) and lichen Cetraria is / andica ( lichenano; Sigma Aldrich), xyloglucan (main chain of β-1,4-glucan; Megazyme) of tamarind and carob galactomannan (Megazyme). Non-cellulosic βglucans are present in significant concentrations in the non-starch polysaccharide component of various plant residues, especially cereal derivatives. Tables 7A-D illustrate differential induction of the respective activities in the strains, the induction pattern being completely different in the more complex (raw) carbon sources.

Glucose is a potent repressor of glucanase and mannanase production in almost all strains (Table 33A). For all samples, activities in dialyzed and non-dialyzed samples were measured.

As the results reveal, carob is a potent inducer of high levels of β-1,3; 1,4-glucanase (against BBG and lichen) in strain 393751, the highest levels for all strains tested (Table 33B). The levels of β-1,4-glucanase (against CMC) produced by strain 393751 were veces10 times lower than the activity against BBG (the level of CMCase was higher for this strain compared to other strains). Xyloglucanase was detected even lower, the levels obtained for strain 393751 being the highest. The zymogram analysis confirmed that the types of endoglucanase components, expression pattern and relative levels of expression of glucanase components in the filtrates of respective T. emersonii cultures are remarkably different. Low levels of (galacto) mannanase were produced by all strains during carob growth.

The TL / PPL mixture was an even more potent inducer of β-1,3,1,4-glucanase (both BBGase and lichennanase), and (galacto) mannanase, by strain 393751 (Table 33C). In general, these results highlight the lack of equivalence of β-glucanase production by T. emersonii strains and confirm that strain 393751 is an excellent source of different β-glucanase activities and the TL / PPL mixture induces a potent cocktail of these activities.

As expected, OSX (Table 33D), induced much lower levels of β-glucanase than locust bean or TL / PPL. The pattern of enzymatic production is different for strains 393751 and CBS 549.92. In Table 33D, levels of β-1,4-glucanase (carboxymethylcellulase activity) were lower for most strains except CBS 397.64, which produced levels twice as high as strain 393751 (in OSX as inducer), and not It was detected in culture strains of four strains (ie, CBS 393.64, CBS 394.64, CBS 395.64 and CBS 549.92). Significant (galacto) mannanase activity occurred during growth in OSX by strain 393751 (lower than with TL / PPL as inducer) and CBS 472.92. Figure 6A-E compares and contrasts the production of glucanase and mannanase under the experimental conditions presented by strains 393751 and CBS814.70.

In conclusion, the results indicate that:

strain 393751 is a potent producer of high levels of a series of very enzymatic activities

Important, strain 393751 is the only strain of T. emersonii that produces very high levels of both xylanase and β-1,3; 1,4-glucanase in two key depolymerizing hemicellulase activities, in low-cost inductors (carob and TL / PPL), and the highest levels of activity were obtained in raw carbon sources thereby demonstrating that strain 393751 is a cost effective source of a potent series of enzyme cocktails.

64 65

Tabaa 31A: Exoglycosidase production after growth for 120 hours in glucose as a carbon source

Gaucosa� as an inductor Strains of Talaromyces emersonii iUI / g�de�inductor)

Exoglycosidase activity

IMI 393751 CBS180.68 (IMI393753) CBS355.92 (IMI393755) CBS393.64 (IMI393756) CBS394.64 (IMI393757) CBS395.64 (IMI393758) CBS397.64 (IMI393759) CBS472.92 (IMI393760) CBS549.92 (IMI393752) CBS759.71 (IMI 393761)

a-arabinofuranosidase

3.69 5.83 6.82 5.17 6.00 6.33 2.64 4.73 4.18 3.08

-xilosidase

0.00 0.00 1.49 0.00 0.00 1.60 0.00 1.63 0.00 2.40

-Glucosidase

31.90 1.96 15.42 0.99 0.19 10.67 2.20 2.15 8.97 9.30

a-Glucosidase

0.44 0.67 0.09 0.43 1.71 0.00 0.00 0.00 0.00 1.71

-Galactosidase

0.28 1.16 0.00 0.55 0.86 0.00 0.00 0.00 0.00 1.49

-Manosidase

1.30 2.64 1.97 1.60 1.51 1.63 1.10 1.63 0.00 2.15

-Fucosidase

0.00 0.11 0.58 0.22 0.00 0.09 0.00 7.37 0.00 1.10

N Acetyl Glucosaminidase

86.08 156.97 27.83 33.72 0.00 43.56 59.57 74.91 6.22 34.87

Tabaa 31B: Exoglycosidase production after growth for 120 hours in carob as a carbon source

Aagarroba� as an inductor Strains of Talaromyces emersonii 1UI / g inductor)

Exoglycosidase activity

IMI 393751) CBS180.68 (IMI393753) CBS355.92 (IMI393755) CBS393.64 (IMI393756) CBS394.64 (IMI393757) CBS395.64393758) CBS397.64 (IMI393759) CBS472.92 (IMI393760) CBS549.92 (IMI393752) CBS759.71 (IMI393761)

a-arabinofuranosidase

0.72 0.22 3.30 1.49 7.04 1.45 1.32 1.05 2.55 1.49

-xilosidase

7.15 0.39 10.29 1.65 0.00 0.77 2.37 16.45 2.60 11.66

-Glucosidase

23.65 17.22 67.54 6.55 0.00 10.04 24.53 85.36 11.25 74.14

a-Glucosidase

0.99 0.00 0.44 2.26 0.00 0.91 6.27 0.22 1.64 1.43

-Galactosidase

2.97 4.24 1.71 3.52 0.00 1.95 3.30 1.49 1.73 3.03

-Manosidase

1.16 0.77 1.32 2.42 0.00 0.98 3.19 11.17 1.31 7.04

-Fucosidase

36.58 0.00 4.79 1.76 0.00 0.50 3.19 10.12 0.62 6.55

N Acetyl Glucosaminidase

31.02 15.95 19.91 3.85 0.00 7.98 14.85 44.55 6.73 18.15

Tabaa 31C: Exoglycosidase production after growth for 120 hours in tea leaves / paper plates as a carbon source. �te leaves / ducks�de� Talaromyces emersonii iUI / g � inductor) papea

Exoglycosidase activity

IMI 393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761)

a-arabinofuranosidase

3.30 1.10 8.53 0.00 25.74 23.93 3.08 0.88 1.87 2.53

-xilosidase

6.93 0.00 3.08 0.00 12.49 12.82 1.98 4.18 1.93 1.43

-Glucosidase

44.61 20.19 22.72 61.16 9.13 41.20 20.74 32.86 15.51 34.32

a-Glucosidase

0.44 0.00 3.41 0.00 18.10 7.65 0.83 0.00 0.61 0.44

-Galactosidase

2.81 0.00 1.01 0.00 7.26 6.27 3.19 0.00 0.88 2.42

-Manosidase

1.16 0.00 3.25 0.00 12.60 12.27 2.48 0.00 1.44 1.98

-Fucosidase

18.87 0.00 3.58 0.00 12.05 10.51 2.15 14.96 6.88 1.82

N Acetyl Glucosaminidase

27.23 18.15 9.85 46.04 13.20 45.05 16.78 56.71 25.65 25.91

Tabaa 31D: Production of exoglycosidase after growth for 120 hours in xylan of oatmeal husks as a carbon source.

�iaano�de�cascariaaas�de Oats Strains of Talaromyces emersonii iUI / g �de� inducer)

Exoglycosidase activity

IMI 393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761

a-arabinofuranosidase

15.18 2.97 4.07 5.83 23.60 8.91 0.00 1.10 0.00 5.39

-Xylosidase

9.63 0.00 2.26 0.66 0.00 2.10 11.77 14.91 12.16 6.93

-Glucosidase

32.84 16.83 42.30 24.97 0.00 26.13 15.73 67.27 3.14 61.04

a-Glucosidase

0.92 0.00 1.05 0.77 0.00 0.17 1.76 0.00 0.44 0.84

-Galactosidase

4.68 0.00 0.83 1.49 0.00 1.30 1.43 0.00 0.00 0.00

-Manosidase

0.00 0.00 2.09 2.97 0.39 2.75 2.53 0.00 0.11 0.48

-Fucosidase

19.64 0.00 0.00 1.60 0.00 1.82 1.65 6.82 0.77 1.26

N Acetyl Glucosaminidase

82.12 20.85 30.86 40.87 0.99 68.15 40.21 118.80 10.89 28.16

Tabaa 32A: Production of extracellular xylanase by strains of T. emersonii in glucose as the only carbon source

Gaucosa� as an inductor Strains of Talaromyces emersonii iUI / g inductor

Xylan Test Substrate

IMI 393751 CBS 180.68393753) CBS 355.92 (IMI 393755) CBS 393.64393756) CBS 394.64393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761

Oatmeal husk xylan

513.10 28.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Arabinoxylanode rye

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Arabinoxylanode wheat

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Birch Xylan

254.49 155.98 736.78 147.79 279.13 779.90 0.00 452.87 116.99 0.00

Tabaa 32B: Extracellular xylanase activity in culture filtrates of T. emersonii of 120 h in carob powder as the only carbon source

Agarroba� as an inductor Strains of Talaromyces emersonii iUI / g �de� inductor)

Xylan Test Substrate

IMI 393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IM I 393761)

