JP2013099335A - Talaromyces emersonii enzymatic system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide enzymatic compositions, especially heat stable enzymatic compositions optimized for growing "syrup" rich in fermentable sugar used in bioethanol plan from biomass.SOLUTION: There is provided a Talaromyces emersonii strain which is thermophilic and encoding heat stable enzymes. The enzymes sustain activity at temperature exceeding 55°C. These stains and enzymes may be used in various processes from reduction of wastes to production of new food components and bio-fuel.

Description

The present invention relates to Talaromyces emerson.
ii) strains and environmental and waste management, chemical and biochemical production and processing, biotechnological processes, test or diagnostic kits, prebiotics and symbiotics, health care products, functional and new food and beverages, interfaces For use in active agent production, agriculture and horticulture applications,
It relates to enzymes and enzyme systems that can be isolated therefrom.

Europe is currently facing a waste management crisis with 2 billion tonnes of waste generated annually. Most of this waste is organic, is derived from plant material (biomass), is rich in carbohydrates (saccharides), and therefore becomes a valuable resource when it is broken down into its constituent saccharides. Other types of waste, such as fruit waste, rot and result in an environmental crisis. This waste is generally combined with pig feed to derive some value from it, however it is considered a low cost waste.

Biomass is a highly variable and variable raw material, but it is the most renewable energy raw material on the planet. If utilized, it could provide a sustainable alternative to the ever-decreasing storage of fossil fuels. There is a global interest in converting energy storage in biomass into usable energy forms. The main focus was the production of biofuels such as bioethanol for transportation purposes, but the market for beneficial by-products (eg CO ₂ , lignin-rich residues, chemical feedstock) generated during biofuel production Also confirmed. Currently, about 3 billion gallons of ethanol are produced from corn annually, primarily for use in the transportation fuel sector. This is only about 1% of total automobile fuel consumption, and by 2010, bioethanol production is expected to increase more than sevenfold and include other biomass substrates such as wood residues Is done. If the woody residue is derived from a rapidly renewable “waste” source, generally regarded as “shrub forest” or undergrowth, it will satisfy processing costs, production targets and local environmental issues. Both can derive considerable value. The advantages of bioethanol as a clean and environmentally friendly alternative to oil and other fossil fuels are obvious. The global adoption of bioethanol as a major automotive fuel offsets many of the air pollution problems that are characteristic of densely populated urban areas. Modern cars are easily configured to run on ethanol / gasoline mixtures (“Gasohol”), and new engines are available that can utilize pure ethanol as the sole fuel source.

Plant biomass, including softwood species, is rich in complex carbohydrates (polysaccharides) that can be broken down into simple fermentable sugars by enzymes or chemical means. For example, softwood such as bee spruce and pine
About 41 to 43% cellulose (polymer of β-1,4-linked glucose units), 20 to 3
Contains 0% hemicellulose (mixture of mannose, galactose xylose and arabinose containing polysaccharide), and 25-30% lignin, high calorific value non-carbohydrate polyphenol polymer (dry weight percentage). Thus, about 65-70% of the dry weight of the wood residue is a complex carbohydrate that can be used to provide a sugar-rich raw material for fermentation to bioethanol.

Fungi are one of the most important microbial life forms that degrade such materials. Tallaromyces emersonii is a thermophilic aerobic bacterium found naturally in compost piles and other ecosystem-degraded biomass-rich materials. Thermostability is a property of many of the Tallalomyces emersonii enzyme systems that have been isolated to date. Since it is considered a “soft rot” species that can target all parts of plant material, this eustoma is a comprehensive carbohydrate-modifying enzyme system including cellulolytic, hemicellulose degrading, pectin degrading and amylolytic enzymes, and oxidase / oxide Generate reductase sequence and proteolytic activity. Hence, Talalomyces emersonii has access to the complex growth substrate encountered in its natural habitat.

PCT publications WO 01/70998 and WO 02/24926 disclose the isolation of cellulases from Talalomyces emersonii, 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 substrate components. Therefore, in order to metabolize a specific substrate, routine components such as recombinant technology are used to identify the components of the substrate, generate polypeptides with appropriate enzymatic activity to metabolize these components. To produce the required amount of the polypeptide, if necessary, for example to modify the polypeptide to alter the thermostability or pH optimum of the enzyme, to express the polypeptide, and to target that component of the substrate In order to do so, it is necessary to use the resulting enzyme. These are very time consuming and expensive techniques.

Furthermore, these approaches do not take into account the fact that some enzymes may exhibit more than one activity or may have a modified efficiency when used in combination with other enzymes. Therefore, these methods can lead to overproduction of enzymes that are not required, and thus also lead to over spending and time costs. Accordingly, there is a need for enzymes and enzyme systems isolated from Talalomyces emersonii and methods for isolating such enzymes and enzyme systems that overcome the above-mentioned disadvantages.

The object of the present invention is the development of an optimized enzyme composition, in particular a thermostable enzyme composition, for generating “syrup” rich in fermentable sugars for use in biomass in the bioethanol concept. Target biomass substrates or raw materials for bioethanol production can be derived from waste streams (eg, VFCW and OFMSW (vegetable fraction of collected waste and organic fraction of municipal solid waste), crops (eg, corn, sugar beet). , Grass, etc.) and woody biomass, it is important to obtain the correct enzyme preparation at low processing costs to obtain the highest yield of fermentable sugar. The cost of the enzyme used for biomass conversion before ethanol production was a major negative factor, so
The goal is to produce a low-cost enzyme preparation for these purposes.

The enzyme preparation of the present invention is derived from a thermophilic, generally regarded as safe (GRAS) fungal source, while fungal enzymes used to date in commercial and research bioethanol applications are mainly mesophilic. From sources (eg, Trichoderma / Gliocladium, Aspergillus, and Penicillium).

A further object of the present invention is to provide enzyme preparations that work at high reaction temperatures, ie thermostable enzymes allow for short reaction times / enzyme treatment steps, allowing for simultaneous sterilization of hydrolysates, Has the potential to cause significant overall hydrolysis / glycation, reduce enzyme loading, and / or reuse enzyme formulations, all of which reduce costs associated with the use of these enzymes Can help.

The present invention is numbered IMI 393751, and on November 22, 2005, Internet
IONAL MYLOGICAL INSTITUTE (CABI BIOSCIE)
nce UK) related to the Tallalomyces emersonii strain deposited.

The advantage of using Talalomyces emersonii as an enzyme source is that it is a "generally considered safe" (GRAS) microorganism and has a long history of use in the food, beverage, agricultural supply and pharmaceutical sectors .

The advantage of this new strain of Talalomyces emersonii is that the enzyme derived from it
It has optimal activity at temperatures between ˜85 ° C. and some enzymes are to maintain activity at temperatures up to 95 ° C. Therefore, these enzymes can be used even when a high processing temperature is desirable or required, for example, a reaction temperature of 65-90 ° C. promotes simultaneous high reaction rates, fast substrate conversion and simultaneous sterilization of raw materials. It also retains activity in producing high sugar feedstocks (ethanol and methane) for chemical, biopharmaceutical, chemical or biofuel production, which enhances storage and transport capabilities. Moreover, because high temperatures can be used for these enzymes, each process has a short reaction time, thus both time and cost savings. A further advantage is that if the temperature is high enough, sterilization occurs and kills any undesirable microorganisms that cannot tolerate such high temperatures. In addition, the enzyme purified from this strain was found to have a long shelf life.

According to the present invention, there is further provided an enzyme system comprising cellobiohydrolase I or cellobiohydrolase II or a mixture thereof, β-glucosidase 1, xylanase, and endo-β- (1,3) 4-glucanase. Is done. The present invention includes coffee, tea, brewing and beverage residues, fruits and fruit peel / peel, plant peel, catering and food processing, leaf / horticultural waste,
Florist waste, grain and grain processing residue, other agricultural and garden waste, bakery and store waste, paper products such as glossy colored magazines and colored newsprint, black and white newsprint, white, colored and recycled paper , Wrapping paper, paper bags, cards and cardboard, paper cups and dishes, tissues, rags,
Unused plant materials of terrestrial and marine origin, including cellophane and cellophap®, biodegradable packaging, cellulose-rich hospital waste such as bandages, paper, rags, bandages, masks, fabrics such as pajamas and toweling And methods of using this enzyme system for biotransformation of plants or plant-derived materials, such as waste streams, and for subsequent use in the production of biofuels (bioethanol and biogas) provide.

The present invention further relates to an enzyme system comprising cellobiohydrolase I or cellobiohydrolase II or a mixture thereof, β-glucosidase 1, xylanase, and endo-β- (1,3) 4-glucanase. The present invention relates to high value products such as food grade ingredients, cosmetic additives, research oligosaccharides and glycopeptides, and antibiotics including functional glycomics,
There is also a method of using this enzyme system in the production of monosaccharide-rich raw materials from plant residues to generate antibiotics and antivirals, carotenoids, antioxidants, solvents and other chemicals and biochemicals. provide.

The present invention has a growth temperature range of 30-90 ° C, an optimal range of 30-55 ° C, and 55
A thermophilic strain of Talalomyces emersonii that actively produces enzymes at temperatures exceeding ℃ is provided. The Talalomyces emersonii strain has been deposited under the deposit number IMI393751.
The present invention relates to a mutant thereof which also encodes a thermostable enzyme, an enzyme produced by strains that retain activity at temperatures above 55 ° C. The enzyme can be selected from the group consisting of carbohydrate modifying enzymes, proteolytic enzymes, oxidases and oxidoreductases.

The present invention also provides an enzyme composition comprising cellobiohydrolase I or cellobiohydrolase II or a mixture thereof, β-glucosidase 1, xylanase, and endo-β- (1,3) 4-glucanase. The enzyme composition comprises 0.5-90% cellobiohydrolase I or cellobiohydrolase II or mixtures thereof, 0.1-33% β-glucosidase 1, 0.6-89% xylanase, 0 4 to 68% endo-β- (1,
3) It can contain 4-glucanase.

CBH I (10-30%), CBH II (10-15%), and β- (1,3) 4
There is still further provided an enzyme system comprising glucanase (20-45%), β-glucosidase (2-15%) and xylanase (18-55%). The composition can further comprise one or more of the following: β-xylosidase, α-glucuronidase, exoxylanase, α-L-arabinofuranosidase, pectin degrading enzyme, hemicellulase, starch modifying enzyme, oxidoreductase / Oxidases and esterases; and proteases.

The present invention also provides methods of using this enzyme system in the processing and reuse of timber, wood and wood-derived products. This enzyme system is effective against highly wooded wood materials and is present in wood residues and processed materials (eg extracts, resins, lignin degradation products, furfural and hydroxyfurfural derivatives) Resistant to potential inhibitory molecules.

The present invention relates to CBH I (15-30%), CBH II (10-40%), β (1
, 3) to an enzyme system comprising 4-glucanase (15-40%), β-glucosidase (2-15%), xylanase (15-30%) and 1-8% β-xylosidase, Also about further. The composition can further comprise one or more of the following: exoxylanase;
α-glucuronidase; α-L-arabinofuranosidase; pectin degrading enzymes including galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases including galactosidase Oxidoreductase / oxidase and esterase; and protease. The present invention also provides a method of using this enzyme system in textile processing and recycling.

CBH I (5-55%), CBH II (8-50%), β (1,3) 4-glucanase (10-30%), β-glucosidase (0.5-30%), Further provided is an enzyme system comprising xylanase (5-30%) and β-xylosidase (0.1-10%). The composition can further include one or more of the following: α-L-arabinofuranosidase; α-glucuronidase; selected pectin degrading enzymes, other hydrolases including esterases; proteases; and oxidases. The present invention also provides a method of using this enzyme system in saccharification of paper waste.

The present invention includes CBH I (2-10%), CBH II (2-10%), β (1,3
) Further relates to an enzyme system comprising 4-glucanase (10-45%), β-glucosidase (5-10%) and xylanase (1-30%). The composition can further comprise one or more of the following: N-acetylglucosaminidase; chitinase; β (1,3) 6-glucanase; β-xylosidase; α-glucuronidase; α-L-arabinofuranosidase; Pectin-degrading enzymes including galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases including galactosidase; oxidoreductase / oxidase and esterase; and protease. The present invention also provides methods of using this enzyme system in antibacterial, biocontrol and slime control strategies in the environment, medical and construction sectors (eg dry rot control), and pulp and paper industry (eg slime control).

CBH I (1-20%), CBH II (1-28%), β (1,3) 4-glucanase (15-40%), β-glucosidase (2-15%), and xylanase ( 1
8-55%), β-xylosidase (0.1-10%), and α-L-arabinofuranosidase (0.5-5.0%) are still further provided. The The composition may further comprise one or more of the following: α-glucuronidase; starch modifying activity; other hemicellulases including galactosidase; oxidoreductase / oxidase and esterase; protease; exoxylanase; pectin degrading enzyme, phenolic acid And other hydrolases including acetyl (xylan) esterase; protease; and lignin modified oxidase activity. The present invention also provides a method of using this enzyme system in the production of new, bioactive compounds for horticultural applications such as growth promotion and disease resistance. For example, this system
Release of bioactive flavonoid glycosides, oligogalacturonides from pectin-rich materials, xyloglucooligosaccharides from xyloglucan, galactooligosaccharides from galactan, substituted xylooligosaccharides from plant xylan, or fungal cell wall β-glucan, or It can be used for the production of 1,3-gluco-oligosaccharides (laminari-oligosaccharides) from the alga laminaran, growth promotion in plants, activation of plant defense response mechanisms against phytopathogenic fungi, and increased resistance to disease. Because some of these oligosaccharides (eg laminari oligosaccharides)
This is because it can initiate and transmit bioactive properties that are antibacterial, antibacterial, and anti-nematode.

The present invention includes CBH I (5-30%), CBH II (1-15%), β (1,3
) 4-glucanase (10-40%), β-glucosidase (2-15%), xylanase (18-48%), 0.1-20% β-xylosidase, 1-10% α −
It further relates to an enzyme system comprising glucuronidase and 0.1-5.0% α-L-arabinofuranosidase. The composition can further comprise one or more of the following: exoxylanase; pectin degrading enzyme comprising galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; galactosidase Other hemicellulases including: oxidoreductase / oxidase and esterases and proteases. The present invention also provides a method of using this enzyme system in animal feed production to enhance the digestibility of cereal-based feed. This system combines the fiber components of grain-based feeds with oligosaccharides and monosaccharides (monosaccharides).
sugars)), some of which are absorbed into the intestine and metabolized. The system can also produce “prebiotic” oligosaccharides (eg, mixed-bound glucooligosaccharides from non-cellulosic cereal β-glucan) that enhance the growth of prebiotic bacteria, and release antioxidants such as ferulic acid, In addition, oligosaccharides (substituted glucurono-xylo-oligosaccharides from cereal xylan) having an antibacterial effect on intestinal microflora species known to produce carcinogenic molecules (eg phenol, amine sugar) can be produced.

CBH I (0.5-10%), CBH II (0.5-10%), and β (1,3)
4-glucanase (15-43%), β-glucosidase (2-10%), xylanase (30-88%), 0.1-2.0% β-xylosidase, 0.1-3 .0%
There is further provided an enzyme system comprising a α-glucuronidase of 0.1 to 4.0% α-L-arabinofuranosidase. The composition can further comprise one or more of the following: pectin degrading enzyme; starch modifying activity; oxidoreductase / oxidase and esterase; protease. The present invention also provides a method of using this enzyme system in the production of a low pentose-containing cereal-based feed for monogastric animals with improved digestibility and low non-cellulosic β-glucan content. Non-cellulosic β-glucan and arabinoxylan from cereals have high water binding capacity and generate highly viscous solutions. Monogastric animals (eg pigs and poultry) reduce the intake and bioavailability of important nutrients, increase the spread of digestive enzymes, impair the intimate mixing of intestinal contents and are present in these feeds These carbohydrates, which act as physical barriers to the degradation of protein and starch, cannot be degraded. Overall, these polysaccharides can lead to poor growth performance characteristics. The enzyme systems in the present invention have some potential probiotic properties without the release of pentose sugars (arabinose and xylose) that are poorly metabolized by monogastric animals and may have a nutritional inhibitory effect.
It can catalyze the degradation of cereal arabinoxylan and non-cellulosic β-glucan to produce oligosaccharides (mainly DP3-10). The present invention therefore provides a method of using alternative grains such as sorghum and corn as feed.