Oatmeal husk xylan

3168.55 177.87 610.23 266.81 17.77 217.53 238.10 727.65 283.20 361.19

Arabinoxylanode rye

1212.20 0.00 0.00 15.07 0.00 0.00 0.00 135.47 90.31 201.30

Arabinoxylanode wheat

2464.00 21.89 116.27 108.08 0.00 0.00 49.28 365.31 9.57 271.15

Birch Xylan

2520.27 474.76 607.48 513.10 373.45 428.23 348.92 751.30 348.92 636.35

Tabaa 32C: Extracellular xylanase activity in culture filtrates of T. emersonii 120 h during growth in tea leaves / paper plates as the only source of

carbon

Sheets / Paatos�de�papea

Strains of Talaromyces emersonii �iUI / g �de� inducer)

Xylan Test Substrate

IMI 393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 391757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761

Oatmeal husk xylan

2696.65 124.52 198.39 618.20 166.93 467.94 280.50 287.32 335.23 264.06

Arabinoxylanode rye

2398.55 102.63 180.62 522.67 260.15 399.52 441.93 281.88 114.95 254.49

Arabinoxylanode wheat

2525.60 53.35 123.15 499.40 123.20 351.62 305.09 261.25 170.34 236.01

Birch Xylan

2127.40 417.29 458.37 771.65 457.00 626.45 727.65 617.10 647.13 521.29

Tabaa 32D: Extracellular xylanase activity in culture filtrates of T. emersonii of 120 h during growth in Xilano de Cascarillas de Avena as the only carbon source

�iaano�de�cascariaaas de�avena Strains�of Talaromyces emersonii �iUI / g �de� inductor)

Xylan Test Substrate

IMI 393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CB S 759.71 (IMI 393761

Oatmeal husk xylan

2725.25 216.15 287.87 295.35 235.35 262.68 389.95 2126.30 789.47 305.09

Arabinoxylanode rye

2009.15 284.51 220.28 384.45 372.13 247.67 351.62 1543.30 723.80 328.35

Arabinoxylanode wheat

2722.50 180.62 265.43 297.00 191.51 197.01 220.28 1641.75 577.50 281.88

Birch Xylan

2196.15 548.68 567.82 695.20 550.00 489.83 584.21 1908.50 871.53 623.92

Tabaa 33A: Extracellular glucanase and mannanase present in 120 h culture filtrates of T. emersonii strains during glucose growth as the only carbon source

Gaucosa� as an inductor Strains of Talaromyces emersonii �iUI / g �de� inductor)

Deglucanase / Mannanase Substrate

IMI 393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 3937551 CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761)

Carboxymethyl cellulose

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

barley β-glucan

0.00 0.00 0.00 0.00 77.00 69.25 137.78 95.98 0.00 0.00

lichen

0.00 0.00 0.00 0.00 202.46 112.64 0.00 0.00 0.00 0.00

Xyloglucan

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Carob galactomananode

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Tabaa 33B: Extracellular glucanase and mannanase present in 120-hour culture filtrates of T. emersonii strains during carob growth as the only carbon source

Agarroba� as an inductor Strains of Talaromyces emersonii �iUI / g �de� inductor)

Deglucanase / Mannanase Substrate

IMI393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761

Carboxymethyl cellulose

318.34 76.89 246.84 34.93 0.00 64.96 109.40 166.54 7.21 221.71

barley β-glucan

3420.45 145.64 1120.35 81.29 0.00 60.06 281.77 1150.05 45.65 1702.47

Lichenano

2162.60 135.52 429.39 47.36 0.00 32.01 203.45 431.97 19.91 914.65

Xyloglucan

180.24 40.15 89.82 45.71 0.00 43.45 39.16 48.68 10.45 112.97

Carob galactomananode

54.18 108.08 90.15 107.42 110.99 61.05 107.09 94.71 49.01 101.86

Tabaa 33C: Extracellular glucanase and mannanase present in 120-hour culture filtrates of T. emersonii strains during growth in Tea Leaves / Dishes

Paper as the only source of carbon

Leaves / footsteps / pappae� Strains of Talaromyces emersonii �iUI / g �de� inductor)

Deglucanase / Mannanase Substrate

IMI393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI3937581 CBS 397.64 (IMI 393759) CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761)

Carboxymethyl cellulose

368.61 90.75 147.02 282.10 0.00 131.56 196.90 396.72 257.95 109.40

barley β-glucan

4341.15 393.47 783.20 1818.30 100.54 783.64 804.65 3113.00 1512.50 626.89

Lichenano

2735.15 452.54 581.90 1239.70 164.56 631.40 611.05 2268.75 1356.85 559.79

Xyloglucan

192.34 88.83 26.46 171.44 6.16 146.63 77.06 177.65 82.94 39.82

Carob galactomananode

825.44 845.68 130.96 347.44 313.78 210.65 321.97 339.90 421.52 846.34

Tabaa 33D: Extracellular glucanase and mannanase present in 120-hour culture filtrates of T. emersonii strains during growth in Xilano of Oatmeal as a single source of carbon

�iaano�de�cascariaaas de�avena Strains of Talaromyces emersonii iUI / g �de� inductor)

Deglucanase / Mannanase Substrate

IMI393751 CBS 180.68 (IMI 393753) CBS 355.92 (IMI 393755) CBS 393.64 (IMI 393756) CBS 394.64 (IMI 393757) CBS 395.64 (IMI 393758) CBS 397.64 (IMI 3937591 CBS 472.92 (IMI 393760) CBS 549.92 (IMI 393752) CBS 759.71 (IMI 393761)

Carboxymethyl cellulose

101.86 32.62 22.22 0.00 0.00 0.00 213.24 64.35 0.00 25.14

barley β-glucan

375.82 100.27 100.87 44.72 21.23 72.49 283.42 282.76 52.58 117.87

Lichenano

477.68 176.33 197.56 114.62 203.72 135.52 353.27 360.80 115.61 840.40

Xyloglucan

35.26 44.39 0.00 0.00 3.91 6.88 230.84 25.14 0.00 38.23

Carob galactomananode

653.84 344.80 56.82 85.53 174.02 95.48 298.38 641.30 110.72 139.10

Example 14: Thermozymes of T. emersonii IMI393751 with potential for bioenergy production

The purpose was to reduce the biodegradable component of sterile cellulose-rich clinical waste, thereby reducing the volume of landfill waste, and recovering the production of liquids enriched with sugars after enzymatic treatment and recovering energy in the form of biofuel.

The waste stream contained a high proportion of cellulose (50%) and consisted mainly of paper, paper handkerchiefs, medical swabs and bandages and fabrics rich in cotton, branch cotton, etc. The main "fiber" in the waste stream is cellulose, but many products are "finished" with polysaccharide coatings, binding agents and fillers, so a mixture of adjuvant enzymes (specifically hemicellulase, pectinase and starch degrading enzymes) is essential ) to enhance the accessibility of cellulose and improve the reduction of residues or conversion to simple soluble sugars (for example, glucose, galactose, xylose, etc.).

Focus on Experiment:

The endohydrolase and exoglycosidase enzyme activity profile was determined in each of the 10 thermozyme cocktails, derived from strain 393751 (Tuohy & Coughlan 1992; Tuohy et al., 1993, 1994, 2002; Murray et al., 2001, 2004 ). Table 34 summarizes the relative levels of key activities determined in a selection of cocktails. Enzyme preparations were added in different concentrations to batches of 100 g of cellulose-rich residues treated with STG, at 50 DC and 70 DC, and incubated for 24-48 h (at 5060% humidity levels). Samples of sugar-rich liquor (and cellulose-rich materials, for example paper tissues, etc.) were periodically removed for 48 hours and analyzed for (i) weight and volume reduction, (ii) volume of rich liquor in recovered sugar, (iii) released reducing sugars, (iv) physical structure of the substrate after enzymatic treatment (using scanning electron microscopy), (v) qualitative analysis of the types of sugars released by TLC, (vi) quantitative analysis of the sugars produced by HPLC, CG-EM and ESI-QTOF-MS, (vii) substances potentially toxic to fermentation microorganisms (bioenergy production),

(viii) sterility of hydrolysates, (ix) bioethanol production and (x) biogas production, as described in Tuohy et al., 1993, 1994, 2002; Murray et al., 2001, 2004, Gilleran (2004) and Braet (2005).

Resolved:

A. Weight and volume / volume reduction, volume of sugar rich in recovered sugars, reducing / reduced sugars

Weights were recorded before and after enzymatic treatment and estimates of the volume reduction recorded for all enzyme preparations were made. The volume reduction and reducing sugar data obtained at 50 DC are illustrated, using the same reaction parameters (enzymatic load, 60% moisture content and a 24 h reaction time) in Figure 7.

In summary, 70 DC for 24 hours at 60% humidity produced the best results with regard to volume reduction for all cocktails. In repeated studies with the 6 cocktails selected (and with repeated trials), the volume reduction was systematically between approximately 60-75% depending on the cocktail (see Figure 8). The reducing reducing sugars were converted to the percentage of hydrolysis or conversion of the cellulose present, which was between 62-82% of the cellulose-rich fraction present in the initial sterilized residues. A volume reduction of 60-75% was obtained in laboratory tests.

For each batch of 100 g, approximately 70-100 ml of added moisture (H2O) was added to bring the final moisture content to 50-60%. The volumes of liquid recovery, after enzymatic hydrolysis, varied from 69-105 ml. Experiments were also completed at reaction temperatures of 75-85 DC and different pH values. Above 70 DC a minimal increase (∼2-5.5%) was observed in the global hydrolysis, compared to values obtained at 70 DC, and although pH 3.5-4.0 produced optimal levels of hydrolysis, the values were not significantly higher (5%) than those obtained using H2O.

B. Physical loss of substrate integrity, sugar products generated and re / active amounts of each type of sugar. The MEB showed significant loss of cellulose fiber structure (Figure 9B)

Hydrogen analysis products: quantitative analysis by TLC; Quantitative analysis by HPLC

76-92% of the sugar released was monosaccharide for the 7 best cocktails, and this consisted mainly of glucose, with some galactose, mannose and xylose. For example, sugar levels varied from 0.2-0.55 g / ml and HPLC monosaccharide concentration ranges were as follows (depending on the thermozyme cocktail and waste batch):

Glucose: 43-70%; Mannose: 5-15%; Galactose: 4-10%; Xylose: 20-30%; Cellobiose: 4-12% and higher oligosaccharides: 5-26%.

C. Exp / oration with respect to substances that are potentially toxic to fermentation microorganisms (bioenergy production), before and after fermentation, ana / isis of hydrophilicity and bioethane production / and biogas

71 5

The recovered liquid fraction did not appear to be toxic to explored yeast species with respect to fermentation of sugar-rich hydrolysates to bioethanol, that is, I do not prevent the growth of S. cerevisiae (baker's yeast), Pachyso / en tannophi / us, Pichia sp., Candida shehatae and � / uveromyces marxianus. In addition, the analysis of ethanol production (using an enzyme-linked test kit) indicated that yeasts produced ethanol.

Agar plates (containing the appropriate agar medium) were inoculated with samples of liquors rich in sugars and residual waste, incubated under recommended conditions (for the microorganism) and analyzed for the presence of colonies (bacteria and yeasts) and radial growth (filamentous fungi). There was no microbial growth in plates inoculated with sugar-rich liquors and residue samples from enzymatic treatments at 70 AD, that is, there was no microbial deterioration (and loss of sugars) of the waste hydrolysates.

Production of bioethane

The production of bioethanol was evaluated from raw materials rich in sugars by different species of yeast such as Saccharomyces cerevisiae, Pachyso / in tannophi / us, Pichia sp., Candida shehatae and

� / uveromyces marxianus (and strains). Valuation criteria measured included yeast growth (and yeast biomass), sugar utilization, evolution of CO2 and ethanol produced. Two of the enzymatic digestions at 70 DC (hydrolysates generated by cocktails 5 and 8 in Figure 8) were selected as the test raw materials for bioethanol production by all yeast species in 1 l laboratory scale cultures.

Figures 10A and B illustrate the ethanol production profiles obtained with S. cerevisiae. The ethanol yields are similar with both raw materials, even though the thermozyme cocktail 8 produces minimally more simple sugars in the hydrolyzate. However, the digestion product of cocktail 8 contains more pentose (not fermented by S. cerevisiae) than that generated by the thermozyme cocktail 5. The digestion product of the thermozyme cocktail 5 contains some cellobiose (and smaller amounts of cellooligosaccharides ) that are easily metabolized by yeast.

Tabaa 34 results for various combinations of digestion products rich in sugars yeast

Species of aevadura

Product of digestion of thermozyme 5 Product of digestion of thermozymes

S. cerevisiae

Ethanol 9.2 g / l Ethanol 9.4 g / l

P. tannophi / us

Ethanol 8.5 g / l Ethanol 8.8 g / l

�. marxianus

Ethanol 9.6 g / l Ethanol 8.4 g / l

A. pu // u / ans

Ethanol 6.7 g / l Ethanol 7.4 g / l

C. shehatae

Ethanol 6.3 g / l Ethanol 5.4 g / l

Ana / isis of finely toxic / fermentation products.

A number of techniques (HPLC, EM), especially CG-EM, were used to determine the presence of potentially toxic end-of-fermentation products. Almost complete use of sugars was achieved (with the exception of digestion products rich in pentose sugars (xylose, arabinose) for S. cerevisiae, and normal end-of-fermentation products, ie glycerol, acetate and some trace by-products were detected (solvents).

Biogas production

Larger batches of Cocktail 5 and 8 digestion products were prepared to feed the anaerobic digesters of the Superior Flow Anaerobic Hybrid Reactor (UAHR) thermophilic and mesophilic. The total carbohydrate levels of the inputs and outputs of both reactors were measured (Dubois et al. 1956; Laboratory protocol). Reducing sugars present in input and output samples were determined (Tuohy et al., 1994). To determine the Chemical Oxygen Demand (COD), a known volume of the hydrolyzate was oxidized using potassium dichromate (concentrated sulfuric acid with silver sulfate as a catalyst), for 2 hours (international test procedure: Laboratory protocol). The remaining dichromate was determined by titration with a standard solution of ferrous ammonium sulfate. The effectiveness of carbohydrate withdrawal and COD (daily samples) throughout the trial was measured. The specific methanogenic activity (SMA) of wastewater was analyzed using the pressure transducer technique (Colleran and Pistilli, 1994; Coates et al. 1996). A sample of wastewater was removed from the wastewater bed through an outlet orifice and tests were carried out at 37 DC (for mesophilic reactor wastewater) or DC 55 (for thermophilic reactor wastewater).

The sugars present in the enzymatically generated digestions were rapidly metabolized by the bacteria in UAHR both mesophilic (37 DC) and thermophilic (55 DC), that is, a 95-97% reduction in carbohydrates at loading rates of 4.5 g COD / m3 / day, in non-optimized conditions. The methane levels in the biogas flow obtained were between 55 and 61%, and the estimated retention time (days) it took to metabolize

all the sugar and reach the maximum methane levels were ,03.0-4.0 days. The pH of the output was monitored and there was no observable change in the pH of the outputs of any of the reactors.

C. Optimization of the thermozyme systems for treatment of faujos de cainicos residues rich in ceauaosa.

A combination of genomics and functional proteomics was used to identify the optimal growth conditions and substrates to use (based on information from the 10 cocktails used in the initial experiments) to obtain an enzyme cocktail that would have optimal levels of all the key enzyme components . Two inductive combinations were selected: a 1: 1 mixture of spent tea leaves and residual paper plates, and a 1: 1 mixture of sorghum and non-molasses beet pulp. Additional mixtures of cocktails selected from the 10 previously used were also prepared. The new cocktail and mixtures were characterized with respect to the component enzymes and their ability to catalyze exhaustive conversion of commercial celluloses, hemicelluloses and cellulose-rich residues sterilized into simple fermentable sugars. The optimal pH and temperature were determined for maximum enzymatic reactivity, thermostability of the optimized enzyme system and mixed cocktails, and potential inhibition by product or final reaction products, simple sugars or potential toxic molecules (phenolic compounds / benzene derivatives). Optimum temperatures: 75-80 DC, with 70-85% of activity remaining at 85 DC, depending on the enzymatic preparation / mixture (the enzymatic activity was detected at 90-95 DC). Thermostabiaity: there was no real loss of activity after 24 h at 50 DC and 5-10% loss of activity after 1 week at the same temperature. At 70 AD, 2-20% loss of activity in the first 24 hours, with 10% additional loss of activity then for a period of 5 days. Optimum pH: Although enzymes were more active at pH 4-5, 60% activity was observed at pH 3.0 and pH 6.8, with all enzymes still showing activity at pH 7.0. Enzymatic preparations were more stable between pH 3.5 and 6.0 (4-50 DC, for a period of 1 week).

Although the rate of reaction / conversion of substrate to monosaccharides was reduced or reached a plateau when high concentrations of glucose and other monosaccharides were present, the enzyme preparations were still quite active in the presence of high concentrations (up to 100 nM) of monosaccharides (glucose, xylose, arabinose, galactose). In addition, although the phenolic compounds / benzene derivatives reduced the overall activity of the cocktails, the concentrations used in the trials were significantly higher than those potentially present in the residues. However, certain cocktails and the optimized enzyme cocktail showed significant activity (50-70%) in the presence of these compounds.

In general, the concentrations of each enzyme required to achieve hydrolysis values of ∼65-75% were surprisingly low (9-16 units of cellulase / 10 g of waste) and, although they were dependent on cocktail / mixture, the higher dosages ( up to 60 cellulase units) did not increase the final degree of hydrolysis significantly. The increase in the scale of the enzymatic treatment to batches of 100 and 10 Kg produced a similar degree of final hydrolysis, and a profile and concentration of similar sugar products. The best volume reduction values obtained for optimized cocktails were 73-80%, while the corresponding% hydrolysis values for conversion of cellulosic fraction to simple sugars were 72-81%. Two of the optimized mixtures produced similar endpoints with respect to volume reduction and cellulose hydrolysis. The yield of ethanol obtained was 195-210 l / ton with S. cerevisiae and 215-220 l / ton with P. tannophi / us. Approximately 80-85% of bioethanol could be recovered by distillation, but this could be improved.

Example 15: thermozymes of T. emersonii IMI393751 for the generation of raw materials rich in sugars, from `` waste '' of `` papapa '' and `` papeaueos '' of `` papai '' in ceauaosa

Measurements of enzymatic activity:

Raw enzyme preparations were made for a number of different lignocellulose hydrolysis enzymatic activities using concentrations of 10-30 mg / ml of the substrates relevant for internally acting enzymes, 50 mg of filter paper / ml of reaction volume for "filter paperasa" (general cellulase) or 1 mM of the appropriate 4-nitrophenyl glycoside derivative (Tuohy et al., 2002; Murray et al., 2001). Triplicate tests were performed. All results are representative of two identical experiments using different crude enzyme preparations.