The present invention comprises 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 Further relates to an enzyme system comprising 1% α-glucuronidase and 0.1-5.0% α-L-arabinofuranosidase. The composition can further comprise one or more of the following: exoxylanase; pectin degrading enzyme comprising galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; galactosidase Other hemicellulases including: oxidoreductases / oxidases and esterases; and proteases. The present invention also provides a method of using this enzyme system in the production of functional feeds with bioactive capabilities for use in veterinary and human health care. This system comprises immunostimulatory β-glucooligosaccharides from terrestrial and marine plants and fungi, xylooligosaccharides with prebiotic and antimicrobial properties from terrestrial and marine plants, growth promotion from crustacean and fungal cell walls, antimicrobial and Bioactive oligosaccharides can be generated from raw materials having a GRAS state for use in animal health care (pets and large animals), including chitooligosaccharides having antiviral ability and phenolic compounds having antioxidant ability.

CBH I (1-15%), CBH II (1-15%), β (1,3) 4-glucanase (10-45%), α-glucosidase (2-10%), xylanase ( 1
Further provided is an enzyme system comprising -55%) and 0.5-12% β-xylosidase. The composition may further comprise one or more of the following: α-glucuronidase, α-
L-arabinofuranosidase; β (1,3) 6-glucanase; N-acetylglucosaminidase; pectin degrading enzyme including chitinase, galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; Starch modifying activity; other hemicellulases including galactosidase; oxidoreductase / oxidase and esterase; protease. The present invention also provides a method of using this enzyme system in the production of specialty dairy or dietary products such as food and beverage formulations for health care of the elderly and children. For example, this system can be used to produce digestible foods that are rich in prebiotics for the nutrition of the elderly and children, and to be lactose-free for individuals with galactosemia or for those who are lactose intolerant Can also be used to produce products.

The present invention includes CBH I (1-10%), CBH II (5-15%), β (1,3
) 4-glucanase (15-40%), α-glucosidase (2-30%), xylanase (15-55%), 1-12% β-xylosidase, and 1-8% α-glucuronidase And an enzyme system comprising 0.5-5.0% α-L-arabinofuranosidase,
Still further. The composition may further comprise one or more of the following: pectin degrading enzymes including galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; others including galactosidase Hemicellulases; oxidoreductases / oxidases and esterases; proteases. The present invention also provides methods of using this enzyme system in the bread and confectionery sector and in the formation of new health food bakery products. The selected enzyme system is suitable for the use of new grains / raw materials such as rye, corn and sorghum. The enzyme system increases the puffed volume and increases the shelf life by selectively modifying the arabinoxylan component of cereal flour, generates prebiotic and immunostimulatory oligosaccharides, and antioxidant molecules (eg, Ferla and Coumaric acid) release and can modify the texture, aroma and sensory properties of bread and confectionery products.

CBH I (1-20%), CBH II (1-40%), β (1,3) 4-glucanase (15-45%), α-glucosidase (2-30%), xylanase ( 1
Further provided is an enzyme system comprising 0-55%), 0.5-10% β-xylosidase, and 0.1-5% α-L-arabinofuranosidase. The composition can further comprise one or more of the following: β (1,3) 6-glucanase; N-acetylglucosaminidase;
Chitinase α-glucuronidase; pectin degrading enzyme including galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; oxidoreductase / oxidase and esterase; protease. The present invention releases monosaccharides and disaccharides that can be fermented into various products including citric acid, vanillin, etc., and fruits / fruit pulp (eg, glucosylated aromatic alcohols found in some fruits including melon, From geraniol, limonene, etc.) to flavored complex carbohydrates (aroma precursors)
Sweet, which is a precursor of rhamnose, having peptides, flavors, scents or sensory properties with umami, bitter and sweet taste, or biotransformable into molecules with such products, e.g. furaneol, strawberry flavor Of flavors, fragrances and sensory precursor compounds in the food industry by releasing amino acids for conversion to foods (eg phenylalanine) and phenol molecules (cinnamate, flavonoid glycosides such as quercetin-3-O-rhamnoside) A method of using this enzyme system in production is also provided. In addition, some of these molecules, such as flavonoid glycosides, have antioxidant and antimicrobial (antiprotozoan) activity.

The present invention relates to CBH I (1-15%), CBH II (1-15%), β (1,3
A) an enzyme system comprising 4-glucanase (10-45%), β-glucosidase (2-30%), xylanase (1-55%), and 0.5-12% β-xylosidase; About. The composition can further comprise one or more of the following: α-L-arabinofuranosidase; β (1,3) 6-glucanase; N-acetylglucosaminidase; chitinase
α-glucuronidase; pectin degrading enzymes including galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; other hemicellulases including galactosidase; oxidoreductase / oxidase and esterase; Protease. The present invention relates to the production of functional foods, specifically foods having enhanced health promoting properties, such as immunostimulatory gluco-oligosaccharides, xylo-oligosaccharides,
Produces foods rich in soy oligosaccharides, (arabino) galactooligosaccharides, (galacto) and / or (gluco) mannooligosaccharides, gentiooligosaccharides, isomaltoligosaccharides and palatinose oligosaccharides, and carotenoids and phenolic substances (cinnamic acid Also provided are methods of using this enzyme system in the modification of plant carbohydrates (terrestrial and slightly marine) to facilitate the release of antioxidant molecules such as catechins and flavonoid glycosides.

CBH I (1-15%), CBH II (1-15%), β (1,3) 4-glucanase (10-45%), β-glucosidase (1-15%), xylanase ( 1
Further provided is an enzyme system comprising ˜30%) and 0.5-20% β-xylosidase. The composition can further comprise one or more of the following: α-L-arabinofuranosidase; β (1,3) 6-glucanase; N-acetylglucosaminidase; chitinase;
α-glucuronidase; pectin degrading enzymes including galactosidase, rhamnogalacturonase, polygalacturonase, exogalacturonase and galactanase; starch modifying activity; oxidoreductase / oxidase and esterase; protease. The present invention also provides methods of using this enzyme system in the production of new designer non-alcoholic and alcoholic beverages, fruit juices and health drinks. This system produces β (1,1) to produce low calorie non-alcoholic and alcoholic beer / lagers, fruit juices and health drinks with improved sensory, antioxidant, immune enhancement, antibacterial, antiviral capabilities. 3) 4-glucan, pectin, arabinan, xylan, lactose, protein and phenolic substances can be modified. For example, a haz comprising a bioactive mixed-bound gluco-oligosaccharide and cinnamic acid (antioxidant)
e) “Light” beer with low residual β-glucan content to prevent formation (but enough β-glucan to provide mouthfeel properties), or pectin fragment (oligosaccharide) with prebiotic effect And juice rich in cinnamic acid and selected flavonoid glycosides that provide antioxidant capacity.

The present invention comprises an enzyme composition CBH I (1-25%), CBH II (1-28%),
β (1,3) 4-glucanase (18-40%) and β-glucosidase (2-30%)
And xylanase (15-55%) and β-xylosidase (0.7-20%). The composition may further comprise one or more of the following: α-glucuronidase (1-1
0%), α-L-arabinofuranosidase (0.1-5.0%), 1-15% exoxylanase, 5-25% pectin degrading enzyme, 2-12% starch modifying activity, 2 ~ 11
% Hemicellulase, 1-15% oxidoreductase / oxidase and esterase, and 2-15% protease. The composition can be used in the production of raw materials rich in monosaccharides from plant residues.

The present invention relates to CBH I (12-55%), CBH II (15-30%), β (1
, 3) 4-glucanase (12-26%), β-glucosidase (5-12%), xylanase (5-30%), β-xylosidase (0.1-10%), α-L -Providing an enzyme composition comprising arabinofuranosidase (0.5-3.0%). The composition can further comprise one or more of the following: α-glucuronidase, pectin degrading enzyme, other hydrolases including phenolic acid and acetyl (xylan) esterase, protease and lignin modified oxidase activity, protease and oxidase. The composition can be used for processing and recycling wood, paper products and paper.

The present invention relates to CBH I (3-15%), CBH II (3-15%), β (1,3
) An enzyme composition comprising 4-glucanase (15-45%), β-glucosidase (1-15%), xylanase (16-55%), and β-xylosidase (0.5-7%) provide. The composition may further comprise one or more of the following: α-glucuronidase, α
L-arabinofuranosidase, exoxylanase, pectin degrading enzyme, enzyme with starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase, and protease. The composition comprises (mixed bonds 1,3 (4) and 1,3 (6) -glucooligosaccharides, galactooligosaccharides, xyloglucooligosaccharides, pectin oligosaccharides, branched and linear xylooligosaccharides, (galacto) glucomannooligosaccharides Can be used in the production of biopharmaceuticals such as glycopeptides and flavonoid glycosides from bioactive oligosaccharides, terrestrial and marine plants rich in monosaccharides, plant residues, fungi and waste streams or by-products.

The present invention includes CBH I (1-20%), CBH II (1-40%), β (1,3
) Further relates to an enzyme composition comprising 4-glucanase (15-45%), β-glucosidase (2-12%), xylanase (1-35%), and β-xylosidase (1-5%). . The enzyme composition can further comprise one or more of the following: α-glucuronidase, α
L-arabinofuranosidase, exoxylanase, pectin degrading enzyme, starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase, and protease. Compositions have natural antibacterial and antiviral activity including flavonoids and cyanogenic glycosides, saponins, oligosaccharides and phenols (including ferula and p-coumaric acid, epicatechin, catechin, pyrogallic acid, etc.) Can be used to increase the bioavailability of a molecule.

The present invention relates to CBH I (3-15%), CBH II (3-15%), β (1,3
) An enzyme composition comprising 4-glucanase (25-45%), β-glucosidase (2-15%), xylanase (10-30%), and β-xylosidase (0.5-8%) Concerning further. The enzyme composition can further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, exoxylanase, pectin degrading enzyme, starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase ,
And proteases. The composition provides bioavailability of natural antioxidant biomolecules such as carotenoids, lycopene, xanthophylls, anthocyanins, phenols and glycosides from all plant materials, residues and wastes including various fruits and berries. Can be used to increase.

The present invention includes CBH I (1-25%), CBH II (1-40%), β (1,3
) An enzyme composition comprising 4-glucanase (15-40%), β-glucosidase (2-15%), xylanase (18-35%), and β-xylosidase (0.5-12%) Concerning further. The enzyme composition can further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, pectin degrading enzyme, starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase, and protease .
The composition can be used for the production of raw materials from raw plant materials, plant residues and wastes for use in the microbial production of antibiotics by fungi and bacteria including Penicillium species and Streptomyces species.

The present invention includes CBH I (1-30%), CBH II (1-40%), β (1,3
) An enzyme composition comprising 4-glucanase (15-40%), β-glucosidase (2-15%), xylanase (18-35%), and β-xylosidase (0.5-8%) Concerning further. The enzyme composition can further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, pectin degrading enzyme, starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase, exoxylanase ,
And proteases. The composition can be used to produce raw materials from raw plant material, plant residues and waste for use in microbial production of citric acid.

The present invention relates to CBH I (2-15%), CBH II (2-15%), β (1,3
) An enzyme composition comprising 4-glucanase (20-45%), β-glucosidase (2-25%), xylanase (1-30%), and β-xylosidase (0.5-8%) Concerning further. The enzyme composition can further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, pectin degrading enzyme, starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase, exoxylanase And proteases. The composition can be used in the production of oligosaccharides from additives derived from algal polysaccharides (eg laminaran and fucoidan) and plant extracts by processes that are generally considered safe in the preparation of cosmetics.

The present invention relates to CBH I (3-30%), CBH II (1-10%), β (1,3
And) an enzyme composition comprising 4-glucanase (10-45%), β-glucosidase (2-12%), xylanase (1-48%), and β-xylosidase (0.1-8%). Concerning further. The enzyme composition can further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, pectin degrading enzyme, starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase, exoxylanase And proteases. The composition is used as a research reagent in biosensor production and as a receptor.
For use as a tool in the generation of substrate libraries for functional glycomics to explore ligand interactions and for profiling enzyme-substrate specificity
Can be used to produce oligosaccharides and glycopeptides.

The present invention provides CBH I (5-15%), CBH II (5-30%), β (1,3
) An enzyme composition comprising 4-glucanase (20-45%), β-glucosidase (1-12%), xylanase (10-30%), and β-xylosidase (0.5-8%) Concerning further. The enzyme composition may further comprise one or more of the following: α-glucuronidase, α-L-arabinofuranosidase, pectin degrading enzyme, enzyme having starch modifying activity, hemicellulase, oxidoreductase / oxidase and esterase And proteases. The composition can be used for the production of modified cellulose and β-glucans, cellooligosaccharides, modified starches and maltooligosaccharides, lactulose and polyols (eg mannitol, glucitol or dulcitol, xylitol, arabitol).

The present invention also provides the use of biofuels and substrates produced by any of the above methods as feedstocks in the production of bioethanol or biogas such as methane or carbon dioxide, whenever produced by the process To do. The advantage of using this enzyme system to produce bioethanol or biogas is that it is a cost-effective way to produce valuable products that can be used in many industries from nearly worthless waste products. It is. At the same time producing valuable products, waste is reduced, which in turn has a positive environmental impact.

All of the above methods can be carried out with the enzyme compositions defined herein or with the microbial strains described herein.

The present invention also provides enzyme compositions and methods of using them, further comprising enzymes derived from other fungal species, including keto thermophiles and Thermoscus aurantiacus.

Preferably, the microbial strain is selected from the group consisting of one or more of Talalomyces emersonii, Ketmium thermophile, and Thermoscus aurantiacus.

More preferably, the microbial strain is Tallalomyces emersonii IMI393751 or a mutant thereof that can similarly produce an enzyme that can be active at temperatures of 55 ° C. or higher.

The present invention further provides a method for obtaining an enzyme system suitable for converting a target substrate,
The method obtains a sample of the target substrate; allows the inoculum of the microbial strain to grow on the target substrate and secrete the enzyme; an enzyme secreted during growth on the target substrate Recovering; determining enzyme activity and properties; constructing gene expression profiles; identifying enzyme proteins and constructing protein expression profiles; comparing gene expression to protein expression profiles; Including purification. The enzyme can then be stored. The method can further include analyzing the enzyme; and / or designing an enzyme system.

1 is a process overview of a method for designing an enzyme system suitable for converting a target substrate. Production of sugar-rich raw material for biofuel production by thermozyme treatment of apple flesh / squeezed straw. Figure 2 is a thin layer chromatogram of the product generated during paper cup hydrolysis. Electron microscopy of pre-treatment (0 hours) and post-treatment (24 hours) paper cups where the Talalomyces emersonii paper cup induced an enzyme cocktail that showed extensive hydrolysis of the target substrate. Comparison of xylanase production by 393751 strain and wild type CBS549.92 (formerly CBS814.70) strain. It is a comparison of glucanase production by 393751 strain and wild type CBS549.92 strain. Comparison of (galacto) mannanase production by 393751 strain and wild type CBS549.92 strain. 24. Volume reduction of sterilized cellulose-rich clinical waste catalyzed by 10 enzyme cocktails at 50 ° C. after 24 hours. 24. Volume reduction of STG sterilized cellulose-rich clinical waste catalyzed by 10 enzyme cocktails at 70 ° C. after 24 hours. Untreated cellulose rich waste (A) and enzyme treated waste (B). S. on the hydrolyzate obtained by treatment of sterilized cellulose-rich clinical waste at 70 ° C. for 24 hours (60% moisture) with cocktail 5 (A) and cocktail 8 (B). It is ethanol production by Celebrige. T.A. It is the effect of pH on cellulase activity in an Emersony cocktail. T.A. Effect of pH on xylanase activity in an Emersony cocktail. T.A. The effect of temperature on cellulase activity in an Emersony cocktail. T.A. The effect of temperature on xylanase activity in an Emersony cocktail. It is the activity of a purified novel xylanase against various xylans. OSX, autospell xylan, WSX, straw xylan, LWX, larch xylan, BWX, birch xylan, RM, Rhodymenan (1,3 of red algae; 1,4-β-D -Xylan). Activity is expressed as% relative to autosperkisilane (100%). FIG. 4 is the activity of purified (A) Xyn IV and (B) Xyn VI against various xylans. Activity is expressed as% relative to autosperkisilane (100%). FIG. 4 is the activity of purified (C) Xyn VII and (D) Xyn VIII against various xylans. Activity is expressed as% relative to autosperkisilane (100%). FIG. 2 is the activity of purified (E) Xyn IX and (F) Xyn X against various xylans. Activity is expressed as% relative to autosperkisilane (100%). Figure 2 is the activity of purified Xyn XI against various xylans. Activity is expressed as% relative to autosperkisilane (100%). Relative activity rate of purified Xyn XII against various purified polysaccharides. OSX, oat spelled silane; BBG, barley β-glucan; LIC, lichenan; CMC, carboxymethylcellulose; LAM, laminarin; D1, dextran; D2, dextrin; INU, inulin; ARA, arabinan; GAL P, galactan (potato); RG, rhamnogalacturonan; LWAG, larch arabinogalactan. FIG. 4 is the specific activity of purified Xyn XII against aryl β-xyloside and aryl β-glucoside. Comparison of specific activities of selected xylanases represented by IMI 393751 and CBS 549.92 against OSX as assay substrate.