PH and temperature requirements for activity and stability:

The pH and temperature requirements for enzymatic activity and stability were evaluated over the pH range of 2.6-7.6, and over a temperature range of 30-90 DC, using normal test procedures.

Studies of low hydrolysis of modem:

An aliquot of individual enzymes was incubated, containing 5-60 UPF (or 2-20 xylanase units, depending on

73 5

appropriate), with 1 g of the target substrate, at the appropriate pH and reaction temperature, for up to 24 h. The samples were withdrawn at time intervals and the content and composition of sugars (reducing sugars) was analyzed.

Ceauaosa-rich substrates investigated: toilet paper and mixed paper tissues, office paper waste, brown paper, mixed newspaper.

Summary of results: thermozyme cocktails showed high activity in a broad spectrum of carbohydrate substrates and therefore reflect the complexity and efficiency of enzyme production by T. emersonii IMI393751. The production of particular thermozymes reflected the composition and variation of inducing substrate (Table 35).

Tabaa 35: relative amounts of cellulose and hemicellulose hydrolyzing enzymes

Activity

C6cteaes MGBG 2 MGBG 3 MGBG 4 UI / g�de�Inductor MGBG 5 MGBG 6 MGBG 7 MGBG 8

β-glucosidase

26.4 240.35 90.75 134.2 114.4 119.9 36.85

Endocellulase

710.6 468.6 310.2 747.45 799.15 70.4 919.6

Endo 1,3,1,4 glucanase

4471.5 3199.9 2067.45 8004.7 9493 313.5 6207.85

Endoxylanase

1234.2 1646.15 1051.6 1429.45 1369.5 653.95 1731.4

Filterpaperase

216.7 205.7 271.15 155.65 146.85 105.6 172.15

β-xylosidase

8.25 4,125 9.24 11.77 11,715 5.17 5,335

αArabinofuranosidase

4.29 33.33 17,105 34.87 20.24 7,205 23,155

β-galactosidase

10,175 137.5 6.82 42.79 13,695 1.98 115.5

Pectinase

173.25 358.6 119.9 168.85 157.3 19.25 429.55

MGBG 2, derived from 108 h T. emersonii cultures grown in a 1: 1 mixture of spent tea leaves / paper plates, MGBG 3, derived from 120 h T. emersonii cultures grown in a 1: 1 mixture sorghum / beet pulp; MGBG 4, derived from 120 h T. emersonii cultures grown in a 1: 1 mixture of wheat bran / beet pulp; MGBG 5, derived from cultures of T. emersonii 120 h. grown in a 1: 1 mix of paper plates / beet pulp; MGBG 6, derived from 120 h T. emersonii cultures grown in a mixture

(2: 1: 1) brown paper / paper plates / beet pulp; MGBG 7, derived from 120 h T. emersonii cultures grown in a 1: 1 mixture of rye flakes / wheat bran; MGBG 8, derived from 120 h T. emersonii cultures grown in a 1: 1 mixture of beet pulp / spent tea leaves.

Cellulase in thermozyme cocktails was more active at approximately pH 4.0 (Figure 11A) and between 70 'C and 80' C (Figure 12). The cellulase component or components in each enzyme preparation are active over a wide pH range (<pH 2.6 -> pH 6.5). The xylanase activity present in the same cocktails was more active at pH 4.0-5.0 (Figure 11B) and between 75-85 'C (Figure 13). However, the xylanase activity in the cocktails was active over a wide pH range (<pH-2.6 to> pH 7.0), while activity levels similar to 90 'C and 50' C were observed .

The thermal stability of several of the MGBG cocktails (listed in Table 35) was reflected in the long half-life values at 50 'C and 70' C, that is, in fact without loss or with minimal loss of endoxylanase activity

or endocellulase at 50 'C after incubation in buffer only (pH 5.0). All cocktails were crude enzyme preparations and no stabilizers or enhancers were added. The xylanase activity present in cocktails 2, 5, 6 and 8 was particularly stable at 70 'C (loss of only approximately <220% of xylanase activity after 25 h). For example, the tY value of cocktail 2 at 70 'C was greater than 6 days. Stabilities at 70 'C of cocktails 3 and 4, and to a lesser extent cocktail 7, were lower due to the presence of high levels of ecolysin protease (tY of 2 h, 12 h and 25 h). In the presence of substrate (i.e. raw waste), the stability of the three cocktails was markedly greater, that is, (tY values of 22 h, 46 h and 72 h). The overall yield of MGBG cocktails on cellulose and hemicellulose substrates was very high (Table 36). The hydrolysis level increased significantly as the reaction temperature increased from 50 'C to 70' C (Table 37). At higher reaction temperatures (for example 70 'C), the percentage of hydrolysis was achieved in 18 h. and was similar to, and in some cases greater than, the percentage of hydrolysis obtained after 24 h at 50 'C (Table 37). This finding highlights the advantage of thermozymes compared to enzymes of mesophilic organisms, which would be active at lower temperatures.

Tabaa 36:% hydrolysis of cellulose rich materials

C6cteaes

% �de�hydr6aisis�i50 ��C)

Substratum

2 3 4 5 6 7 �

Toilet paper

37.9 85.6 9.8 9.7 47.4 22.4 22.6

Paper cups

17.6 20.0 16.1 27.5 34.2 16.3 26.2

Filter paper

18.3 23.7 7.2 17.4 18.8 0.0 20.7

β- barley glucan

72.6 90.1 60.2 83.3 80.4 78.7 61.9

74 5

35 Tabaa 37:% of cellulose-rich paper pantry hydrolysis

MGBG 2

MGBG 3 MGBG 4 MGBG 5 MGBG 6 MGBG 7 MGBG �

24 hours at 50 DC

70 87 51 79 80 66 71

18 hours at 70 AD

68 89 68 66 74 61 67

The products of the time cycle hydrolysis of cellulose and hemicellulose, which were analyzed by TLC, have confirmed the high conversion efficiency of MGBG thermozymes. The polysaccharide substrates were initially degraded in oligosaccharides and finally in glucose, which was almost the only product of hydrolysis, while cellulose present in cellulose-rich residues, for example paper tissues, hydrolyzed similarly almost completely to glucose.

The implications of these results for the bioethanol-based industry are significant and indicate the potential of T. emersonii thermozyme systems.

Example 16: Bioconversion of beet puapa in sugar-rich hydrogenates for the production of Bioethane.

Eight thermozyme cocktails were selected from an initial series of 20 cocktails. The profile of endohydrolase and exoglycosidase activities was determined in each of eight thermozyme cocktails, derived from strain 393751 (Tuohy & Coughlan, 1992, Tuohy et al., 1993, 1994, 2002; Murray et al., 2001, 2004 ).

Enzyme preparations were combined in different concentrations or dosages with batches of 1 g of sugar beet fractions, prepared using different extraction procedures. The fractions were as follows:

A. Upper parts and stalks of sugar beet (1 g dry weight / 10 ml total volume)

B. Sugar beet pulp (1 g dry weight / 10 ml total volume)

C. Fruit of the sugar beet (1 g dry weight)

D. Sugar beet skin (1 g dry weight)

The A-D sugar beet fractions were homogenized in a Waring mixer (2 x 30 30 second bursts). Aliquots of enzyme, ie 2 ml and 5 ml of enzyme solutions (1-8) were added to a final total reaction volume of 10 ml (with tap water, pH 7.2) and incubated at 63 'C initially. Additional experiments were performed to optimize the reaction temperature (75 'C), pH (pH 4.0) and to reduce the incubation time (16 hours). Samples were taken at time intervals during incubation for 16-48 hours, centrifuged, and the boiling enzymatic action was terminated at 100 ° C for 10 min. The supernatant fraction was analyzed with respect to reducing reducing sugars.

The qualitative analysis of the types of sugars released by TLC was analyzed, and the quantitative analysis of the sugars produced by HPLC was determined. Sugar-rich raw materials were evaluated with respect to bioethanol production (Tuohy et al., 1993, 1994, 2002; Murray et al., 2001, 2004; Gilleran, 2004; Braet, 2005).

Resolved:

The optimal reaction conditions were 75 'C and pH 4.0. Table 38 shows the reducing sugars released after 16 hours in optimal conditions. The increase in incubation at 48 hours increased the percentage of hydrolysis in the case of skin and fruit fractions. The optimization of the enzymatic loading conditions allowed the incubation time to be reduced by half (ie 24 hours) with the yields shown in Table 39.