The invention will be more clearly understood from the following description of embodiments thereof, given by way of example only with reference to the accompanying drawings.

Referring to FIG. 1, a process overview for designing an enzyme system for converting a target substrate is provided. In step 1, the target substrate is obtained. In step 2, the target substrate is inoculated with a microorganism that is cultured on the target substrate to provide a culture. Microorganisms secrete enzymes, and these enzymes are recovered in step 3 by obtaining a sample of the culture. The culture sample is
In step 4, it is separated into cell fraction and culture filtrate. The cell fraction (mRNA) is analyzed in step 5 to determine enzyme activity and properties. At step 6, a gene expression profile is constructed based on the analysis of step 5. At step 7, the culture filtrate is screened for protein activity and a protein expression profile is constructed at step 8. In step 9, the gene and protein expression profiles are compared. Step 10
The enzyme is then purified. The enzyme can be stored at step 11 and at step 12, the enzyme can be further analyzed, and the enzyme system is designed at step 13.

It has been discovered that when fungi are used as microorganisms, better results are obtained when the substrate is inoculated with fungal mycelium. This cultivation is preferably carried out in a fermenter and the reaction conditions vary depending on the type of microorganism and substrate used. The culture may be in the form of a liquid or solid state fermentation. For Talalomyces emersonii, a culture temperature in the range of 45-65 ° is preferred, and the enzyme is optimally active up to 85-90 ° C.

The enzyme is separated from the target substrate by separation of cell (fungal) biomass from the extracellular culture filtrate, using centrifugation in the case of enzymes produced by liquid fermentation, or using a cell separation system for large cultures. Collected. The enzyme is then recovered from the cell biomass fraction by homogenization of the known weight of 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 enzyme extraction of solid state cultures, the cultures were homogenized by shaking for 2 hours at 140 rpm at room temperature and extracted 0.01% (v / v
10 volumes of 100 mM citrate phosphate buffer, pH 5.0
Mixed with. The enzyme rich extract is then recovered by centrifugation.

Enzymatic system comparisons are made at both genomic and proteomic levels, i.e. genes,
It is essential that the expression product (mRNA) and the identified protein are compared. This is due to the fact that there may be genetic information that is expressed at the mRNA level but not translated into functional protein at the protein level. Transcription, genomic and proteomic analysis is important for determining the relative abundance of a particular enzyme.

An isolated population of filamentous strains from which some of the enzymes of the present invention have been isolated is for the purpose of the patent procedure of Nov. 22, 2005, International Myological Inst.
itute (IMI) (CABI Bioscience UK), Bakeham L
ane, Englefield Green, Egham, Surrey TW20 9
Deposited with TY, United Kingdom-Deposit Number IMI393751. After purification of the enzymes, they can optionally be stored or analyzed directly, (a) detailed information on individual thermal stability / thermal activity in general catalyst and functional properties, (b) individual and Detailed information on their mode of action and catalytic ability in combination, (c) information on synergistic interactions, (d) partial sequence information to aid gene cloning, and (e) optionally, three-dimensional structural data Obtain sufficient protein acquisition to facilitate collection. Analysis of these enzymes is then utilized to identify the key enzyme systems and optimal harvest time for these systems.
The system can be optimized with respect to key activity and key enzyme mix, performance characteristics and target application conditions (laboratory scale) levels.

Example 1 Emersonii IMI 393751, Isolation Freshly harvested (˜200 g) clean grass (lawn) cuts and other mixed plant biomass were placed in closed containers to simulate a composting environment and about 65 hours at 65 ° C. so,
Incubated in a constant temperature chamber (combined sterilization and equilibration of substrate)
. The humidity / moisture content was maintained at ˜65-70%. The containers were fitted with wires that were spaced apart to provide a low pulse of wet filtered air. After 2 hours, T.W. A spore suspension (1 × 10 ⁸ spores in 2% sterile water) of Emersony (originally a laboratory stock of a 12-year isolated population from CBS 814.70) was used to inoculate the central area of biomass Used. The temperature was maintained at 65 ° C. for 2 days and then increased at 2 ° C. intervals every 24 hours until an air temperature of 70 ° C. was reached internally in the chamber. The culture is aseptically removed from the inoculated “hot spot” or central area sample and transferred to an agar plate (Emerson Agar for thermophilic fungi) to ensure culture purity, and They were grown for an additional 7 days before being subcultured. Purity was verified by microscopic analysis. Liquid medium containing basic nutrients (Tuohy et al., 1992; Moloney et al., 198)
3) and 2% (w / v) glucose were inoculated with a 1 cm ² fragment of fungus from an agar plate culture after 36 hours. The liquid culture was grown at 55 ° C. for 36 hours in a 250 mL Erlenmeyer flask (containing 100 mL growth medium) with shaking at 220 rpm. An aliquot (2.0 mL) of mycelium suspension is removed after 36 hours, aseptically washed with sterile water, transferred to a sterile petri dish (10 mm diameter), and timed intervals (10-10).
60 seconds). Sterilized agar (noble agar containing 0.2% w / v individual catabolite repressor such as 2-deoxyglucose) was inoculated with a sample of mycelium of irradiated fungi. Homotypic cultures were incubated at 45 and 58 ° C. Single colonies were carefully selected (soft white appearance), transferred aseptically to Sabouraud glucose agar plates, and further purified through several transfers. Strain IMI393751 is an isolated population taken from plates incubated at 58 ° C. Mutants were evaluated in a comparative study with parental organisms for enhanced thermal stability and enzyme production, which was the same growth condition, culture appearance, sporulation ability (strain IMI393751 does not sporulate) Clear differences were apparent between both strains in terms of thermophilicity and stability, enzyme production patterns, differential expression and levels of individual enzyme activity.

Physiological / mycological differences between CBS 814.70 and IMI 393751 Appearance of growing cultures on various agar media-when cultured on Emerson agar
CBS 814.70 has typical characteristics of this kind (Stolk & Samson, 1
972), i.e. pale cream / pale yellow, with a pale yellowish shadow near the agar surface (around the inoculation zone), which turns into a dark dark pale yellow / reddish brown as the culture matures, and It consists of a high density of fungal cones. In contrast, IMI393751
Is much whiter and the culture has a very “soft” appearance.

Differences with respect to sporulation-CBS 814.70 produces conidial spore, ascosis and ascospores. A sporulating strain of Emersony. The conidial pattern appears as a vertical branch on the mycelium (light yellow in color and septum). Ascosas, or spores containing sac, are somewhat ellipsoid in shape and can often be found in the chain; ascospores are smooth and elliptical in shape (green in electron micrographs) Looks). In contrast, IMI393751 mycelium produces very few classic conidial spore, ascomyta and ascospores. In stark contrast to CBS 814.70, IMI393751 only produces a small number of spores under extreme and quite specific conditions, while strain CBS 814.70 is on several different media. Produces spores.

Differences in thermophilicity and optimal growth temperature range. CBS 814.70 has a strict growth range of 35-55 ° C., with lower or higher temperatures with little growth and an optimal growth range of 40-45 ° C. On the other hand, IMI393751 has a wide growth temperature range of 30-65 ° C., good growth to 80 ° C., 85 ° C., and 90 ° C., with the optimum being 48-55 ° C. IMI393751 grows very well at> 55 ° C., continues to produce high levels of fungal biomass (mycelium), and actively secretes significant amounts of various proteins.

Differences regarding growth at high pH values. CBS 814.70 has a p above pH 6-6.5.
Does not proliferate very much at H level (actually starts to die) (previous data, Tuohy & C
oughlan, 1992), while IMI393751 grows well and secretes considerable levels of enzymes at pH values up to 9.0.

Growth under nutrient limited / depleted conditions. IMI393751 survives well in nutrient-depleted cultures up to day 55, whereas CBS 814.70 undergoes autolysis after day 7 and after 15 days, a few viable mycelia / Cells remain. The mycelium harvested from the 55-day nutrient-depleted liquid culture of IMI393751 could be revived by transfer to Sabouraud glucose agar, while the rest of the mycelium of CBS 814.70 strain (55 days) was The same approach could not be restored.

Example 2: Enzyme system from Talalomyces emersonii for converting hemicellulose materials. Complete hydrolysis of xylan requires the synergistic action of xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase and esterase.
Table 1 shows examples of selected target substrates (including waste / residues) and the percent carbon source induction used in this example. % Induction refers to the weight of carbon source per volume of medium (g / 100 ml)
.

Based on the results obtained, the enzyme system was designed using enzymes purified from Tallaromyces emersonii for degradation of hemicellulose (xylan) or wood-derived products, residues or wastes rich in xylan. . The relative amounts of key enzymes present in the enzyme system are tabulated in Table 2.

The relative amount of central activity, collateral activity profile and relative amount will vary depending on the composition of the target substrate. Table 3 outlines the enzyme system from Talalomyces emersonii designed for specific applications.

Example 3: System from Talalomyces emersonii to convert cereals, beet pulp, and other materials (including waste) rich in arabinoxylan and acetylated hemicellulose.
Table 4 shows Talalomyces emersonii IMI393751 and two previously identified mutations designed to convert arabinoxylan and acetylated hemicellulose rich grains, beet pulp, and other materials (including waste) The relative amounts of various enzyme activities in the designed enzyme system from the strain are shown.

Example 4: System from Taralomyces emersonii for converting non-cellulosic materials such as tea leaves and carob powder. Important for both selective modification and degradation of various grain residues, including potential candidates for brewing and animal feed, such as sorghum, corn and rye,
Characterization of three novel endoglucanases (EG). These three enzymes were purified from Tallalomyces emersonii IMI393751.

The total carbohydrate content of the purified enzyme preparation is the glucose or mannose standard curve (20
~ 100 μg. mL ^-1 ) and determined by the phenol sulfate method (Dubo)
is et al. ).

Protein Separation Technology Sequential gel filtration, ion exchange, hydrophobic interaction and lectin affinity chromatography and chromatofocusing (pseudo ion exchange)
Required to obtain highly purified preparations of VI and EG VII. Non-denaturing gel and isoelectric focusing followed by Coomassie blue staining is performed by standard methodologies known to those skilled in the art and provides relevant references (eg, Murray et al.
al, 2001; Tuohy & Coughlan, 1992, Tuohy et a.
l. 2002; Maloney et al. 2004). The initial experiment was EG V-V
The pi value of II was found to be <pH 3.5. Therefore, chromatofocusing on PBE94 pre-equilibrated with 0.025 M piperazine-HCl, pH 3.5 was used to determine the correct pi value for EG V-VII. The pH corresponding to the elution peak of β-glucanase was determined to be pI. A difference of 0.01 pH units could be detected by thus generating the correct pi value for each enzyme.

pH and temperature optimum conditions and stability The optimum pH value is determined by observing enzyme activity at a pH value between 2.5 and 9.0 at 50 ° C. in a constant ionic strength citrate phosphate buffer. It was. To evaluate the effect of pH on the activity of EG V-VII, purified samples of each enzyme were incubated at 50 ° C., pH 4.0, 5.0 and 6.0. An aliquot is taken after 0, 1, 2, 3, 6, 9, 12, 24, 36, 48 hours and 336 hours (1
4 days) and 100 mM NaOAc buffer, pH 5.0 (normal assay pH)
) Appropriately diluted and assayed for residual activity according to standard assay methods. 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 was not a regulatory protein in that none of the three enzymes was adsorbed to cellulose (or other insoluble carbohydrates) (ie they did not contain carbohydrate linkages).

Substrate specificity for various polysaccharides and synthetic glycosides is determined by measuring activity against a wide variety of carbohydrates (BBG, lichenan, CMC, other polysaccharides and synthetic glycosides) using normal β-glucanase assay procedures. It was evaluated.

Effect of metal ions, chemical modifying reagents and potential inhibitors The range of monovalent, divalent, heavy metal ions at a final assay concentration of 1.0 mM is determined by incubating the enzyme with chemicals and then normal enzyme assays. By performing EG V, EG VI and EG VII
It was investigated with respect to their potential effects on the activity of Finally, the inhibitory effects of glycosides such as disaccharides, lactones, and flavonoid glycosides on purified enzymes were examined.
The specific concentration of each glycoside was included in the normal assay cocktail and residue activity determined by comparison with the control (no glycoside present).

Purification parameters such as summary of EG V, EG VI and EG VII purification and% yield are given in Table 5.

Yield, and final specific activity values were similar for EG V and EG VII, while EG VI was obtained in low yield and had high specific activity.

Enzyme homogeneity M _r and pI value Schiff's reagent by positive staining (results not shown), all 3
It was shown that the seed enzyme is a single subunit glycoprotein. The homogeneity of each enzyme preparation is
Confirmed by IEF. The pI values obtained were as follows: EG V, 2.4
5; EG VI, 3.00; EG VII, 2.85.

Evidence for glycosylation and estimation of molar extinction coefficient The total carbohydrate content (w / w) is calculated as EG V
, EG VI and EG VII 22.6 ± 0.1%, 14.7 ± 0.1, respectively
%, And 65.9 ± 0.04%. Calculated molar extinction coefficient (ε ₂₈₀ ) value (mo
l. l. cm ⁻¹ ) is 1.04 × 10 ^{−5 for} EG V and 8.4 for EG VI.
80 × 10 ⁻⁶ , and 7.05 × 10 ⁻⁶ for EGVII.

pH and temperature optimums and stability The three enzymes were active over a relatively broad pH range, but acidic pH of 5.7, 5.4 and 5.7 for EG V, EG VI and EG VII, respectively. The optimum value is shown. A pH optimum of 4.5 is Obtained for BBGase activity in the crude Emersony extract. The optimal activity of about 75% is between pH 3.0 and 7.0 (
EG V), pH 2.5-7.5 (EG VI) and pH 2.8-7.5 (EG VII)
) Was obvious.

Release of reducing sugar from BBG, pH 5.0, at 30-90 ° C. for 10 minutes. The optimum temperature values for activity are 78.0 ° C., 78 for EG V, EG VI and EG VII, respectively.
. 0 ° C and 76.0 ° C. Activation energy estimated from Arrhenius plot (
E _a ) is 21.2 ± 0.05 kJ. mol ⁻¹ , EG VI 23.5 ± 0.02 kJ. 26.7 ± 0.05 k for mol ⁻¹ and EG VII
J. et al. mol ⁻¹ .

The effect of pH on enzyme stability is at pH 5.0, pH 5.5 and pH 6.0 (50 ° C.
And at 70 ° C.). The pH stability is maximal over the 1 hour incubation period in the range of pH 4.0-7.0, with a marked decrease observed at pH <4.0 and> 7.0, with EG V of 15 days Without losing activity at pH 5.0 and 50 ° C. over a period of
On the other hand, EG VI and EG VII lost minimal activity (13.0% and 11.5%, respectively). However, at 70 ° C. and the same pH, EG V, EG VI and EG
VII lost 42%, 35% and 33% of the original activity in each sample over a period of 60 minutes, respectively. At pH 5.5 (50 ° C.), EG V, EG VI and EG VII are 1
Loss of original activity of 30%, 22% and 8% respectively over a period of 5 days but at 70 ° C
Thus, EG V was further destabilized (58% decrease in original activity after 60 minutes), while 60%
The activity of EG VI and EG VII after minutes was very similar to that obtained after incubation at pH 5.5 and 70 ° C. EG V was considerably less stable at pH 6.0 and 50 ° C. and lost 63% of its original activity on day 15. EG VI and EG VI
I is considerably more stable at the latter pH and has that similar to the stability determined at pH 5.5 (30% loss of activity for both enzymes). By raising the incubation temperature to 70 ° C., the activity of EG V is increased over an incubation period of 60 minutes (
Significantly reduced (65%) (at pH 6.0), on the other hand, EG VII remained very stable and only lost 22% of its original activity. Half-life (T1 / 2) value is pH 5.0
And at 50 ° C for more than 15 days, on the other hand at 70 ° C the value goes from 67 minutes (EG V) to> 8
0 min (EG VI and EG VII).

Evidence of module structure EG V, EG VI and EG VII are pH 5.0 and 4 ° C.
Weakly adsorbed on Avicel (microcrystalline cellulose) (8-15% adsorption); however, this adsorption range does not indicate the presence of a carbohydrate binding module (CBM).

Effects of metal ions, chemical modifiers and potential inhibitors on enzyme activity EG V, EG
The effect of a final concentration of 1 mM in the range of monovalent, divalent, and multivalent cations on the activity of VI and EG VII was investigated. Activity was expressed as a percentage of the control (no metal ions present in the incubation mixture). In general, heavy metal ions are thought to inactivate enzymes by forming covalent salts with cysteine, histidine, or carboxyl groups. A number of metal ions such as Mg ²⁺ , Zn ²⁺ and Mo ⁶⁺ enhance the activity of all three enzymes, and ions such as Ag ⁺ , Fe ²⁺ and especially Hg ²⁺ are EG V
Although the activity of -VII was markedly reduced, significant differences between enzymes were observed with respect to the effects of other metal ions. Na ⁺ and K ⁺ chlorides had no effect on or slightly stimulated the activity of each enzyme, for example K ⁺ increased the activity of EG VII by 20%, while N
a ⁺ enhanced the activity of EG V by> 39%. Ba ²⁺ and Ca ²⁺ decreased the activity of EG V by 16.5% and 21.5-24.6%, respectively, while both ions were
Increased EG VI activity by 37.6% and 10%. Cd ²⁺ , Co ²⁺ and Cu ²⁺
Other divalent cations such as had a significant inhibitory effect on EG V (˜26.
5-77.4% loss of activity). Almost complete inhibition of the activity of all three enzymes by Hg ²⁺ suggests the presence of essential thiol groups involved in catalysis. It is noted that the valency regulates the activity, for example Fe ³⁺ has a stronger negative effect on the activity of all three enzymes (35.4-88.0% activity decrease), Fe ²⁺ actually increases the activity of EG V 2
0% enhancement, with minimal effect on EG VII, and 38.
In contrast to the 6% reduction (Fe ³⁺ reduced the activity of EG VI from 53.6 to 59
. 3% reduction). Counter anion, i.e. Cl ^- versus SO ₄ properties of ^2-, salts of both specific anion has a profound effect on activity when investigated. Ca ²⁺ and Fe ³⁺
Of SO ₄ ^2- salt selectively enhanced the activity of EG VII by ˜2-fold and ˜4.5-fold, respectively, over the activity observed for the corresponding Cl ^- salt. In contrast, Mg ²⁺ SO ₄ ^2
- salt, Cl same cation ^- with respect to salts, EG V (54.2%) and EG VI (5
0.1%) activity was significantly reduced. The selection of reagents known to modify amino acid R groups in proteins was tested for their effects on EG V, EG VI and EG VII both in the absence and presence of substrate ( Thus, the substrate protecting effect could be observed) (see Table 6).

The strong inhibitory effect of N-bromosuccinimide (NBS) suggests the involvement of tryptophan in binding and / or catalysis. However, another tryptophan modifying reagent, o-phthaldialdehyde, has no net effect on the activity of EG V, EG VI or EG VII, and therefore NBS may thereby have side reactivity. Other known amino acid residues such as cysteine could be modified. Furthermore, the absence of a substrate to prevent enzyme inactivity excludes the direct role of tryptophan in binding or catalysis. Cysteine and dithiothreitol (DTT) both prevent inactivation, so NB
The effect of S can be attributed to side reactions such as the oxidation of cysteine. Sulfhydryl (sulp
hydryl) reagents iodoacetamide, p-hydroxymercurybenzoic acid, and N-ethylmaleimide cause little inhibition in the presence or absence of substrate,
It seems to suggest that EG V, EG VI and EG VII do not have the essential thiol group. However, cysteine, DTT and dithioerythritol are all 3
Activating certain enzymes, especially EG VI and EG VII, which could possibly suggest reduction of oxidized disulfides during extraction and / or enzyme purification, and thus the enzyme molecule, or the active site of the enzyme as a whole Restore the native conformation of the region. Sodium borohydride, a strong reducing agent, inhibits three enzymes (especially EG VI) in the presence of a substrate. In contrast, the oxidation of other reactive groups at the active site by the action of strong oxidants such as sodium periodate, iodine and thioglycolic acid significantly enhances the activity of all three enzymes and the effect is Most obvious for sodium periodate and EG VI in the presence of substrate. Woodward reagent K, a carboxylic acid modifying reagent, enhanced the activity of EG V, EG VI and EG VII and was very effective in the absence of substrate.

In general, to name just a few examples of tested compounds, m-phenylphenol, (-
Phenolic substances such as epicatechin, (+) catechin, o-coumaric acid, caffeic acid, ferulic acid, syringic acid and tannic acid, except for tannic acid, which is a potent inhibitor for its protein precipitation function, There was no significant inhibitory effect on enzyme activity (in the absence of substrate). In fact, some compounds, such as protocatechuic acid, syringic acid, caffeic acid and polyvinyl alcohol, markedly activated all three enzymes (results show that pre-enzyme pretreatment with inhibitors in the absence of substrate. Obtained during incubation). However,
When co-incubated with enzyme and substrate, some of the phenols are ~ 7-3
It was noted that it resulted in a 2% decrease in activity (the substrate did not protect any of the enzymes from the potent effects of tannic acid).

In general, the majority of detergents tested did not cause loss of enzyme activity. However, taurocholic acid, taurodeoxycholic acid, Tween 20 and Tween 80 all have an activity of EGV of 47.7%, 22.7% and 12.12, respectively, when preincubated by the enzyme prior to addition of the substrate. 7% and 30.8% reduction. Tween 8
Except for 0 (34.2% loss of activity), co-incubation with substrate restored total activity. Enhanced activity is observed when substrates, enzymes and detergents (deoxycholic acid, CHAPS, taurodeoxycholic acid and chenodeoxycholic acid) are incubated simultaneously, eg, the activity of EG VI is in the presence of substrate and deoxycholic acid. Increased by a factor of> 2.

Glycosides such as salicin, esculin and arbutin had no apparent effect on the activity of EG V-VII, as did the disaccharides melibiose, maltose, sucrose and alditol, sorbitol. However, cellobiose concentrations from 50-75 mM markedly inhibited EG V, which was also inhibited by all three enzymes, particularly lactose at concentrations> 75 mM. Lactose is also EG VI at a concentration of ~ 120 mM.
And ˜50% inhibition of EG VII BBGase activity. Glucono-δ-lactone and glucoheptono-1,4-lactone are also much higher concentrations (50% for 50% inhibition).
0 to> 750 mM) but inhibits EG V-VII.

Substrate specificity The crude extract of Emersony catalyzes the hydrolysis of various polysaccharides including spectra of cellulose, CMC, BBG, laminaran, lichenan, xylan, pectin and synthetic glycoside derivatives. None of the three purified enzymes were active against 4-nitrophenyl derivatives of β-D-xylopyranoside or α-D-galactopyranoside (Table 7).

Furthermore, EG V, EG VI and EG VII did not show any activity against filter paper, Avicel, locust bean gum galactomannan and did not catalyze the oxidation of cellobiose upon prolonged incubation with the substrate. The result is the control (B
The activity against BG is assigned in terms of% activity against (assigned a value of 100%). All three enzymes showed maximal activity against mixed-bound β-glucan, BBG and lichenan, and markedly more activity against the latter substrate. With extended incubation periods, the trace activity exhibited by all three enzymes with respect to xylan can be explained by the fact that the used autospertoxylan preparation contained a small amount of contaminating β-glucan. .

After performing a similar analysis for each of the expressed enzymes, the enzyme system was purified from Tallalomyces emersonii for degradation of non-cellulosic materials such as tea leaves, carob powder and other similar materials. Designed using

  Table 8 gives examples of the relative amounts of various enzyme activities of this enzyme system.

Example 5: T for converting cellulose, cellulose-rich waste and cellooligosaccharides
. System from Emersonii Talalomyces emersonii IMI393751 was grown on various paper waste and paper products as substrates. Secreted enzyme was extracted and enzyme expression was observed and quantified by proteome and transcriptome analysis and by full spectrum of functional assays. Some paper wastes have been found to be excellent inducers of cellulases (and complementary activities such as starch-hydrolyzing enzymes for which coated / finished paper products have been used). The difference was clearly evident for the relative amount / type of cellulase enzyme induced by various paper wastes / products. The data obtained is cellobiohydrolase I (
CBH I) and cellobiohydrolase II (CBH II) isoenzymes are the most important cellulase activity and if more complete saccharification / bioconversion of cellulose in the target substrate is desired (eg for biofuel production) Β-glucosidase I (BG I) was also found to be very important for the production of monosaccharide-rich raw materials.

The paper plate has a remarkable level of filter paper (FP) degradation activity (1573 IU / g, where IU is
Product formed, μmol / reaction time, minutes / induced substrate, g), low endocellulase level (27.5 IU / g) and low β-glucosidase level (6.05 IU / g)
Induced. At the transcriptome level, CBH I is the most abundant / highly expressed cellulase, with observations supplemented at the functional level, 242.0 IU / g CBH
Type I activity was detected. The enzyme produced during growth on paper cups is significantly more exo-acting, with FP activity of 495.0 IU / g and 53.
9 IU / g β-glucosidase was detected. In the latter example, gene expression and functional assays indicated that CBH II was a key cellulase (CBH II transcription and enzyme levels were ˜2 times the corresponding levels of CBH I enzyme). CBH II was again a key cellulase induced by wrapping paper, corrugated paper and white office paper. (
Separate enzyme systems (eg, for amplification of exo or accessory / accompanying activity) and combinations thereof can be used in a wide variety of cellulose-rich unused secondary and waste materials (and hemicellulose / Other carbohydrates) have been shown to be an effective tool for conversion.

The enzyme was isolated and analyzed by conventional procedures (Walsh, (1997) and
et al, 2002).

CBH IA containing trace amounts of xylanase as the only contaminating activity is 115-170 mM.
Elute at a NaCl concentration of between. Fractions 52-66 were pooled and dialyzed for 16 hours against 4 changes of 100 mM ammonium acetate buffer, pH 5.0 and replaced with p-aminobenzyl-1-thio-cellobioside. -Sepharose 4B column (1.
4 × 11.3 cm). Residual contaminating xylanase activity did not bind and eluted in the application and wash buffers. CBH IA was eluted using 0.1 M lactose in 100 mM ammonium acetate buffer, pH 5.0.
Fractions 13-21 were pooled and dialyzed against distilled water to remove lactose and stored at 4 ° C. until used.

CBH IB (DE-52 at pH 5.5) from the anion exchange step is 100 mM.
Dialyzed against ammonia acetate buffer, pH 5.5 and applied to an affinity column for CBH IA. Residual contaminating activity, mainly endoglucanase, did not bind to the affinity matrix and eluted in the wash. CBH IB was specifically eluted 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 further use.

Based on the above analysis, the following enzyme system was designed using enzymes purified from Tallaromyces emersonii for the degradation of paper waste and paper products, and other wastes containing cellooligosaccharides.

Example 6: System from Thermophilic Fungus Species, Ketomium Thermophile and Thermosuscus Orantiacus Strategies outlined for the design of enzyme system / thermozyme compositions from Emersonii IMI 393751 and previously known mutants are configured to produce carbohydrate modified compositions with more than 23 mesophilic and thermophilic fungal species It was done. Special attention was paid to the generation / induction of potent hemicellulases (xylanases, mannanases) and pectinase-rich enzyme systems by these fungal species.

Ketomium thermophiles and Thermoscus aurantiacus, as previously described, contain 1 to 6% of an induced carbon source Individually cultured in liquid fermentation on an Emersony nutrient medium (enzyme production by solid fermentation was also investigated). A powerful mannan degrading enzyme system is available for 96-120 hours of C.I. Obtained by culture of thermophilic bacteria. This composition of the system is characterized and contains 45-60% mannan hydrolysis activity, 0.7-4.0% pectin modifying enzyme, 35.2-52.0% xylan modifying activity And the rest considered to belong to cellulase activity (very low or trace amounts of CBH and β-glucosidase were observed).

Incubation of the same fungus on soybean bran resulted in a powerful xylan-degrading enzyme system (70.
2-86.5% total activity was considered to belong to the xylan modifying enzyme);
The remaining carbohydrate modifying activity of 8.0% and ˜8.6-22.5% was considered to belong to cellulase, pectin degradation and low levels of mannan degrading enzymes.

Similarly, a strong pectin-modifying enzyme system is available for Th. On bran and beet pulp (1: 1).
A total carbohydrate modification activity profile of> 56.5-80.0% elicited during aurantiacus culture was represented by pectin degrading activity; this enzyme system is ˜22.1-40
. It also contained 1% xylan modifying enzyme, the rest being mainly cellulase / β-glucan modifying activity. In contrast, Th. Aurantiacus culture induces a powerful xylan-degrading enzyme system (> 62.1 to 85.8%), supplemented by ˜3.5 to 11.0% pectin modifying enzymes, the remaining activity is Mainly β-glucan modification. C. In contrast to thermophiles, Th. Orantiacus did not produce significant levels of mannan degrading enzymes during culture on either substrate.

These systems, alone and in combination with each other or with other enzyme systems (eg, T. emersonii), are a wide variety of agricultural, food / vegetable, beverage, wood / paper and other carbohydrate-rich unused And production of sugar-rich feedstocks that have been shown to perform extensive saccharification (sugar release) of waste materials and for specialized oligosaccharide products or for a wide range of biotechnological applications (eg biofuel production) Can be used for

Example 7: T. for the production of sugar-rich raw materials from food / beverage, paper and wood waste used for biofuel production. A system from Emersony.
(A) Biotransformation of food / beverage waste: apple flesh / squeezed straw (removed pulp) waste apples, apple flesh and squeezed straw are local fruit suppliers,
Obtained from food processing and apple juice (alcohol) / beverage manufacturers and distributors. T. T. et al. Emersony (
Cultured on 2-6% apple flesh / squeezed straw (by both solid and liquid fermentation) and a high level of carbohydrate modifying enzyme range was measured. This system consists of high levels of β-glucan hydrolase, mainly non-cellulose β-glucanase (˜27.
4%), especially high levels of α-arabinofuranosidase (13. of total carbohydrase activity).
3%) and a significant amount of key exo-glucosidase with β-galactosidase (22.6%). Additional esterases, pectin and xylan modifying enzymes were also detected (> 7.2-33.5%). Therefore, using the above analysis, it is possible to design an enzyme system suitable for degrading apple waste.

Early work showed 2344 nkat xylanase, 5472 n per 3.6 Kg substrate.
An enzyme charge containing kat mixed-bound β-glucanase and 8529 nkat lichenanase was used and a reaction temperature of 70 ° C. was used. Complete sterilization of the hydrolyzate was achieved at 70 ° C. and the hydrolyzate was used to feed a mesophilic and thermophilic upflow anaerobic reactor (UAHR). 100% utilization of the sugar feedstock was observed and methane was incidentally produced (50-70% in the biogas stream). Subsequent optimization studies were conducted, which was about 2/3
80 ° C. with gentle stirring (˜120 rev / min) for a period of 24 hours with the original enzyme dose of
Incubation with a simple, proved to achieve ˜87% saccharification of existing carbohydrates to fermentable sugars.

The sugar produced by this enzyme system can be used as a raw material rich in monosaccharides for biofuel production.