Tabaa 3�:% carbohydrate hydrolysis present after 16 hours at 75 AD

C6cteaes

% �de�hydr6aisis�i75 ��C) �after �16�hours

Substratum

2 3 4 5 6 7 �

Pulp

35.5 91.1 48.8 38.5 59.8 37.4 48.6

Skin

12.8 32.7 5.8 14.8 7.8 9.8 20.0

Fruit

13.8 6.4 3.0 26.0 11.5 3.0 20.7

75 Tabaa 39:% carbohydrate hydrolysis present after 24 hours at 75 DC with optimized enzyme loads

C6cteaes

Hydr6aisis�i75��C) -mg�RS� after 4�� hours

Substratum

Pulp

84.4

90.8

85.4

88.4

69.9

82.5

90.0

Skin

86.3

79.8

38.5

53.2

77.5

81.6

67.5

Fruit

66.6

75.5

59.1

104.1

79.5

85.4

102.2

Development and optimization of an enzyme cocktail was undertaken to perform hydrolysis using a combination of genomics and functional proteomics (based on information on the 8 cocktails used in the initial experiments). 5 Two cocktails were produced, labeled MGBG SB # 1 and MGBG SB # 2. The hydrolysis of the total sugar beet plant and the total fruit component were compared by the two optimized cocktails with cocktails 3 and 5 of the previous experiments. Reactions were analyzed under the optimal conditions for the two new cocktails, that is 71 'C and pH 4.5. Table 40 provides the relative levels of enzyme included in each reaction per gram of substrate (ie, total sugar beet plant or total fruit component). Tables 41 and 42

10 summarize the hydrolysis results obtained.

Tabaa 40: Enzymatic loads per gram of sugar beet substrate

Activity

C6ctea3 C6ctea5 MGBG�SB�1 MGBG�SB�2

Xylanase

4.8 4.98 0.68 0.20

Cellulase (FPase)

1.64 1.12 0.12 1.22

Pectinase

0.96 2.26 0.30 0.56

The main differences between optimized cocktails and cocktails 3 and 5 were in the relative amounts of key activities, especially exoglycosidases.

Tabaa 41:% hydrolysis based on the reducing sugars released (71 'C, pH 4.5) after 24 hours.

C6cteaes �i% de�Hidr6aisis) · RS

Substratum

3 5 MGBG�SB�1 MGBG�SB�1

Beet fruit

56.8 46.9 65.6 58.3

Total plant

51.8 68.3 97.5 87.8

Tabaa 42:% of the total sugar released corresponding to glucose

C6cteaes

Substratum

3 5 MGBG�SB�1 MGBG�SB�1

Beet

116.8 90.3 112.0 97.9

Total plant

95.0 65.6 67.1 43.2

Both new cocktails released high levels of reducing sugar from the total plant homogenate. Of the

15 reducing sugars released approximately 88-90% were monosaccharides of which approximately 4367% was glucose.

Abiobioethane Fermentation

Studies were conducted to investigate bioethanol production from beet and total plant hydrolysates. More than 90% of the glucose present in the total plant hydrolyzate was converted to bioethanol by S. cerevisiae

20 (from fermentable sugar 40 g / l, ethanol was obtained at approximately 9.0 g / l). Other yeast species were investigated with respect to bioethanol production (aerobic conditions), for example Pachyso / in tannophi / us. The last yeast used glucose and pentose sugars released (xylose and arabinose) in repeated fermentations (> 92-95% of metabolism). However, ethanol yields were slightly lower than with S. cerevisiae (from fermentable sugar 40 g / l, ethanol was obtained approximately 6.7 g / l).

25 Biochemical properties that are selected from new years.

Optimum temperatures: 75-80 'C, with> 75-87% of the remaining activity at 85 DC, depending on the cocktail.

Thermostabia: no real loss of activity after 24 hours at 50'C and <10% after 3 weeks at 50'C. At 71 'C, approximately 4-9% loss of activity in the first 24 hours, with <5-7% additional loss for 5 days.

Optimum pH and stability: although both cocktails were more active at pH 4.5,> 55% of the activity was observed at pH <2.6 and> 50-60% at pH 6.8; Both enzymes showed significant activity (> 48-58%) at pH 7.0. Enzymatic preparations were more stable between pH 3.5-6.0 (4-50 'C, for 1 month).

Example 17: Identification and properties selected from a new bifunctional iiaanasa produced by Talaromyces emersonii IMI393751.

Ta / aromyces emersonii secretes between 14 and 20 different endoxylanase components when grown in the appropriate carbon source. Thirteen of these endoxylanases have been purified to homogeneity and have been characterized with respect to catalytic properties. The molecular weights of purified endoxylanases vary between 30-130 kDa. The expression of xylanase and glucanase is not equivalent between 10 strains of T. emersonii grown under identical conditions in the same nutrient medium and carbon inducers. A new low molecular weight xylanase, Xyn XII (17.5 kDa) has been identified from the strain of T. emersonii IMI393751 of the xylan degrading system. Xylanase secretion with Mr values less than or equal to 20 kDa has been indicated for several other fungal and bacterial species, but not for T. emersonii before this.

Purification and characterization of enzymes

T. emersonii IMI393751 was grown in a 1: 1 mixture of wheat bran and beet pulp for 120 hours at 45 'C, 210 rpm (or alternatively for 11 days in solid (static) fermentation, substrate 33%: humidity 67 %; at 45 'C). The xylanase and protein contents of crude and fractionated enzyme samples were analyzed as previously described (Tuohy & Coughlan, 1992; Tuohy et al., 1993, 1994; Murray et al., 2001). Crude enzyme extract was collected as previously described (Tuohy & Coughlan, 1992). Ultrafiltration was used using an Amicon DC2 system, equipped with a HIP 10-43 hollow fiber dialyzer to separate the new xylanase (permeate fraction) from the higher molecular weight xylanases (retained). Xyn XII was purified to homogeneity using a combination of fractionation techniques, including "precipitation by salt addition" or precipitation with (NH4) 2SO4 (0-90% cut), gel permeation chromatography (GPC) in Sephacryl S200 SF (100 mM NaOAc buffer, pH 5.0 as eluent), ion exchange chromatography (IEC) in Whatman DE-52 (equilibrated with 30 mM NaOAc buffer, pH 5.0; eluted xylanase by applying a gradient of 0.0-0.3 M linear buffered NaCl), followed by hydrophobic interaction chromatography (HIC) in Phenyl Sepharose CL4B (equilibrated with 15% (NH4) 2SO4 in 30 mM NaOc, pH 5.0). Prior to HIC, the xylanase sample was "dissolved by salt addition" with (NH4) 2SO4 to a final concentration of 15% (w / v). Buffer salts and (NH4) 2SO4 were removed by application of the sample to Sephadex G-25 (not shown here). Finally, the xylanase-rich fractions were further fractionated by application to a second anion exchange column of DE-52, at pH 7.0, followed by gel permeation chromatography on Sephacryl S-100 HR (100 mM NaOAc buffer, pH 5 , 0 used as equilibration and irrigation buffer) and a final fractionation stage in DEAE-Sepharose, pre-equilibrated with 50 mM NH4OAc buffer, pH 5.5 (0.0-0.2 M NaCl gradient used to elute xylanase) was used ). The pooled enzyme was dislodged by application to Sephadex G-25 or BioGel P-6 and lyophilized before electrophoretic analysis.

Properties�acted from�aa�new�iiaanasa�bifuncionaa

Physicochemical properties are selected

The purity of the new enzyme was confirmed by electrophoresis in polyacrylamide-SDS gel in 15% (acrylamide / bis-acrylamide) gels, according to the Laemmli procedure. The SDS-PAGE revealed a unique protein band in silver staining that corresponded to an estimated Mr of 17.5 kDa. In addition, a unique protein band in IEF corresponding to a pI value of pH 5.0 for Xyn XII was obtained. The optimum temperature for the Xyn XII catalyzed degradation of OSX was determined to be 75 'C, and the optimum pH for the activity was pH 4.0 -4.5. However, unlike Xyn I-XI, which lost between 25-81% of the respective original activities during a 10-minute incubation period at pH 3.0, Xyn XII was remarkably stable against acid and retained more than 91% of its original activity at pH 3.0 even in prolonged incubation.

Cataaitic properties are selected

Suitably diluted aliquots of Xyn XII were incubated with a series of polysaccharides, including various xylanes, β-glucans, pectic polymers and fructane (all at a concentration of 1.0% (w / v)). The reducing sugars released during a prolonged incubation period of 30 minutes were quantified as described above. Activity against aryl glycosides (1.0 mM) was determined using a microassay procedure (Murray et al., 2001). Preliminary studies were carried out to determine the kinetic constants by varying the [substrate], xylan, between 0.2 and 25 mg / ml, under normal test conditions.

The results presented in Figure 14 illustrate the relative reactivity of the new xylanase (Xyn XII) against different xylanes. This enzyme is more active in an unsubstituted mixed bond xylan (1,3; 1,4-β-D-xylan)

known as rhodimenan of the red algae Pa / maria pa / mata. On the contrary, of the two cereal arabinoxylans, that is, OSX and WSX, the enzyme exhibited greater activity against the substituted WSX. The overall reactivity pattern was: RM> WSX> LWX> OSX> BWX.

As Figures 15A · G show, the substrate preferences of several of the other xylanases (Xyn IV to Xyn XI) are quite different from Xyn XII.

With Xyn IV, OSX> RM> WSX> LWX >>> BWX (Figure 15A). The order of reactivity with Xyn VI is OSX> LWX> RM> BWX> WSX (Figure 15B), while that represented by Xyn VII is RM> LWX> WSX> BWX >> OSX (Figure 15C). The reactivity of Xyn VIII was RM> BWX> WSX >> LWX> OSX (Figure 15D), and Xyn IX was RM> LWX> BWX> OSX> WSX (Figure 15E). Finally Xyn X presented the following preference RM >> BWX-WSX> OSX >>> LWX (Figure 15F) and Xyn XI RM> LWX> WSX> BWX> OSX (Figure�15G).