(B) Paper waste: Bioconversion of paper cups and paper products. Emersony was cultured without supplementation on various paper wastes in liquid fermentation (
See Example 5). Paper cups are highly effective carbohydrase inducers resulting in a powerful multi-component enzyme cocktail with high levels of xylanase and amylolytic enzyme activity, and the level of cellulase activity is reported for conventional growth substrates Turned out to be higher. The potential of this enzyme system to effectively release reducing sugars and decompose paper waste was clearly explained by biochemical tests and scanning electron microscopy. The effect of thermozyme treatment on substrate integrity and morphology using scanning electron microscopy confirms the potential of these cocktails as powerful biotechnological tools for paper waste conversion. SEM is a T.W. Clear evidence of significant cellulose fiber degradation (complete loss of fiber structure in certain samples) following treatment of cellulose-rich substrates with an Emersony cocktail was submitted.

After 108 hours of growth on a paper cup, The enzyme cocktail produced by Emersony contains a series of cellulose, hemicellulose, and amylolytic enzymes, and saccharification studies performed on this multi-component cocktail have shown that glucose and other reductions from traditional cellulose and paper waste substrates Demonstrate its ability to effectively release sugar. This enzyme cocktail was found to be active against all paper wastes analyzed and conventional cellulose substrates. All substrates were gradually degraded over time, but different biodegradability was shown in response to different substrate compositions.

Prior to any pretreatment, the biodegradable packaging showed the strongest potential for enzymatic hydrolysis, followed by tissue paper, paper cups and cardboard. The paper cup-inducing enzyme system works optimally, with an enzyme dose of substrate at a temperature of 50 ° C., pH 4.5, 4 mL / g, and 37 revolutions /
Releases maximum sugar levels from paper waste during minutes of shaking. The homogenization of the paper cup increased the hydrolysis level by a factor of 2.3. Under these experimental conditions (36 FPU enzyme dose) (filter paper unit)
85% total hydrolysis was achieved and glucose accounting for -80% reducing sugars was released.
Glucose and xylose were the major products released (see Figure 3). However, by reducing the enzyme dose to 9 FPU, ˜76% overall hydrolysis was performed. Electron microscopy demonstrated the excellent hydrolysis properties of this cocktail (Figure 4).

Heat treatment increased cardboard conversion by the same enzyme system by a factor of 34% (overall carbohydrate hydrolysis based on ~ 88% released reducing sugar), while the combination of both heat treatment and homogenization is The released reducing sugar is increased by 80% and 1.47 mg / ml glucose (
Yielded 31.7% of the total sugar released). The paper dish was rapidly degraded by the paper cup-inducing enzyme cocktail, releasing glucose that accounted for ~ 67% of all sugars.

Enzymatic saccharification of paper and food waste involves sequential hydrolysis and saccharification (SHF), an enzyme pretreatment followed by yeast fermentation to produce ethanol, and the raw material is continuously produced and immediately fermented by the yeast. Simultaneous hydrolysis and saccharification (SSF). The enzyme pretreatment reaction temperature is different in both processes, i.e. high in SHF as the hydrolyzate is cooled before fermentation, and in SSF is near ambient temperature for yeast growth and fermentation.
T. T. et al. The Emersonii IMI393751 enzyme is a more effective and higher reaction rate and sterilization is achieved (and fewer enzymes are required). Emersony enzyme is 25
It still works very well at ˜37 ° C. and is well comparable to commercial enzyme preparations from other fungal sources. The raw materials rich in sugar produced are biofuels (bioethanol and biogas)
It was found to be suitable for production.

(C) Bioconversion of softwood residue for bioethanol production Wood residue and waste from primary and secondary sources (eg bark, decimation, and processing waste such as shavings and sawdust) are in lignin Hemicellulose (~ 19-28)
%, And mainly xylan and mannan, and some other polysaccharides) and cellulose (~ 39-4)
6%) is a vast resource with a dry weight of 65-70% containing complex carbohydrates.

T. T. et al. Emersonii IMI 393751 is used to produce bee spruce sawdust and ash shavings to produce an enzyme system with an appropriate profile of enzymes for target waste conversion.
grown in liquid or solid state fermentation on woody residues such as Various reaction / pretreatment temperatures and enzyme doses were investigated. The enzyme system evaluated is T on various substrates.
. The cocktail obtained during the growth of the Emersony was included. 50 ° C., 60 ° C., 70 ° C. and 80
A reaction temperature of 0 C was investigated and a number of different substrates were used, namely untreated and pretreated wood residues. Enzyme loading was also investigated, with initial studies starting at 60 FPU and later 200 FP
Increased to U (FPU: filter paper units, a measure of total cellulase activity).

  The effect of enzyme dose on theoretical ethanol yield is shown in Table 11.

Example 8 Preparation of Saccharification Test Substrate for Woody Biomass Spruce chips (2-10 mm in diameter) were sulfur dioxide (3% w / v moisture) at room temperature for 20 minutes to 2.5% w / w moisture absorption. ). The SO ₂ treated spruce was treated with steam at 215 ° C. for 2-5 minutes. The hemicellulase content was almost completely hydrolyzed; the solids recovery was 60-65% of the starting raw material.

Enzymes MGBG enzymes: MGBG1, MGBG2, MGBG3 and MGBG4 were numbered. The commercial enzyme used is T.W. Celluclast 2L and A. from reesei. Novozym 188 from Niger (Novo Industry)
A / S, Bagsvaard, Denmark).

Enzymatic hydrolysis evaluation Standard enzymatic hydrolysis was performed at 300 mL, 1 L and 10 with ~ 130 rev / min stirring.
Performed at 37 ° C., 50 ° C. and 60 ° C. in the L reactor. The enzyme dose was 32 FPU of each enzyme preparation per gram of cellulose in a buffered substrate solution (Gilleran, 20
04). The pH of the reaction buffer was adjusted to pH 5.0 for the MGBG enzyme and pH 4.8 for commercial preparation. Samples were removed at timed intervals and the enzymatic action was terminated by boiling each reaction mixture (and control) for 10 minutes. At the lowest reaction temperature (37-50 ° C.), the two inventive enzyme preparations perform as well as the commercial Celluclast preparation, and (ii) the performance of the three MGBG enzymes is commercially Cell
It is in the same range as uclast (and Celluclast / Novozym blend).

Glucose yields using the compositions of the present invention were similar to optimized commercial preparations, but they yield higher levels of additional fermentable sugars than commercial enzymes.

The enzyme preparation of the present invention outperformed commercial enzyme / enzyme blends at high reaction conditions (60-70 ° C.) in terms of overall hydrolysis range, product yield and enzyme stability. Lower enzyme doses could be used at higher reaction temperatures to achieve similar hydrolysis performance (only 62.5-78% of commercial enzyme loading was required, depending on enzyme preparation). They were also less affected by inhibitors present in the steam pretreated substrate and were high in glucose and cellobiose in sugar rich hydrolysates. They produce larger amounts of sugar at 60 ° C. for 24 hours than commercial enzymes achieve at commercial enzyme's optimal working temperature of 50 ° C. for 72 hours.

The key results for enzyme hydrolysis are shown in Table 12, while the best reaction temperature / reaction time combination for the optimum% hydrolysis of each commercial and inventive enzyme composition examined on the other hand is Are given in Table 13.

Simultaneous saccharification and fermentation (SSF)
Batch SSF experiments with spruce hydrolyzate were performed to compare the performance of various enzyme preparations. Fermentation was performed at a spruce fiber concentration of 4% and the pretreatment material was diluted to the desired concentration with sterile water. The pH was maintained at 5.0 by the addition of 2M NaOH. The fermentation temperature was 37 ° C. and the stirrer speed was 500 revolutions / minute. The reaction medium was sparged with nitrogen (600 ml / min) and the CO ₂ content was measured by a gas analyzer. The enzyme preparation was added directly to the fermentor with a load of 25 filter paper units (FPU) / g cellulose. The fermentation medium was supplemented with nutrients: 0.5 g / l (NH ₄ ) ₂ HPO _4.
0.025 g / l MgSO ₄ . 7H ₂ O and 1.0 g / l yeast extract. The concentration of added yeast (baker yeast, Saccharomyces cerevisiae) cell population was 5 g / l and all SSF experiments were performed for 72 hours at 37 ° C. Samples are withdrawn at various time intervals and centrifuged in a 1.5 ml microcentrifuge tube at 14000 g for 5 minutes (Z160
M; Hemle Labtechnik, Germany), the supernatant is then HPLC
Prepared for analysis.

The hydrolysis product generated during the degradation of the lignocellulosic material was degraded by HPLC. Cellobiose, glucose, xylose, galactose, mannose, HMF and furfural were separated on a polymer column (Aminex HPX-87P) at 85 ° C. and the mobile phase was Millipore water at a flow rate of 0.5 ml / min. Ethanol, glycerol and acetate concentrations were determined using a Shimadzu HPLC system using an Aminex HPX-87H column at 60 ° C. and equipped with a refractive index detector. The mobile phase was a 5 mM H ₂ SO ₄ aqueous solution with a flow rate of 0.5 ml / min. The ethanol produced was similarly determined using an enzyme binding assay (r-Biopharm, Germany).

Yeast growth takes place in two phases. Carbon dioxide is an important byproduct of the ethanol fermentation process because anaerobic fermentation of 1 mole of glucose yields 1 mole of ethanol and 2 moles of carbon dioxide. Therefore, measuring the carbon dioxide concentration in the outlet gas is an indirect measurement of the fermentation rate. In the first growth phase, the available glucose is consumed and ethanol is formed and the initial fast response to the glucose present is indicated by a sharp rise in CO ₂ evolution.

The results obtained during SSF are given in Table 3. As previously mentioned, the total time taken by the SSF for each enzyme preparation was 72 hours, and the temperature used for the SSF was 37
° C. Fermentation efficiency was generated if the actual ethanol concentration produced (g / L) was converted if all available substrate was converted to soluble fermentable sugar and all sugar was converted to ethanol. Determined by dividing by the total theoretical ethanol (g / L) and multiplying by 100.

Based on the data obtained, the ethanol yield for each enzyme / SSF combination is given in ethanol, L / ton dry and the corresponding US units ethanol, gallon / dry US ton.

Example 9 Comparison of Commercial and MGBG Enzymes in Sequential Hydrolysis and Fermentation (SHF) Strategies for Bioethanol Production The bioethanol yields obtained by sequential hydrolysis and fermentation were investigated for commercial and inventive enzyme preparations. It was. One advantage of SSF is that the process consists of an initial fast fermentation and metabolism of monomeric sugars resulting from pretreatment steps. Once glucose is released from the substrate by the action of the added hydrolase, the fermentation is fast, which means that SS
In F, it means that the concentration of free sugar always remains low. In SSF, the fermentation rate ultimately decreases as a result of the rate-limiting one of reducing the substrate conversion rate by the enzyme or inhibiting yeast metabolism. The data obtained in these experiments is summarized in Table 15.

HPLC analysis shows that the main product formed is a monosaccharide (single sugar) and a very small amount of high oligomers (disaccharides, the main or only high chain present in the hydrolyzate. It was confirmed that cellobiose (sugar) was formed.

Example 10 Animal Sample Application Each enzyme composition is evaluated in a separate target application and model studies have shown a final reaction volume of 50-250 mL at pH 2.5-7.0 and 37-85 ° C. with or without shaking. 5-25 of them
Performed on a laboratory scale with g substrates (cereals, flour, or other plant residues). Enzyme performance is evaluated with and without substrate pretreatment, ie, gentle steam pretreatment (105 ° C, 8 psi for 5 minutes), polishing using a mortar and pestle, homogenization in a Parvalux or Ultraturrax homogenizer. It was. For soft fruit and vegetable tissues / residues, the substrate is
Coarse by mixing and incubated with the enzyme without pretreatment. Substrate hydrolysis consists of (i) measurement of released reducing sugars and assays to detect and quantify individual sugars, (ii) confirmation TLC and HPLC analysis of hydrolysis sugar products, (iii) weight of residue /
Analysis of volume reduction, (iv) Comparison of cellulose, hemicellulose, starch and pectin content before and after enzyme treatment, and (v) Substrate degradation by scanning electron microscopy (SEM) of fibrous substrates such as paper and wood waste Was observed by physical analysis.

For example, for feeds rich in key oligosaccharides
The galactooligosaccharide-MGBG16 enzyme cocktail is the best, the glucooligosaccharide-MGBG16 and 22 is the best, and the fructooligosaccharide-MGBG21 cocktail is the best

For feed preparations rich in antioxidants, the best cocktail used for processing is MGBG13
, 16 and 23.

Example 11: Monogastric animal feed applications:
The study was done with raw grain fraction (1-5 g lot) (with shaking at 140 rev / min) 50
Performed at temperatures ranging from ~ 85 ° C. Hydrolysis of carbohydrates in the substrate with each enzyme preparation (maximum dose of 0.5 IU per gram of substrate) was observed over 24 hours by quantifying the reducing sugar released, and the product formed in the sample was Removed from the reaction mixture at regular intervals.

Significant hydrolysis of the xylan and non-cellulose β-glucan fractions was observed, which led to the formation of the medium into a longer chain oligosaccharide product of hydrolysis, which was also for probiotic microorganisms Would be a suitable fermentation substrate.

Example of biogas production Two 10 L upflow anaerobic hybrid reactors (UAHR; Reynolds, 1986), one maintained at 37 ° C. and the other at 55 ° C., each of mesophilic and thermophilic bacteria Used for biogas production by mixed populations. The sugar-rich hydrolyzate was pumped through the sludge bed and degraded by the existing microbial community. A separator at the top of the reactor was used to separate the gas produced from any sludge particles that could be removed during anaerobic digestion. Both reactors have COD removal (APHA
1992), total carbohydrate reduction (Dubois method), and by observing the efficiency of methanogenesis, was evaluated continuously throughout the 650 day operating period. Fatty acid production and methane production, which are indicators of sugar metabolism, were observed by gas chromatography (GC).

Biofuels can be generated from a number of feedstocks. Many of them require the use of different enzyme cocktails. MGBG16 is the best cocktail for raw material production from food and vegetable waste listed below for biogas production by anaerobic digestion.

The data in Table 18 was achieved with a retention time of only 3 days, in contrast to reports in the literature where the retention time is typically between 9-30 days. The retention time indicates a period (or induction period) required for metabolism of the carbohydrate raw material and generation of biogas.

Example 12 Bioethanol Production Standard enzymatic hydrolysis was performed at 37 ° C., 50 ° C. and 60 ° C. in 300 mL and 1 L vessels with agitation of ˜130 revolutions / minute. The enzyme dose was 32 FPU of each enzyme preparation per gram of cellulose in a buffered substrate solution (Gilleran, (NUI, G
always, Ph. D. As described in Thesis, 2004), in a laboratory scale study, the total processing weight = 100 g). The pH of the reaction buffer was adjusted to pH 5.0.
Samples were removed at timed intervals and the enzymatic action was terminated by boiling for 10 minutes. The following were measured over a period of 0 to 72 hours:
Release of reducing sugars and detection and quantification of individual sugars (expressed in g / L)
HPLC analysis of hydrolysis sugar products (product amount expressed in g / L)
・ Residue weight / volume reduction ・ Cellulose fiber content before and after enzyme treatment

Scanning electron microscopy (SEM) of substrate integrity before and after enzyme treatment The production of key fatty acids during methane metabolism and methanogenesis by anaerobic bacteria was observed by gas chromatography (GC). Bioethanol produced by yeast fermentation was measured using two approaches, an enzyme-linked assay kit for ethanol quantification (r-Biopharm, Germany) and also by high performance liquid chromatography (HPLC).

Bee spruce hydrolyzate, paper waste and woody residues (mixture of coniferous residues, mainly bee spruce) were examined in a laboratory scale study. SHF (sequential hydrolysis and fermentation), and SSF (
Two ethanol production formats (simultaneous saccharification and fermentation) were investigated.

Some of the cocktails listed above have uses in a wide range of other applications. In these applications, the key differences in cocktail use are (a) the enzyme dose or amount used in each treatment, and (b) the duration of the incubation. For example, MGBG18
Is very suitable for the treatment of certain waste streams as well as nutritional applications. "waste"
In the treatment / saccharification step, a high dose of enzyme is used and the reaction time is ~ 18 ~
24 hours (~ 25-32 filter paper units). The ultimate goal is to achieve extensive degradation of the target residue into fermentable monosaccharides. In contrast, if the production of bioactive oligosaccharides (either gluco-oligosaccharides or xylo-oligosaccharides) is required and / or tissue changes to bread, low enzyme concentrations are required and the desired end point is reached The modification (or reaction) time can take up to 1 hour (up to 2 hours).