In addition, the difference of Xyn I-XI that were strictly active against xylanes only, the new bifunctional xylanase (Xyn XII) exhibited substantial activity against barley mixed bond β-glucan (1,3; 1,4-β-Dglucan) , that is, more than 55% activity in relation to the observed OSX, the normal test substrate (Figure 16). Unlike Xyn I-XI, the new bifunctional xylanase showed activity against aryl-β-xylosides 4nitrophenyl β-D-xyloside (4NPX) and chloronitrophenyl β-D-xyloside (CNPX), with greater activity against the second substrate (Figure 17). This finding may reflect the effect of electron removal from the rest of the chlorine, making the chloronitrophenyl group a better "leaving group." However, other new endoxylanases from strain CBS814.70 of T. emersonii (Tuohy et al., 1993; also produced by strain IMI393751, although expressed differently) are significantly active against CNPX and have little or no activity against 4NPX . This phenomenon reflected the partial "external" action characteristics of these enzymes, and may also reflect a similar characteristic for Xyn XII. In addition, low activity was observed against 4NP β-glucoside and CNP-β-cellobioside, which was not unexpected since Xyn XII is active against 1,3; 1,4-β-D-glucan and if it has some action properties external, as observed with aryl β-xylosides, one might expect that it would have corresponding activity against aryl β-glucosides. No reactivity was observed against any other aryl glycoside linked to α or β.

Therefore, "in vivo", this new bifunctional xylanase can play a very important role for T. emersonii IM I393751 by providing access to hemicellulose from the plant cell wall, for example, by degrading mixed bond glucans in cereals and others. plants, remains and plant residues.

Example 1: `` Epiration '' of `` hydroaases '' and `` other '' adjuvant enzymes through `` erasonii '' strains.

The anticipation of cavity enzymes in the same source of carbon is different.

The number, type and relative abundance of xylanase or xylanases produced by T. emersonii IMI393751 and other strains are different. As shown above, enzyme levels in culture filtrates are remarkably different and suggest that strains of T. emersonii produce different levels of the same xylanases, or express different isoforms. To illustrate this point, zymogram staining was performed after SDS-PAGE gel electrophoresis (renatured as described in Tuohy & Coughlan (1992)) and IEF with the IMI393751 and CBS549.92 strain culture filtrates. These studies confirmed that (i) different xylanases are expressed and (ii) the number of xylanase isoforms expressed is markedly different. The results obtained for xylanase expression in carob are summarized as follows: xylanase isoforms detected (see Tuohy et al. (1994) for reference to pI and Mr of isoforms) IMI393751: Xyn IV, Xyn V, Xyn VI, Xyn IX and Xyn XI, Xyl I (βxilosidase) and Xyl II CBS549.92: Xyn II, Xyn VII (note: Xyn VII is identical to the sequence published in a previous patent (GenBank Accession Number AX403831) and very low amounts of Xyl I (β-xylosidase).

In addition, IMI393751 is the only strain produced by Xyn XII during growth on tea leaves / paper plates (and tea leaves only).

The pressure pattern in different sources of carbon is different.

The pattern of xylanase expression in different carbon sources is not equivalent, that is

IMI393751:

Gaucosa as a carbon source: Xyn I, Xyn VIII, a smaller amount of Xyn XI and without β-xylosidase Agarroba: Xyn IV, Xyn V, Xyn VI, Xyn IX and Xyn XI, Xyl I (β-xylosidase) and Xyl II TL / PPL: Xyn III, Xyn V, Xyn IX, Xyn X, Xyn XII, Xyl I and Xyl II

CBS549.92:

Gaucosa: low levels of a component that could be equivalent to Xyn IV and without β-xylosidase Aagarroba: Xyn II, Xyn VII and very low amounts of Xyl I (β-xylosidase). TL / PPL: proteins similar to Xyn I, Xyn VII and Xyn IX, some of Xyl I

In addition, other clear examples of differences in expression were observed, for example, expression of Xyl I. Xyl is not expressed by IMI393751, CBS549.92, CBS180.68, CBS393.64, CBS394.64 or CBS397.64 during growth in glucose. On the contrary, under identical conditions, Xyl is expressed by CBS355.92, CBS395.64, CBS472.92 and CBS759.71.

Similarly in carob, Xyl is expressed by all strains, although at remarkably different levels, with the exception of CBS394.64. Xyl is also expressed by all strains, except CBS180.68 and CBS394.64 during growth in TL / PPL. With OSX as a carbon source, Xyl was not expressed by CBS 180.68 and CBS394.64, but was expressed by CBS393.64 (low levels) and all other strains. In general, notable differences were observed in the levels of expression of Xyl I and the pattern of expression (ie, the strains expressing the greater or lesser amounts of Xyl I) among all the inducing substrates.

Differences of specific activity � from�hom6aogos� produced� by� different strains.

The different strains were compared with respect to the expression of key xylanases as described above. However, in addition the xylanases present in induced cultures of IMI393751 and CBS549.92 were fractionated and the specific activity of the purified xylanase components was compared. In the first case, only IMI393751 seems to produce Xyn XII. In addition, when specific activities were compared (results are presented for OSX as a test substrate later), some differences in specific activity of individual enzymes were clearly observed (Figure 1).

Production of new components by T. emersonii IMI393751 during growth in different carbon sources

T. emersonii IMI393751 was cultured in a number of carbon sources and its profile was performed by renaturing SDS-PAGE and IEF, followed by zymogram staining, as presented above. The following are a sample of some of the results obtained:

(i)

Leaves of inducer: Xyn III, Xyn V, Xyn IX, some of Xyn X and Xyn XII; Xyl I and Xyl II

(ii)

Aagarroba: Xyn IV, Xyn V, Xyn VI, Xyn IX and Xyn XI, Xyl I (β-xylosidase) and Xyl II

(iii) Rye flakes: Xyn IV, Xyn V, Xyn VI, Xyn IX, with a new "xylanase" component of pI 6.5; Xyl I (β-xylosidase) and Xyl II

(iv)

Selling flour: Xyn III, Xyn VI, Xyn VIII, Xyn IX, Xyn X, Xyl I (β-xylosidase) and Xyl II, with a new additional "xylanase" component of pI 5.8

(v)

Sorghum: Xyn I, Xyn VIII, Xyn IX, Xyn X, Xyn XI, some Xyl I (β-xylosidase) and Xyl II

(saw)

Ìiaosa: Xyn I and Xyn VIII, Xyl I

(vii) Gaucosa: Xyn I, Xyn VIII, a smaller amount of Xyn XI and without β-xylosidase

Example 19: Eipresi6n differentiates from other adjuvant enzymes by strains of T. emersonii, gauation peroiidase

Culture filtrate samples (unconcentrated and concentrated samples) were processed in 7.5% native PAGE gels, soaked in glutathione reduced to 50 'C, followed by incubation with 0.002% H2O2. The gel was then mixed with 1% ferric chloride / 1% potassium ferrocyanide. Zymogram staining is also complemented by enzyme assays in the culture filtrates.

IMI393751 produces extracellular glutathione peroxidase (Mr ∼45 kDa), with differential expression observed in several carbon inductors (numbers 1-18 represent different inductors). On the contrary, no extracellular activity was observed for any of the other strains of T. emersonii, even in the concentrated samples. The IMI393751 strain also produces significant levels of extracellular glutathione peroxidase during growth in TLIPPL.

Cataaasa

Culture filtrate samples (unconcentrated and concentrated samples) were processed in 7.5% native PAGE gels, followed by incubation with 3% H2O2. The gel was then mixed with 1% ferric chloride / 1% potassium ferrocyanide (catalase bands appear as intense yellow bands). Zymogram staining is also complemented by enzyme assays in the culture filtrates, using the conventional published procedure.

IMI393751 produces extracellular catalase (Mr 30230 kDa), with differential expression observed in several carbon inductors (numbers 1-18 represent different inductors). On the contrary, no extracellular activity was observed for any of the other strains of T. emersonii, even in the concentrated samples.

The presence of these enzymatic activities in the IMI393751 culture filtrates would potentially be important in affecting the structure of the substrate, but also in removing any compound that could oxidize key hydrolase activities, for example, xylanase or glucanase.

79 5

Example 20: Comparison of phenotype of 10 strains of T. emersonii in different solid media (agar)

B.� Means� of �agar

one.

 Sabouraud dextrose agar (Oxoid Ltd., United Kingdom)

2.

 Potato maltose agar (Oxoid Ltd., United Kingdom)

3.

 Czapek Dox (Oxoid Ltd., United Kingdom; pH not adjusted)

Four.

 Cornmeal agar (Oxoid Ltd., United Kingdom)

5.

 Malt Extract Agar (Oxoid Ltd., United Kingdom)

6.

 Nutrient agar (Difco Ltd., United Kingdom)

7.

 Emerson agar (yeast potassium soluble starch; YpSs) The following ingredients were added to 1 L of distilled H2O

15.0 g of soluble starch (Sigma-Aldrich, Dublin, Ireland) 4.0 g of yeast extract (Oxoid Ltd., United Kingdom) 1.0 g of potassium di-hydrogen phosphate (KH2PO4) 0.5 g magnesium sulfate heptahydrate (MgSO4.7H2O) 20.0 g of ND 1 agar (Oxoid Ltd., United Kingdom)

8.

 Yeast glucose agar (YGA) The following ingredients were added to 1 L of distilled H2O

20.0 g of glucose (Sigma-Aldrich, Dublin, Ireland) 10.0 g of yeast extract (Oxoid Ltd., United Kingdom) 15.0 g of ND 1 agar (Oxoid Ltd., United Kingdom)

Agar plates (each type of agar) were inoculated with the 10 different strains of T. emersonii. A batch of plates was incubated at 45 'C and the second at 55' C (the moisture content of the air was -3020-30%). The cultures were checked daily, and the following results were recorded:

(to)

 culture diameter measurement (using calipers)

(b)

 photographic record using a 7.0 M pixel digital camera

(C)

 record of visible phenotypic differences / changes

(d)

 signs of spore formation (microscopic analysis) or others

Resolved:

The culture of the T. emersonii strains in the different agar media revealed clear differences between strain 393751 and the other 9 strains with respect to the following:

Tabaas 43 and 44 Summary of the speed and extent of growth at 45 DC - culture diameter measurements taken on day 2 and day 5 Tabaa 45: Growth speed at 55 'C - culture diameter measurements taken on day 5 Tabaa 46: Summary of visible phenotypic changes and spore formation tests.

Important observation 1: Many of the IMI / CBS strains produced multi-colored cultures. However, the purity of these cultures was verified. The colors / pigments produced and the production pattern is typical of many Penici // ium species, for example, P. pinophi / um.

Important observation 2: As indicated in Table 20, there are clear differences between strain 393751 and the other with respect to spore formation.

Tabaa 43: Speed and range of growth at 45 'C - Measurements of culture diameter taken on day 5

Strain

ADS AHM AMP AEM YpSs Yg Nutr agar CzapeD �Doi Aha

IMI 393751 (the inventors strain)

++++++

+++++ ++  ++++++ ++  ++++++ ++

++++++

++++++

IMI393752 (CBS 549.92)

++++++ +++++ ++ +++ ++ ++++ ++ +++ +++++

80 (continued)

Strain

ADS AHM AMP AEM YpSs Yg Nutr agar CzapeD �Doi Aha

IMI393753 (CBS 180.68)

+++ ++++ + +++ + ++++ ++ +++ +++

IMI393755 (CBS 355.92)

++++ +++++ ++ ++++ + ++++ + ++ ++++

IMI393756 (CBS 393.64)

++++ +++++ +++ ++++ ++ ++++ ++ +++ ++++

IMI393757 (CBS 394.64)

+++ +++ + ++++ + +++ + ++ +++

IMI393758 (CBS 395.64)

+++++ +++++ ++ +++ + ++++ +++ ++ ++++

IMI393759 (CBS 397.64)

+++++ +++++ ++ +++ ++ +++++ ++ +++ ++++

IMI393760 (CBS 472.92)

+++++ +++++ ++++ +++++ ++ ++++ ++ +++ ++++++

IMI393761 (CBS 759.71)

+++++ +++++ nd ++++ ++ +++ ++ ++ ++++

ADS, Sabouraud dextrose agar; AHM, cornmeal agar; AMP, potato maltose agar; AEM, malt extract agar; YpSs, yeast potassium soluble starch; YG, yeast glucose agar; AN, nutrient agar; AHA, oatmeal agar. Culture parameters: full plate, ++++++; 65-80 mm, +++++; 50-65 mm, ++++; 35-50 mm, +++; 20-35 mm, ++; and <20 mm, +.

81 82 83 (continued)

Tabaa 44: Speed and extent of growth at 45 DC - culture diameter measurements taken on day 2

Strain

ADS AHM AMP AEM YpSs Yg Nutr agar CzapeD�Doi Aha

IMI 393751 (the inventors strain)

++++

++ +  ++++ + +++ + +++

++++

IMI393752 (CBS 549.92)

+++ +++ + + + ++ + + +++

IMI393753 (CBS 180.68)

++ ++ - + + ++ + + +

IMI393755 (CBS 355.92)

++ ++ + ++ - + - - +

IMI393756 (CBS 393.64)

++ + + + + ++ - + +

IMI393757 (CBS 394.64)

+ + - ++ - + - + +

IMI393758 (CBS 395.64)

++ ++ + + - + + + ++

IMI393759 (CBS 397.64)

++ ++ + + + + + + +

IMI393760 (CBS 472.92)

++ ++ ++ ++ + ++ - + +++

IMI393761 (CBS 759.711

++ ++ nd + +/- + +/- + +

ADS, Sabouraud dextrose agar; AHM, cornmeal agar; AMP, potato maltose agar; AEM, malt extract agar; YpSs, yeast potassium soluble starch; YG, yeast glucose agar; AN, nutrient agar; AHA, oatmeal agar. Culture parameters: full plate, ++++++; 65-80 mm, +++++; 50-65 mm, ++++; 35-50 mm, +++; 20-35 mm, ++; and <20 mm, +; no growth tests, -.

Tabaa 45: Growth reach speed at 55 'C - culture diameter measurements taken on day 5

Strain

ADS AHM AMP AEM YpSs Yg Nutr agar CzapeD�Doi Aha

IMI 393751 (the inventors strain)

++++++

+++++ ++  ++++++ +++  ++++++ ++

++++++

++++++

IMI393752 (CBS 549.92)

++ ++ + + + ++ + + +++

IMI393753 (CBS 180.68)

+ + - + - + - + +

IMI393755 (CBS 355.92)

+ ++ +/- + - + - - ++

IMI393756 (CBS 393.64)

+ ++ + - - + - - - +

IMI393757 (CBS 394.64)

+ + - + - + - - +/-

IMI393758 (CBS 395.64)

+++ +++ + +++ + ++ + + +++

IMI393759 (CBS 397.64)

+ - - - - ++ - +/- +/-

IMI393760 (CBS 472.92)

+ ++ ++ ++ - +/- - - ++

Strain

ADS AHM AMP AEM YpSs Yg Agar�nutr CzapeD �Doi Aha

IMI393761 (CBS 759.71)

+ - - +/- - +/- - - +

ADS, Sabouraud dextrose agar; AHM, cornmeal agar; AMP, potato maltose agar; AEM, malt extract agar; YpSs, yeast potassium soluble starch; YG, yeast glucose agar; AN, nutrient agar; AHA, oatmeal agar. Culture parameters: full plate, ++++++; 65-80 mm, +++++; 50-65 mm, ++++; 35-50 mm, +++; 20-35 mm, ++; and <20 mm, +; no growth tests, -.

Tabaa 46: Summary of visible phenotypic changes and spore formation tests (cultures 5 days old, 45 'C).

Strain

ADS AHM AMP AEM YpSs Yg Agar�nutr CzapeD�Doi Aha

IMI 393751 (the inventor strain)

Appearance Growth, very dense, spongy, baba, creamy; coaor, orange, in the center Light, translucent growth, dense growth at the point of failure Appearance of cooking and spongy; Growth bamboospongeous; coercing before on the edge of attack Center of Darkness; Dense Growth of Bank in Attack Edge `` Dark '' Center; dense growth of caaro antecedents `` on '' edge of the attack Translucent growth; edge of dark attack Growth translucent; appearance "pathetic" Baanco ycentro�deante

Sporulation

X something X X X X X X Yes

Appearance

Baanca / cream; green�aima; before dark & green · in the center Growth, translucent, pale, spores, green Dense growth · centercream · antey and front edges Centro�crema�caaro; �verde�aima Cream Antecacaaro Green�aima; edge�of cream attack Antecacaaro; flank viscous attack Translucent growth; green center Dense green growth; pink center; Edge of Attack Creme

IMI393752 (CBS549.92)

Sputtering

Yes Yes � Yes Aug Yes � Yes Yes

Appearance

Baanco Esponjoso�con�cen�deverde�a�ante� dark. Growing translucent�cremabaanco Very dense growth; cream · suede Dense growth; border, cream, yellow center, green, orange, pale Cream · suede Green edges; Center and cream green and green Dense cream edge of visataquevisco Growing translucent; center �verdecaaro Edge of a cream attack; `` Center '' of `` green '' caaroa�cremaoscuro

IMI393753 (CBS180.68)

Sputtering

Yes Yes Yes Yes Aug Aug � Yes Yes

(continuation)

Strain

ADS AHM AMP AEM YpSs Yg Agar�nutr CzapeD�Doi Aha

Appearance

Dark center; edge of attack Growth, translucent, cream Dense growth; cream · suede Bordes�crema; medium�verde�aima; Center orange Suede Center · Creme · Dark Cream face; viscous attack edge Very translucent growth Front edges; centro�verde caaro�crema and anteoscuro

IMI393755 (CBS355.92)

Sputtering

Yes Yes Yes Yes Aug Yes Aug Aug Yes

Appearance

Growth� very �dense; cream · suede Growth�caaro, �transauced Dark Ante�caaro · cream · green�caaro · orange�and � center�crema Ante, dense growth Front, green, green, dark, center Ante · baanco Growth� very caaro and translucent Crema�en�aos�bordes; � center� green · orange

(IMI393756 (CBS393.64)

Sputtering

Yes Aug Yes Yes Yes Yes Yes Aug Yes

Appearance

Cream en�aos�bordes; center green�aima · orange; growth� very dense Transaucido; caaro green Dark before; sporangium Cruciform; edges� green�paid; center� orange · suede Cream Green Dense Growth Cream · suede; viscous appearance� Caaro; spores in front of them on the edges Green border, cream, cream, green center, darker