If the target substrate was inherently rich in cellulose, the enzyme concentration was based on “filter paper units or FPU”. When bioactive oligosaccharides (eg, non-cellulose β-gluco-oligosaccharides) are produced, the enzyme concentration is determined by fractionation of the target substrate (eg, non-cellulose, mixed-bound β1,3 from fungal or algae sources; 1,4- Glucan or β-1,3; 1,6-glucan).

Example 13 T. during liquid fermentation Enzyme production by Emersonii strains Talaromyces emersonii strains tested were:
IMI (Imperial Myological Institute (CABI)
Bioscience)) 393751 (patented), IMI 393753 (CBS (Ce
ntraal Bureau floor Schemes) 180.
68), IMI393755 (CBS355.92), IMI393756 (CBS39)
3.64), IMI393757 (CBS394.64), IMI393758 (CBS)
395.64), IMI 393759 (CBS 397.64), IMI 393760 (C
BS472.92), IMI393752 (CBS549.92), IMI393761
(CBS 759.71).

Liquid fermentation; Emersony's isomorphic liquid medium is in a non-pH controlled condition.
Grown at 45 ° C. in the medium described by y et al, (1983) and Tuohy & Coughlan (1992). The four carbon sources selected are: glucose (
Monosaccharide), oat spelled xylan (arabino glucuronoxylan), carob powder and 1: 1 tea leaf / paper dish mixture (˜15-20 seconds, fragmented in blender, culture supernatant is Recovered (Tuohy & Coughlan, 1992; Murray et a
l, 2002), and used to analyze extracellular enzyme production).

Enzyme assay; enzyme activity was expressed in international enzyme units (IU) per gram of induced carbon source. One unit IU releases 1 micromole of product (reducing sugar, 4-nitrophenol, etc.) per minute. All exoglycosidase and endohydrolase enzyme assays were performed as previously described (Tuohy & Coughlan, 1992; T
uohy et al, 1994, 2002; Murray et al, 2002; G
illeran, 2004). Unless otherwise stated, all initial activity measurements were made at 50 ° C. and pH 5.0. The exoglycosidase activity includes β-glucosidase, α-glucosidase, β-xylosidase, β-galactosidase, β-mannosidase, β-fucosidase, α-arabinofuranosidase, N-acetylglucosaminidase, α-rhamnopyranosidase, α-galactosidase, α-fucosidase, α-arabinopyranosidase, α-mannosidase and α-xylosidase were included. α-Glucuronidase activity was assayed by the reducing sugar method using a mixture of aldouronic acids as substrate (Megazyme International Ltd). This substrate contained reduced aldtriouron, aldtetrauron and aldopenauronic acid in an approximate ratio of 40:40:20. Activity was measured at pH 5.0 with this mixture of 5 mg / ml stock. Reducing groups released during the 30 minute incubation period were detected by the DNS method. Since some enzyme samples contain significant β-xylosidase activity that was able to release xylose residues from alduronic acid, the assay was repeated, and xylose was included in the reaction mixture to inhibit β-xylosidase activity. It was.

Additional exogenous xylan-degrading enzymes such as: α-arabinoxylan arabinofuranohydrolase (release of arabinose from gale arabinoxylan was measured using an enzyme binding assay), acetylesterase (4-nitrophenyl and 4-methylumbelliferyl acetate substrate is used), acetyl xylan esterase activity (observation of acetate release from acetylated beechwood xylan), ferulic acid esterase (spectrophotometric and HPLC assay) is measured as well It was done.

Endohydrolase activity includes: β-D- (1,3; 1,4) -glucanase (β-glucan from barley (BBG) or lichenan as assay substrate), xyloglucanase (tamarindoxyloglucan), laminarinase (laminaria Laminaran from Diguita), endo-1,4-glucanase (called CMCase), based on activity against the commercial substrate carboxymethylcellulose, β-mannanase (Carob galactomannan) pectinase, and polygalacturonase, rhamno Galacturonase (soybean rhamnogalacturonan), galactanase (lupine and potato pectin galactan as substrates), arabinanase (sugar beet arabinan), amylase, glucoamylase,
And dextrinase.

Temperature / pH optimal conditions and stability; the optimal temperature for activity is the usual assay buffer (10
It was determined by performing the appropriate standard assay in 0 mM NaOAc, 5.0) with increasing temperature over the range of 30-100 ° C. Changes in pH with temperature were taken into account. The pH optimum was determined using the following buffers pH 2.2 to 7.6: McKilben type constant ionic strength citrate phosphate buffer; pH 7 to pH 10 Tris-HCl buffer. All buffers were adjusted to the same ionic strength for KCl regardless of pH.

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

Protein determination; protein concentration in enzyme samples (raw culture samples) was measured using BSA fraction V as a standard (Murray et al., 2001) Bensado of Lowry method.
estimated by un and Weinstein's modification (Bensadoun and W
einstein, 1976; Lowry et al. , 1951).

Electrophoresis and enzyme electrophoresis; to determine the profile of proteins present in the culture filtrate, the known volume of each sample is condensed by lyophilization and is native and / or reconstituted SDS-PAGE or isoelectric Point electrophoresis (IEF; Tuohy & Coughlan,
1992). The endoglycanase activity band in reconstituted SDS-PAGE gels and IEF gels is based on the gel overlay technology of MacKenzie and Williams (1
984) (Tuohy & Coughlan, 1992)
. To detect exoglycosidase activity, the gel was immediately incubated in a 50-100 μM solution of the appropriate 4-methylumbelliferyl glycoside derivative (2-
30 minute reaction period). The enzyme activity zone is Fluor-S ™ Multiimager (B
visualized with UV light using ioRad).

result:
The liquid culture of the strain was repeated at least three times in independent experiments performed at various periods. Culture filtrates (120 hours) taken from replication flasks (for each strain / carbon source combination) were assayed for enzyme activity; multiple replicates were assayed at a range of enzyme dilutions. Moreover, the results were demonstrated within and between assay measurements and with independent volumetric testing. There was a clear difference between cultures in terms of culture appearance and growth pattern. For example, IMI
The 393751 strain resulted in a rapidly very dense filamentous culture on glucose, while on the other hand several other strains (eg CBS 180.68, CBS 355.92, CBS 3
93.64, CBS 395.64, CBS 397.64, CBS 549.92 and CBS
759.71) showed limited proliferation and atypical morphology. That is, a limited culture population was formed that did not have normal filamentous growth and appeared mucous. Strain CBS 394.64 resulted in low mycelial biomass but grew as a filamentous culture while CBS 472
. 92 took the form of pellets under the same growth conditions. Previous studies have shown that extracellular exoglucosidase and endoglycanase activities are T. cerevisiae on most carbon sources. A 120 hour growth time point was chosen because it was shown to be present in significant amounts during the growth of the Emersony ("maximum and minimum" of key activity was observed in a pH uncontrolled state. However, maximum activity is generally detected towards the end of the fermentation cycle).

The use of glucose was only about 15-25% up to 120 hours for many strains.
Strain CSB 394.64 used ˜50-55% glucose in the medium, while CBS 472.92 (which showed pellet morphology) used ˜20-25%. In contrast, ˜95% glucose in the medium was used by the strain of the present invention (IMI 393751) for up to 72 hours, and no glucose was detected in the medium at the 120 hour collection point.

Tables 31A-D show the production of selected exoglycosidases by strains. As the results reveal, a clear distinction is made with respect to the production of exoglycosidase in the strains of the present invention and other T. coli. Seen among Emersony strains.

Glucose is T. The Emersony strain does not completely suppress exoglycosidase production (Table 31A). Strain 393751 produces significantly higher levels of β-glucosidase (BGase) than the other strains, and the second highest level of N-acetylglucosaminidase (NAGase) during growth on glucose. The production pattern obtained for strain 393751 is in sharp contrast to that of strain CBS549.92 (formerly CBS814.70). The production of some exoglycosidase activity by the latter strain appears to be inhibited by glucose. It should be noted that when residual glucose in the medium inhibits BGase and / or NAGase present, exoglycosidase activity levels were not dialyzed and were measured in dialyzed culture filtrates (dialysis A similar pattern was noted in the prepared samples).

Carob bean induced differential production of extracellular glycosidase by strain (Table 31B)
. Strain CBS 394.64 produces relatively little exoglycosidase activity apart from α-arabinofuranosidase. Low levels of all exoglycosidases are found in strain CBS39.
3.64, CBS 395.64 and CBS 549.92. 39375
One strain produced a significant level of a wide range of exoglycosidases (the highest level of some activity, such as pectin modified exoglycosidase β-fucosidase).

T. T. et al. Endoglycanase production by an Emersonii strain Xylanase production: Two of the major types of endohydrolase activity required for conversion of waste residues rich in plant biomass and non-starch polysaccharides are glucanase and xylanase. Of all the macromolecular glycan degradation activities assayed, glucanase and xylanase were the predominant glycanase activities present.

Tables 32A-D show values for xylanase production by all strains on the same carbon source. Previous studies have shown that wild type (CBS 814.70) and other mutant strains produce a complex xylan degrading enzyme system with multiple endoxylanases (Tuohy).
et al. 1993; 1994). Several isolated xylanases are available for various types of xylan, such as arabinoxylan, arabinoglucuronoxylan, glucuronon xylan, (from previous and ongoing results for enzymes from the strains of the present invention). It exhibits selective specificity for more substituted xylan relative to substituted xylan. As the results show, glucose is an essential component of all T. coli. It is a strong suppressor of xylanase expression in the Emersonii strain. A significant level of xylanase activity that is active against autosperzarabinoxylan (OSX) is expressed by strain 393751. This component is not active against rye or wheat arabinoxylan.

Carob that is rich in galactomannan (including some xylan) is 393751
It is a strong inducer of very high levels of xylanase activity against all xylan substrates by the strain. The role of carob bean as an inducer of strong xylanase activity is unexpected based on knowledge of its composition. The appearance of the cultures obtained in a large number of CBS strains (after 120 hours) is markedly different and a clear morphological difference is observed between 393751 and CBS549.92 strains, ie dense mycelium with respect to IMI393751 A limited (non-filamentous) culture population was observed that appeared (filamentous) growth and mucous with respect to CBS 549.92. As shown in Table 31B, xylanase levels are significantly higher than any other for strain 393751. In contrast to the 393751 strain, locust bean is CBS 394.64, CB
It is a very weak inducer of xylanase in S395.64 and CBS549.92. Another striking difference is seen in the type of xylanase activity induced by carob. In strain 393751 a strong activity is generated against all xylans. In other strains, very little activity is generally produced against rye and wheat arabinoxylan. Only two strains other than 393751 produce significant levels of activity against both of these xylans, namely CBS 472.92 and CBS 759.71. Enzymatic electrophoretic image analysis of the 393751 strain culture filtrate revealed a high level of multiple xylanase activity bands including the isolated novel bifunctional xylanase.

The tea leaf / paper dish (TL / PPL) mixture also proved to be a strong inducer of xylanase activity in strain 393751 (Table 32C), and this mixture induced xylanase expression in other strains. However, the level was very low. As observed for carob,
Strong activity was generated by strain 393751 for all xylans, nearly 1.8 times higher activity was obtained against lime gear rabinoxylan (RyeAX), and activity against OSX and birch xylan was low. This suggested differential expression of individual xylanases by TL / PPL and carob inducers, which was subsequently supported by enzyme electrophoresis image analysis. TL / PPL is a multi-component xylan-degrading enzyme system and 39
In 3751 strain, it also induces complementary esterase and oxidase / peroxidase activity as in other strains. These complementary activities enhance the effectiveness of polysaccharide hydrolases in enzyme cocktails optimized for key biomass degradation applications (eg cereals, plant waste, wood residues, paper products). Compared to carob, TL / PPL induces higher xylanase production in all strains (especially activity against arabinoxylan), but the level is 393751
Much lower than stocks. OSX is a known inducer of xylanase in fungi and induced enzyme production by all strains, with the most prominent induction being by strain 393751. However, in contrast to the 393751 strain, only OSX induced high xylanase activity and TL / PPL was a low inducer of xylanase production by the 472.92 strain. OS
The pattern of enzyme production on X is different from that obtained for carob and TL / PPL. Overall, the results of xylanase production on OSX suggest that strain 393751 can metabolize the raw substrate very quickly and effectively to generate a soluble inducer of xylanase. Hemicellulose in the more complex raw matrix is more accessible to this strain. The results also suggest that the enzyme cocktail produced by strain 393751 on such a complex substrate is more suitable for hydrolysis of complex raw plant material and residues. Model studies and applications investigated to date (eg woody biomass conversion, carbohydrate-rich food and vegetable waste and OFMSW and cereal saccharification) confirmed the ability of these cocktails.

Finally, FIG. 5 shows the 393751 strain and the parent strain CBS 549.9 on all four carbon sources.
2 (also CBS 814.70) various assay substrates (eg OSX, Rye)
The production of xylanase active against AX etc. is compared and contrasted.

B. Glucanase and mannanase production: According to previous studies, wild type (CBS814.7
0) and other mutant strains have been shown to produce complex glucan-degrading enzyme systems containing cellulases and a series of non-cellulolytic β-glucan modifying activities (Murray et a
l, 2001, 2004; Tuohy et al, 2002; McCarthy et
al, 2003, 2005). As noted for the xylan degradation system, multiple endoglucanases are produced depending on the carbon source. Some of the isolated β-glucanases exhibit selective specificity for various types of β-glucan.

Tables 7A-D show modified commercial β-1,4-glucan CMC (Sigma Aldr
ich), β-1,3; 1,4-glucan (BBG; Megazyme) from barley
, And lichen Setraria islandica (
Rikennan; Sigma Aldrich), xyloglucan from tamarind (β-1,
Activity against galactomannan (Megazyme) from 4-glucan backbone (Megazyme) and locust bean. Non-cellulose β-glucan is present in significant concentrations in the non-starch polysaccharide component of many plant residues, particularly those derived from cereals. Table 7A
~ D shows differential induction of each activity in the strain, and the induction pattern is completely different on more complex (raw) carbon sources.

Glucose is a potent inhibitor of glucanase and mannanase production in almost all strains (Table 33A). For all samples, activity was measured against dialysis and non-dialysis samples. As the results reveal, locust bean is the highest level of all strains tested, high levels of β-1,3 (relative to BBG and lichenan) in strain 393751; 1
, 4-glucanase is a potent inducer (Table 33B). The level of β-1,4-glucanase produced by strain 393751 (to CMC) was 10 times lower than the activity against BBG (the level of CMCase was higher for this strain when compared to other strains) ) Even if low xyloglucanase was detected, the level obtained for strain 393751 was the highest. Enzyme electrophoretic image analysis was carried out by analyzing the type of endoglucanase component and the T. glucanase component. It was confirmed that the expression pattern and the relative expression level in the Emersony culture filtrate were markedly different. Low levels of (galacto) mannanase were produced by all strains during the growth of carob.

The TL / PPL mixture was obtained as β-1,3,1,4-glucanase (B
It was a more potent inducer of (both BGase and lichenanase) and (galacto) mannanase (Table 33C). Overall, these results are consistent with T.W. Β- by Emersony strain
Emphasizing that glucanase production is not equivalent and confirming that strain 393751 is an excellent source of various β-glucanase activities and that the TL / PPL mixture induces a strong cocktail of these activities.

OSX (Table 33D) elicited much lower levels of β-glucanase than carob or TL / PPL, as expected. Enzyme production patterns are 393751 and CBS
Different for 549.92 strain. In Table 33D, β-1,4-glucanase levels (
Carboxymethyl cellulase activity) (with respect to OSX as inducer) 393751
Lower than most strains, except for CBS 397.64, which was produced at a level twice as high as the stock,
And four strains (ie CBS 393.64, CBS 394.64, CBS 395.6).
4 and CBS 549.92) were not detected in the culture filtrate. Significant (galacto) mannanase activity was generated during the growth of OSX by strain 393751 (lower than by TL / PPL as an inducer) and CBS 472.92. 6 and 7 show 39375.
Glucanase and mannanase production at the experimental conditions outlined by strain 1 and strain CBS 814.70 are compared and controlled.