IMI393757 (CBS394.64)

Sputtering

Yes Yes Yes Yes Container No. Yes Aug Aug Yes

Appearance

Edge of the bank, center, dark center Poor growth; transduced; spore. Dense growth; green cream Dense, spongy growth; before · green Before · cream; growth very low Front edges; green spores Ante · un�poco�baanco; viscous Very caaro and translucent; very scarce Dense growth; center

IMI393758 (CBS395.64)

(continuation)

Strain

ADS AHM AMP AEM YpSs Yg Agar�nutr CzapeD �Doi Aha

Sputtering

Yes Yes Yes Si�iamariaao) Yes Yes Aug Container No. Yes

Appearance

Bordes�baancos, �verde · before�en�ea center Transacted; spores�baancas Antedense Cream edge; green center orange Growth�escaso; center�deante, �baanco Bordes�verde · suede; center�denso�verde · suede Scarce growth; ante, viscous �en�aos �bordes Very caaro and translucent; antepapaido Bordes�baancos�amariaao · green

IMI393759 (CBS397.64)

Sputtering

Yes Yes Container No. Yes Container No. Yes Container No. Yes Yes

Appearance

Center�denso�ocre · suede · green�aima Baanco; light growth Baanco Border edges, center green, fluorescent Baanco�paaido · cream Dense center · dark green; leading edges Suede, viscous appearance Transacted; some�spores�baancas Transacted Yante

IMI393760 (CBS472.92)

Sputtering

Yes Yes Yes Yes Aug Yes Container No. Aug Yes

Appearance

Dark, dark border, cream, green, green, ocher Growing case · green shade nd Bordes�baancos, �centro�amariaao · dark ocher · dark Debabacocoaapapaido en�aos�bordes Growth�dense�cream · suede Cream · suede, viscous appearance Growth�escaso; spores�baancas Cream �en�aos�bordes, interior �de� green aima�paaido �a�verde

IMI393760 (CBS472.92)

Sputtering

Yes Yes Yes Container No. Container No. Container No. Yes Yes

ADS, Sabouraud dextrose agar; AHM, cornmeal agar; AMP, potato maltose agar; AEM, malt extract agar; YpSs, yeast potassium soluble starch, YG, yeast glucose agar; AN, nutrient agar; AHA, oatmeal agar

Example 20: Reduction of the volume and increase of the value of waste years before incineration

Ia Steriaized in Canicic � Ceauaose wastes

Clinical waste has a calorific value of ∼14 GJ / ton. In this way, they contain ∼50-55% of cellulose-rich material, which has a high water binding capacity and lower calorific value compared to plastics present in the waste stream. The treatment of the cellulose-rich component of these wastes using the thermostable enzyme cocktails of the invention, for example, cocktails 5 and 8, can reduce the volume of waste by ∼73-8% and convert the present cellulose (79- conversion). 84%) and generate ,22.25-2.61 GJ / ton (calorific value). The rest, which is enriched in plastics could be used as a fuel derived from "selected" wastes with a potential calorific value of up to 34-38 GJ / ton.

Organizational Fraction of Municipal Waste

The organic fraction of municipal solid waste has a calorific value of ∼9.2-10.2 GJ / ton. In this form, it contains ∼50-65% of carbohydrate-rich material (mainly food and paper waste), which has a high capacity for water binding and lower calorific value compared to other components, for example, polystyrene, plastics and rubber present in the waste stream. The treatment of the cellulose-rich component of this residue with the thermostable enzyme cocktails of the invention can reduce the volume of residues by ∼50-70% and convert the present carbohydrate (conversion of 71-91%) and generate .83.8-7 , 8 GJ / ton (calorific value). The rest, which is enriched in non-biodegradable components (for example, polystyrene, plastics and rubber), could be used as a fuel derived from "selected" wastes with a potential calorific value of up to 26-38 GJ / ton.

It is therefore possible to treat municipal waste and other forms of waste before incineration. Treating such raw materials in this way can reduce, increase or alter their calorific value. Therefore, when a treatment produces a mixture of liquid or leached sugars that is collected or removed, it would be possible to reduce the calorific value. The alteration of the calorific value could facilitate the incineration of such raw materials: for example, reducing the calorific value to an "established" level would be easier to control the incineration procedure; by increasing the calorific value (that is, treating the sugars present as simple sugars in the treated raw material) the calorific value could be increased which means that the amount of energy required for the incineration itself is reduced. For example, by converting the cellulose present in the flow of clinical residues rich in cellulose into simple sugars and removing these, the calorific value of the rest would be quite low since it would mainly contain plastics, some metals, etc. The enzymatic conversion of complex carbohydrates in paper residues to simple sugars and recovery of simple sugars would significantly reduce the calorific value of the residues, the rest would still have calorific value, but this would be much lower (and would depend greatly on the lignin content).

Therefore, in a further aspect the invention provides a method for altering the calorific value of a waste stream by treating the waste stream with a strain of Ta / aromyces emersonii of the invention or an enzyme

or enzymatic composition of the invention.

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Claims (26)

one.

 A thermophilic strain of Ta / aromyces emersonii, with the deposit ND IMI 393751 or a mutant thereof with a growth temperature range of 30 to 90 'C and that actively secretes enzymes at temperatures above 55' C.

2.

 Use of a strain or mutant according to claim 1 to produce an enzymatic composition.

3.

Use of the microorganism according to claim 1, for bioconversion of plant materials or plant derivatives or waste streams including hospital waste.

Four.

Use of the microorganism according to claim 1, in the production of raw materials rich in monosaccharides from plant residues.

5.

Use of the microorganism according to claim 1, in the processing and recycling of wood paper products, paper and textiles.

6.

 Use of the microorganism according to claim 1, in the saccharification of paper waste.

7.

 Use of the microorganism according to claim 1, in the control of sludge, antifungal and biocontrol.

8.

 Use of the microorganism according to claim 1, for horticultural applications.

9.

Use of the microorganism according to claim 1, in the production of animal feed to enhance the digestibility of cereal-based feed.

10.

Use of the microorganism according to claim 1, in the production of cereal-based feedstuffs with low pentose content for monogastric animals with improved digestibility and low content in non-cellulosic β-glucans.

eleven.

Use of the microorganism according to claim 1, in the production of functional feeds with bioactive potential for use in veterinary medical assistance.

12.

Use of the microorganism according to claim 1, in the production of specialized dairy or dietary products, for example, and food formulations, of beverages for geriatric and infant medical assistance.

13.

Use of the microorganism according to claim 1, in the bakery and confectionery sectors, and in the formulation of new dietary food bakery products.

14.

Use of the microorganism according to claim 1, in the generation of flavor, aroma and sensory precursor compounds in the food industry.

fifteen.

 Use of the microorganism according to claim 1, for the generation of functional foods.

16.

Use of the microorganism according to claim 1, for the production of new alcoholic and non-alcoholic design beverages, fruit juices and dietary drinks.

17.

Use of the microorganism according to claim 1, in the production of biopharmaceutical compounds, such as bioactive oligosaccharides (including 1,3 (4) and 1,3 (6) -glucooligosaccharides mixed bond, galactooligosaccharides, xyloglucooligosaccharides, pectose oligosaccharides, xylooligosaccharides branched and linear, (galacto) glucomanooligosaccharides), glycopeptides and flavonoid glycosides of terrestrial and marine plants, plant debris, fungi and waste streams or secondary products rich in simple sugars.

18.

Use of the microorganisms according to claim 1, to increase the bioavailability of biomolecules with natural antibacterial and antiviral activity, including flavonoid and cyanogenic glycosides, saponins, oligosaccharides and phenolics (including ferulic and p-coumaric acids, epicatechin, catechin, pyrogallic acid and the like).

19.

Use of the microorganism according to claim 1, to increase the bioavailability of natural antioxidant biomolecules, for example, carotenoids, lycopenes, xanthophylls, anthocyanins, phenolics and glycosides of all materials, residues, plant residues, including various fruits and berries.

twenty.

Use of the microorganism according to claim 1, for the generation of raw materials from unprocessed plant materials, plant debris and waste for use in the microbial production of antibiotics by fungi and bacteria, including Penici // ium sp. and Streptomyces sp.

twenty-one.

Use of the microorganism according to claim 1, for the generation of raw materials from unprocessed plant materials, plant debris and waste for use in the microbial production of citric acid.

22

Use of the microorganism according to claim 1, for the production of oligosaccharides from algae polysaccharides (for example, laminaran and fucoidane) and additives derived from plant extracts, by procedures generally considered safe, in the formulation of cosmetic products.

2. 3.

Use of the microorganism according to claim 1, for the production of oligosaccharides and glycopeptides

5 for use as research reagents, in the production of biosensors and as tools in functional glycolics to explore receptor-ligand interactions and in the production of substrate libraries to perform enzyme-substrate specificity profiles. 24. Use of the microorganism strain according to claim 1, for the production of cellulose and β modified glucans, celooligosaccharides, starches and modified maltooligosaccharides, lactulose and polyols (for example, mannitol, glucitol or dulcitol, xylitol, arabitol).

25.

Use of the microorganism strain according to claim 1, in a method for altering the calorific value of a waste stream.

26.

Use according to any of claims 3-25, further comprising the use of enzymes derived from one or both of the Chaetomium thermophi / e and Thermoascus aurantiacus strains.

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