In conclusion, the results show that:
Strain 393751 is a high level powerful product with a range of very important enzyme activities,
Strain 397551 has very high levels of xylanase and β-1,3 on low cost inducers (carob and TL / PPL) for two key depolymerizing hemicellulase activities.
The only T. coli that produces both 1,4-glucanases; It is an Emersony strain and the highest activity level is obtained on a raw carbon source, thus demonstrating that strain 393751 is a cost effective source of a powerful series of enzyme cocktails.

Example 14 Thermozyme from Emersonii IMI393751 The purpose is to reduce biodegradable components of sterilized cellulose-rich clinical waste, thus reducing the amount of landfill waste and recovering sugar-rich liquid effluent after enzymatic treatment And recovering energy in the form of biofuel.

The waste stream was a bandage and cloth, cotton wool, etc., containing a high percentage of cellulose (> 50%) and mainly rich in paper, tissue, medical sanitized cotton and cotton. The main “fibers” in the waste stream
Although cellulose is a cellulose, many products are “finished” by polysaccharide coatings, binders and fillers, so a mixture of accessory enzymes (ie, hemicellulase, pectinase and amylolytic enzyme) is cellulose. There is a need to enhance accessibility and improve waste reduction or conversion to simple soluble sugars (eg glucose, galactose, xylose, etc.).

Experimental approach:
The profile of endohydrolase and exoglycosidase enzyme activity in each of 10 thermozyme cocktails derived from strain 393751 was determined (Tuohy).
& Coughlan 1992; Tuohy et al, 1993, 1994, 20
02; Murray et al, 2001, 2004). Table 34 summarizes the relative levels of key activity determined in cocktail selection. Enzyme preparations were made at 10 different concentrations.
To a 0 g batch of STG treated cellulose rich waste was added at 50 ° C. and 70 ° C. and incubated for 24-48 hours (at a moisture level of 50-60%). Samples of sugar rich solutions (and cellulose rich materials such as tissues) are periodically removed over a 48 hour period and Tuohy et al, 1993, 1994, 20
02; Murray et al, 2001, 2004, Gilleran (2004)
and Braet (2005) as described in (i) weight and volume reduction, (ii)
A) the volume of the sugar-rich solution recovered, (iii) the released reducing sugar, (iv) the physical structure of the substrate after enzyme treatment (using scanning electron microscopy), (v) released by TLC (Vi) Quantitative analysis of sugars produced by HPLC, GC-MS and ESI-Q-TOF-MS, (vii) Substances potentially harmful to fermenting microorganisms (bioenergy production), ( viii) hydrolyzate sterility, (ix) bioethanol production, and (x)
Analyzed for biogas production.

result:
A. The weight and volume reduction, the volume of recovered sugar-rich solution, the weight before and after the released reducing sugar enzyme treatment were recorded, and the volume reduction estimates recorded for all enzyme preparations were made. Volume reduction data and reducing sugars obtained at 50 ° C. using the same reaction parameters (enzyme loading, 60% water content, and 24 hours reaction time) are shown in FIG.

In summary, 60 ° C. for 24 hours at 70 ° C. gave the best results in terms of volume reduction for all cocktails. In repeated studies with 6 selected cocktails (and by replicate testing), the volume reduction was always between ˜60-75% depending on the cocktail (FIG. 9).
reference). The reducing sugar released was converted to a conversion of cellulose present, or% hydrolysis, which is 66-82% of the cellulose rich fraction present in the initial sterilized waste.
It was between. A reduction of 60-75% by volume was obtained on laboratory inspection.

Per 100g batch water added about 70~100mL _(H 2 O) is the final moisture content was added to 50 to 60%. Liquid recovery after enzymatic hydrolysis is 69-105m
L. The experiment was also carried out at a reaction temperature of 75-85 ° C. and different pH values. 70 ° C
Above, a near-limit increase in overall hydrolysis (~ 2 to 5.5%) is noted, 70 ° C
When compared to the values obtained with and at pH 3.5-4.0 resulted in optimal levels of hydrolysis, the values were not significantly greater than those obtained using H ₂ O ( <5%).

B. Physical loss of substrate integrity, generated sugar products and relative amounts of each type of sugar SEM is
A significant loss of cellulose fiber structure was revealed (FIG. 10B).

Hydrolysis products: Qualitative analysis by TLC; Quantitative analysis by HPLC> 76-92% of the released sugars are monosaccharides in the seven best cocktails, and this is mainly from glucose, with some There were galactose, mannose and xylose. For example, sugar levels ranging from 0.2 to 0.55 g / mL, and monosaccharide concentration ranges determined by HPLC are as follows (depending on thermozyme cocktail and waste batch):
Glucose: 43-70%; Mannose: 5-15%; Galactose 4-10%; Xylose: 20-30%; Cellobiose: 4-12%, and High oligosaccharide: 5-26%.

C. Screening for potentially harmful substances to fermenting microorganisms before and after fermentation (bioenergy production), sterility analysis of hydrolysates, and bioethanol and biogas production. The recovered liquid fraction is sugar to bioethanol. Did not appear to be harmful to yeast species screened for fermentation of abundant hydrolysates, ie, S. cerevisiae. Celebrige (baker's yeast), Pachysolen tanophilus
lus), Pichia species, Candida shehatae, and Kluberomyces marxianus
) Did not interfere with growth. Moreover, analysis of the ethanol product (using an enzyme binding assay kit) showed that the yeast was producing ethanol.

Agar plates (including appropriate agar media) are inoculated with a sugar-rich solution and a sample of residual waste, incubated under recommended conditions (for microorganisms), and colonies (bacteria and yeast)
Were analyzed for the presence and radial growth (filamentous fungi). Microbial growth did not occur in dishes inoculated with sugar-rich solutions from 70 ° C. enzyme treatment and waste samples, ie no microbial damage (and sugar loss) of waste hydrolysates occurred. .

Bioethanol production Bioethanol production from sugar-rich raw materials by various yeast species such as Saccharomyces cerevisiae, Paxorene tannofilus, Pichia species, Candida shechate and Kluberomyces marxianus (and strains) was evaluated. The endpoints measured included the use of sugar yeast growth (and yeast biomass), CO ₂ generation, and ethanol produced. Two 70 ° C. enzyme digests (hydrolysates generated by cocktails 5 and 8 in FIG. 8) were selected as test raw materials for bioethanol production of all yeast species in 1 L laboratory scale cultures.

11A and B show the S.I. The ethanol production profile obtained by Celebrige is shown. Although Thermozyme Cocktail 8 resulted in slightly more monosaccharide in the hydrolyzate,
The ethanol yield is similar for both raw materials. However, the digest from cocktail 8 contains a higher pentose (not fermented by S. cerevisiae) than that generated by thermozyme cocktail 5. The Samzyme Cocktail 5 digest contains some cellobiose (and a small amount of cellobiooligosaccharide) that is easily metabolized by yeast.

Analysis of potential harmful fermentation end products Technical scope (HPLC, MS), in particular GC-MS, was used to determine the presence of potential harmful fermentation end products. The use of almost perfect sugar (except for digests rich in pentose sugars (xylose, arabinose)) Achievable for cerevisiae and normal end-of-fermentation products were detected: glycerol, acetate and traces of by-products (solvents).

Biogas production Large batches of cocktails 5 and 8 digests were prepared for feeding to a mesophilic and thermophilic upflow anaerobic hybrid reactor (UAHR) anaerobic digester. The total carbohydrate levels in the influent and effluent of both reactors were measured (Dubois et al. 19
56; Laboratory protocol). Reducing sugars present in the influent and effluent samples were determined (Tuoh et al, 1994). Chemical oxygen demand (CO
To determine D) a known amount of hydrolyzate was oxidized over 2 hours using potassium dichromate (concentrated sulfuric acid with silver sulfate as catalyst) (international test method; laboratory procedure) ). The remaining dichromate was determined by titration with a standardized solution of ammonium ferrous sulfate. COD and carbohydrate removal efficiency was measured during the test (daily sample).
The specific methanogenic activity (SMA) of sludge was analyzed using pressure conversion technology (Col
leran and Pistilli, 1994; Coates et al. 199
6). Sludge samples were removed from the sludge bed through the outlet and the test was performed at 37 ° C (for mesophilic reactor sludge) or 55 ° C (for thermophilic reactor sludge).

Sugars present in enzymatically generated digests are mesophilic (37 ° C) and high temperature (55 ° C) U
Both AHRs were rapidly metabolized by bacteria, ie 95-97% carbohydrate reduction at non-optimized conditions at a loading rate of 4.5 g COD / m ³ / day. The resulting methane level in the biogas stream is between 55-61% and the estimated retention time (days) required to metabolize all sugars and reach the maximum methane level is ˜3. 0 to 4.0 days. The pH of the effluent was observed and there was no noticeable change in the pH of the effluent from either reactor.

C. Optimization of thermozyme systems for treatment of cellulose-rich clinical waste streams In order to obtain enzyme cocktails where the combination of genomics and functional proteomics has optimal levels of all key enzyme components (in initial experiments) Used to identify optimal growth conditions and substrates to use (based on information from the 10 cocktails used). A combination of two inducers was selected: 1 of used tea leaves and waste paper texture
1 mixture and a 1: 1 mixture of beet pulp without sorghum and molasses. Additional blends selected from the 10 cocktails used above were also prepared. The novel cocktails and blends are characterized with respect to the component enzymes and their ability to catalyze the extensive conversion of commercial cellulose, hemicellulose, and sterilized cellulose-rich wastes into simple fermentable sugars. It was done. Optimal pH and temperature for maximum enzyme reactivity, optimized enzyme system and thermal stability of blended cocktail, and reaction end product,
Potential inhibition by monosaccharides or potentially harmful molecules (phenolic compounds / benzene derivatives)
It has been determined.

Temperature optimum: 75-80 ° C.,> 70-85% activity, depending on enzyme preparation / blend,
It remains at 85 ° C. (enzyme activity was also detected at 90-95 ° C.).

Thermal stability: no real loss of activity after 24 hours at 50 ° C, <5-1 after 1 week at the same temperature
There was 0% loss of activity.

At 70 ° C, there was <2-20% loss of activity in the first 24 hours, and <10% further loss of activity over the next 5 days.

pH optimum: the enzyme was most active between pH 4 and 5 but> 60% activity was pH 3
. Observed at 0 and pH 6.8, all enzymes were also active at pH 7.0. The enzyme preparation was most stable between pH 3.5 and 6.0 (4-50 ° C. over a period of 1 week).

When high concentrations of glucose and other monosaccharides are present, the reaction / substrate conversion rate to monosaccharide is
Although reduced or reached level, the enzyme preparation was still very active in the presence of high concentrations of monosaccharides (up to 100 mM) (glucose, xylose, arabinose, galactose). Moreover, although the phenolic compound / benzene derivative reduced the overall activity of the cocktail, the concentrations used in the test were significantly higher than those that might be present in the waste. Nevertheless, certain cocktails and optimized enzyme cocktails
Significant activity (> 50-70%) was shown in the presence of these compounds.

Overall, the concentration of each enzyme required to achieve a hydrolysis value of ˜65-75% is surprisingly low (9-16 cellulase units / 10 g waste) and depends on the cocktail / blend However, high doses (up to 60 cellulase units) did not significantly increase the final degree of hydrolysis. Enlargement of the enzyme treatment to 100 and 10 Kg batches resulted in similar final degrees of hydrolysis, as well as similar profiles and sugar product concentrations. The best volume reduction values obtained for the optimized cocktail were 73-80%, while the corresponding hydrolysis% values for conversion of the cellulose fraction to monosaccharides were 72-81% . The two optimized blends resulted in similar endpoints in terms of volume reduction and cellulose hydrolysis. The ethanol yield obtained was S. cerevisiae. 195-210 L / ton for Celebije and P.I. It was 215 to 220 L / ton for Tannofils. Approximately 80-8
5% bioethanol is recovered by distillation, but this can be improved.

Example 15: T. to produce a sugar-rich raw material Thermozyme from paper and tissue waste rich in cellulose from Emersonii IMI393751
Enzyme activity measurement:
The raw enzyme preparation is a relevant substrate at a concentration of 10-30 mg / mL for the internal acting enzyme, 50 mg filter paper / reaction volume 1 mL for “filter paperase” (general cellulase), or 1 mM of the appropriate 4-nitrophenyl-glycoside derivative was analyzed for a range of different lignocellulose hydrolase activities (Tuohy et al., 2002; Murray et al., 2001).
). The assay was performed in triplicate. All results show two identical experiments using different raw enzyme preparations.

PH and temperature requirements for activity and stability:
The pH and temperature requirements for enzyme activity and stability are determined using normal assay procedures.
It was evaluated over a range of 2.6-7.6 and over a temperature range of 30-90 ° C.

Model-scale hydrolysis studies:
Aliquots of individual enzymes containing 5-60 FPU (or 2-20 xylanase units if necessary) were incubated with 1 g target substrate for up to 24 hours at the appropriate pH and reaction temperature. Samples were removed at timed intervals and the sugar content (reducing sugar) and composition were analyzed.

Investigated cellulose-rich substrates: mixed tissue and toilet paper, office paper waste, wrapping paper, mixed newsprint.

Summary of results: Thermozyme cocktails are highly active against a broad spectrum of carbohydrate substrates and are therefore Reflects the complexity and efficiency of the enzyme product by Emersonii IMI393751. The production of a particular thermozyme reflects the composition and variation of the inducing substrate (
Table 35)

Cellulase in the thermozyme cocktail has a pH around 4.0 (FIG. 12A) and
It was most active between 80 ° C. (FIG. 13). Cellulase components in each enzyme preparation have a wide pH
Active over a range (<pH 2.6 to> pH 6.5). Xylanase activity present in the same cocktail is between pH 4.0 and 5.0 (FIG. 12B) and between 75 and 85 ° C. (FIG. 14).
Was the most active. However, xylanase activity in the cocktail was active over a wide pH range (<pH 2.6 to> pH 7.0), while similar activity levels were observed at 90 ° C. and 50 ° C.

Some thermal stability of the MGBG cocktail (listed in Table 35) is 50 ° C and 70 ° C.
Is reflected in the long half-life at 50 ° C. after incubation with buffer (pH 5.0) alone, and there is virtually no loss of endoxylanase or endocellulase activity at 50 ° C.
Or minimal. All cocktails were raw enzyme preparations and no stabilizers or enhancers were added. Xylanase activity present in cocktails 2, 5, 6 and 8 is 7
It was particularly stable at 0 ° C. (only <2-20% loss of xylanase activity after 25 hours).
For example, the t1 / 2 value for Cocktail 2 at 70 ° C. was> 6 days. The stability of cocktails 3 and 4, and less than that of cocktail 7 at 70 ° C. is high.
low) due to the presence of protease (2 hours, 12 hours and 25 hours t1 /
(Binary). In the presence of substrate (ie raw waste), the stability of all three cocktails is
Remarkably high, ie (t1 / 2 values of 22 hours, 46 hours and 72 hours). The general performance of the MGBG cocktail on cellulose and hemicellulose substrates was very high (Table 36). As the reaction temperature increased from 50 ° C. to 70 ° C., the level of hydrolysis increased significantly (Table 37). At high reaction temperatures (eg 70 ° C.), the% hydrolysis was achieved at 18 hours and was similar to or possibly higher than the% hydrolysis obtained after 24 hours at 50 ° C. (Table 37). ). This discovery highlights the benefits of thermozymes compared to enzymes from mesophilic organisms that would be active at low temperatures.

The product of time-dependent hydrolysis of cellulose and hemicellulose analyzed by TLC confirmed the high conversion efficiency of MGBG thermozyme. The polysaccharide substrate was initially broken down into oligosaccharides and eventually into glucose, which was almost the only hydrolysis product, while cellulose-rich waste, such as cellulose present in tissue paper, Similarly, it was almost completely hydrolyzed to glucose.

The implications of these results for the bioethanol-based industry are important,
And T. Shows the ability of the Emersony Thermozyme system.

Example 16: Bioconversion of beet pulp to sugar rich hydrolyzate for bioethanol production
Eight thermozyme cocktails were selected from the original range of 20 cocktails. 393
The profile of endohydrolase and exoglycosidase enzyme activity in each of the eight enzyme cocktails derived from strain 751 was determined (Tuohy & Coughla).
n, 1992, Tuohy et al. , 1993, 1994, 2002; Murra
y et al, 2001, 2004).

The enzyme preparation was combined with 1 g batch of sugar beet fractions prepared using different extraction methods at various concentrations or doses. The fractions are as follows:
A. Sugar beet top and stem (dry weight 1g / total volume 10ml)
B. Sugar beet pulp (dry weight 1g / total volume 10ml)
C. Sugar beet fruit (dry weight 1g)
D. Sugar beet skin (dry weight 1g)

Sugar beet fractions A to D were homogenized in a Waring blender (2 × 30 second burst). Enzyme aliquots, ie 2 ml and 5 ml enzyme solutions (1-8), (tap water,
Added to a final total reaction volume of 10 ml according to pH 7.2) and initially incubated at 63 ° C. Further experiments were performed to optimize the reaction temperature (75 ° C.), pH (pH 4.0) and reduce the incubation time (16 hours). Sample is 16
Taken at timed intervals and centrifuged over a ~ 48 hour incubation period,
And the enzyme activity was terminated by boiling at 100 ° C. for 10 minutes. The supernatant fraction was analyzed for released reducing sugars.

A qualitative analysis of the type of sugar released was analyzed by TLC, and a quantitative analysis of the sugar produced was determined by HPLC. Sugar-rich raw materials were evaluated for bioethanol production (Tuohy et al, 1993, 1994, 2002; Murra).
y et al, 2001, 2004; Gilleran, 2004; Braet, 20
05).

result:
The optimal reaction conditions were 75 ° C. and pH 4.0. Table 38 shows the reducing sugars released after 16 hours under optimal conditions. Increasing the incubation to 48 hours increased the percent hydrolysis in the case of skin and fruit fractions. Optimization of enzyme loading conditions allowed the incubation time to be reduced by half (ie 24 hours) with the yields shown in Table 39.

The development and optimization of enzyme cocktails for performing hydrolysis (8 used in the original experiment)
This was done using a combination of genomics and functional proteomics (based on information from two cocktails). Two cocktails are produced, MGBG SB # 1 and MGBG SB
Labeled # 2. Hydrolysis of all sugar beet plants and all fruit components by two optimized cocktails was compared to cocktails 3 and 5 from previous experiments. The reaction was performed at the optimal conditions for the two new cocktails, namely 71 ° C. and pH 4.5. Table 40
Gives the relative level of enzyme contained in each reaction per gram of substrate (ie whole sugar beet plant or whole fruit component). Tables 41 and 42 summarize the hydrolysis results obtained.

The main difference between the optimized cocktail, and cocktails 3 and 5, is in the relative amount of key activity, especially exoglucosidase.

Both novel cocktails released high levels of reducing sugar from the whole plant. Of the reducing sugar released, approximately 88-90% were monosaccharides, of which about 43-67% were glucose.

Fermentation to bioethanol A study was conducted to investigate the production of bioethanol from whole plants and sugar beet hydrolysates. More than 90% of the glucose present in the hydrolyzate of the whole plant is S. cerevisiae.
Converted to bioethanol by Celebrige (from 40 g / L fermentable sugar,
About 9.0 g / L of ethanol was obtained). Other yeast species, such as Paxolene tannophyllus, were investigated for bioethanol production (anaerobic conditions). The latter yeast
Glucose and pentose sugars (xylose and arabinose) released in repeated fermentations (> 92-95% metabolism) were used. However, the ethanol yield is S
. Slightly lower than with Celebrige (about 6.7 g from 40 g / L fermentable sugar)
/ L ethanol was obtained.

Selected biochemical properties of the new cocktail Temperature optimum: 75-80 ° C, depending on the cocktail,> 75-87% activity remains at 85 ° C.

Thermal stability: There was no real loss of activity after 24 hours at 50 ° C. and <10% loss after 3 weeks at 50 ° C. At 71 ° C., ˜4-9% loss of activity in the first 24 hours, 5 over 5 days
Further loss of ~ 7%.

pH optimum and stability: Both cocktails were most active at pH 4.5, but> 5
5% activity is observed at pH <2.6 and> 50-60% at pH 6.8; both enzymes show significant (> 48-58%) activity at pH 7.0. It was. The enzyme preparation is pH 3.
Most stable between 5 and 6.0 (4-50 ° C over 1 month).

Example 17: Identification and selected properties of a novel bifunctional xylanase produced by Talalomyces emersonii IMI393751 Talaromyces emersonii secretes 14-20 distinct endoxylanase components when grown on a suitable carbon source To do. Thirteen of these endoxylanases were purified to homogeneity and characterized with respect to catalytic properties. The molecular weight of the purified endoxylanase varies between 30 and 130 kDa. Xylanase and glucanase expression is expressed in 10 T. cerevisiae grown in the same conditions on the same nutrient medium and carbon inducer. Not equal among Emersony strains. A new low molecular weight xylanase has been identified and the xylan-degrading system T.W. Xyn XII (17.5 kDa) from Emersonii IMI393751 strain. Secretion of xylanase having the following M _r values 20kDa, in a number of other bacterial and fungal species has been reported, T. No report has been made about Emersony before this.

Enzyme purification and characterization Emersonii IMI 393751 is a 1: 1 ratio of wheat bran and beet pulp at 120 hours, 45 ° C., 210 rev / min (or 11 days in solid (static) fermentation, 33% substrate, 67% moisture, 45 ° C.) On the mixture. The xylanase and protein content of the raw and divided enzyme samples were analyzed as previously described (Tuohy
& Coughlan, 1992; Tuohy et al, 1993, 1994; M
urray et al, 2001). The raw enzyme extract was harvested as previously described (Tuohy & Coughlan, 1992). Ultrafiltration using an Amicon DC2 system equipped with a H1P 10-43 hollow fiber dialyzer was used to separate the novel xylanase (permeate fraction) from the high molecular weight xylanase (concentrated water). X
yn XII is precipitated by (NH ₄ ) ₂ SO ₄ (0-90% block) or “salting out”, Se
Gel permeation chromatography (GPC) on phacry S-200 SF (100 mM NaOAc buffer, pH 5.0 as eluent), ion exchange chromatography (IEC) on Whatman DE-52 (30 mM NaOAc buffer, pH 5.0) The xylanase was purified to homogeneity using a combination of fractionation techniques comprising a linear buffered 0.0-0.3M NaCl gradient) followed by Phenyl. Hydrophobic interaction chromatography (HIC) on Sepharose CL-4B followed by equilibration with 15% (NH ₄ ) ₂ SO _{4 in} 30 mM NaOc buffer, pH 5.0. Prior to HIC, the xylanase sample was 15% (w / v
) To a final concentration of (NH ₄ ) ₂ SO ₄ . Buffer salts and (NH ₄ ) ₂ SO ₄
Was removed by application of the sample to Sephadex G-25 (not shown). Finally, the xylanase-rich fraction was applied at pH 7.0 to DE-52 on a second anion exchange column, followed by gel permeation chromatography on Sephacryl S-100 HR (used as equilibration and irrigation buffer). 100 mM NaOAc buffer, pH 5.
0), and DEAE-Se pre-equilibrated with 50 mM NH ₄ OAc buffer, pH 5.5
Final fractionation step with pharose (0.0 used to elute xylanase
Further fractionation by ~ 0.2M NaCl gradient). The stored enzyme is Sephad
Desalted by application to ex G-25 or BioGel P-6 and lyophilized prior to electrophoretic analysis.

Selected characterization of the new bifunctional xylanase Selected physicochemical properties The purity of the new enzyme was confirmed by SDS-polyacrylamide gel electrophoresis in a 15% (acrylamide / bisacrylamide) gel according to the Laemmli method. S
DS-PAGE revealed a single protein band on the silver staining which corresponds to the estimated _{M r} of 17.5kDa. In addition, a single protein band corresponding to the pH 5.0 pI value of Xyn XII was obtained on IEF. The optimum temperature for the degradation of OSX with the Xyn XII catalyst was determined to be 75 ° C., and the optimum pH of activity was pH 4.0-4.5. However, unlike Xyn I-XI, which lost between 25 and 81% of each original activity during a 10 minute incubation period at pH 3.0, Xyn XII is remarkably stable to acids, And even with prolonged incubation, it retained its original activity of over 91% at pH 3.0.

Selected catalytic properties Appropriately diluted aliquots of Xyn XII (all at 1.0% (w / v) concentration)
Incubated with a series of polysaccharides including various xylans, β-glucans, pectin polymers and fructans. The reducing sugar released during the extended 30 minute incubation period was quantified as described above. Activity against aryl glycosides (1.0 mM) was determined using a microassay (Murray et al., 2001).
Preliminary studies to determine rate constants have been performed with [substrate] xylan, 0.2
This was done by varying between -25 mg / ml.

The results shown in FIG. 15 show that the novel xylanase (Xyn XII) for various xylans
The relative reactivity of is shown. This enzyme is very active on mixed-bond unsubstituted xylan (1,3; 1,4-β-D-xylan) known as rhodimenane from the red alga Dulse. In contrast, of the two cereal arabinoxylans, OSX and WSX, the enzyme showed the greatest activity against the more substituted WSX. The overall pattern of reactivity is: R
M>WSX>LWX>OSX> BWX. As FIGS. 16-19 show, the substrate preferences of many other xylanases (Xyn IV to Xyn XI) are very different from Xyn XII.

For Xyn IV, OSX>RM>WSX> LWX >>>> BWX (FIG. 16A).
). The order of reactivity of Xyn VI is OSX>LWX> RM ≧ BWX> WSX (FIG. 1).
6B), on the other hand, indicated by Xyn VII: RM>LWX> WSX ≧ BWX
>> OSX (FIG. 17C). The reactivity of Xyn VIII is: RM>BWX>WSX>
>LWX> OSX (FIG. 17D) and Xyn IX is RM>LWX> BWX ≧ 0
SX> WSX (FIG. 18E). Finally, Xyn X is the next priority trend RM >> BW
X to WSX >> OSX >>>> LWX (FIG. 18F), and Xyn XI is RM ≧ LW
X>WSX> BWX ≧ OSX was shown (FIG. 19). Furthermore, unlike Xyn I-XI, which was strictly active against xylan only, a new bifunctional xylanase (Xyn XII)
Is substantially active against mixed-bound β-glucan (1,3; 1,4-β-D-glucan) from barley, ie 55% relative to that observed for normal assay substrate OSX The activity was higher than that (FIG. 20). In contrast to Xyn I-XI, the new bifunctional xylase is aryl-β-xyloside 4-nitrophenyl β-D-xyloside (4NP
X) and chloronitrophenyl β-D-xyloside (CNPX) showed activity, with the latter substrate being more active (FIG. 21). This discovery may reflect the electron withdrawing effect of the chloro moiety, making the chloronitrophenyl group a better “leaving group”. However, T.W. Other novel endoxylanases from the CBS 814.70 strain of Emersonii (Tuohy et al., 1993; differentially expressed, but strain IMI39
Is also significantly active against CNPX and 4NPX
Show little activity against. This phenomenon reflects the partial “external” working properties of these enzymes, and may also reflect similar properties of Xyn XII. Moreover, low activity was observed for 4NPβ-glucoside and CNP-β-cellobioside, which is not surprising. Because Xyn XII is active against 1,3; 1,4-β-D-glucan and has some external action properties as observed for aryl β-xyloside This is because it can be expected to have a corresponding activity on aryl β-glucosides. No reactivity was observed for any other α or β linked aryl glycosides.

Thus, this novel bifunctional xylanase “in vivo” provides access to plant cell wall hemicellulose, for example by breaking down mixed-bound glucans in cereals and other plants, plant residues and wastes. T. EMERSONY IMI393751
Can play a very important role.

Example 18: T.M. Expression of key hydrolases and other accessory enzymes by the Emersony strain.
Expression of key enzymes on the same carbon source is different. The number, type and relative abundance of xylanases produced by Emersonii IMI 393751 and other strains are different. As indicated above, enzyme levels in the culture filtrate are markedly different and It suggests that Emersony strains produce different levels of the same xylanase or express different isoforms. To illustrate this point, Gel S
DS-PAGE electrophoresis and enzyme electrophoresis staining with IEF (Tuohy & Coug)
restored as described by hlan (1992)).
And CBS549.92 strain culture filtrate. These studies confirmed that (i) different xylanases were expressed and (ii) the number of expressed xylanase isoforms was significantly different.

The results obtained for xylanase expression on carob bean are summarized as follows:
Xylanase isoforms were detected (Tuohy et the reference isoforms -pI and _{M r}
al. , (1994))
IMI 393751: Xyn IV, Xyn V, Xyn VI, Xyn IX and Xy
n XI, Xyl I (β-xylosidase) and Xyl II
CBS 549.92: Xyn II, Xyn VII (Note: Xyn VII is equivalent to the sequence published in the prior patent (GenBank Accession Number
AX403831), and very low amounts of Xyl I (β-xylosidase).
Moreover, IMI393751 is the only strain that produces Xyn XII during growth on tea leaves / paper plates (and only on tea leaves).

Expression patterns on different carbon sources are different Xylanase expression patterns on different carbon sources are not equal, ie IMI393751:
Glucose as carbon source: Xyn I, Xyn VIII, a small amount of Xyn XI, and β-xylosidase, no carob: 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 XI
I, Xyl I and Xyl II
CBS 549.92:
Glucose: a low level component that may be equal to Xyn IV, and β-xylosidase is none
TL / PPL: proteins similar to Xyn I, Xyn VII and Xyn IX, some Xyl I

Moreover, other clear examples of expression differences, such as Xyl I expression, were observed. Xyl I
During growth on glucose IMI 393751, CBS 549.92, CBS 180
. 68, CBS393.64, CBS394.64, or CBS397.64. In contrast, under the same conditions, Xyl I is CBS355.92, CBS395.
. 64, CBS 472.92 and CBS 759.71.

Similarly for locust bean, Xyl I is expressed by all strains at significantly different levels except CBS 394.64. Xyl I was also expressed by all strains except for CBS 180.68 and CBS 394.64 during growth on TL / PPL. On OSX as the carbon source, Xyl I is CBS 180.68 and CBS 394.6.
Was not expressed by 4, but was expressed by CBS393.64 (low level) and all other strains. Overall, striking differences in expression levels and expression patterns of Xyl I were noted among all induced substrates (ie strains expressing the highest or lowest amount of Xyl I).

Differences in specific activity of homologues produced by different strains Various strains were compared for the expression of key xylanases as described above. However, the xylanase present in the induced cultures of IMI393751 and CBS549.92 was fractionated and the specific activity of the purified xylanase component was compared. First
In this case, only IMI393751 appears to generate Xyn XII. Furthermore,
When specific activities are compared (OSX results as assay substrate are shown below), slight differences in the specific activities of the individual enzymes are clearly observed (FIG. 22).

Growing T. coli on various carbon sources. Generation of new components by Emersonii IMI 393951 Emersonii IMI393751 is cultured on a series of carbon sources and SDS-P
The profile is generated by restoring AGE and IEF, followed by the enzymatic electrophoresis staining outlined above. The following are some examples of the results obtained.
(I) Tea leaves as inducers: Xyn III, Xyn V, Xyn IX, slightly Xyn
X and Xyn XII; Xyl I and Xyl II
(Ii) Carob: Xyn IV, Xyn V, Xyn VI, Xyn IX and Xy
n XI, Xyl I (β-xylosidase) and Xyl II
(Iii) Rye flakes: Xyn IV, Xyn V, Xyn VI, Xyn IX
, By a particularly novel “xylanase” component of pI6.5; Xyl I (β-xylosidase) and Xyl II
(Iv) Retail flour: Xyn III, Xyn VI, Xyn VIII, Xyn
(V) Sorghum: Xyn I, Xyn VIII, Xyn IX, Xyn X, Xyn with additional novel “xylanase” components of IX, Xyn X, Xyl I (β-xylosidase) and Xyl II, pI5.8
XI, slightly Xyl I (β-xylosidase) and Xyl II
(Vi) Xylose: Xyn I and Xyn VIII, Xyl I
(Vii) Glucose: Xyn I, Xyn VIII, less Xyn XI, and no ββ-xylosidase

Example 19: T.W. Differential expression of other accessory enzymes by Emersonii strains Glutathione peroxidase Culture filtrate samples (non-concentrated and concentrated samples) were run on 7.5% native PAGE gels and soaked in reduced glutathione at 50 ° C. Followed by incubation with 0.002% H ₂ O ₂ . The gel was then stained with 1% ferric chloride / 1% potassium ferricyanide. Enzymatic electrophoretic staining was also supplemented by enzyme assay on the culture filtrate.

IMI393751 produces extracellular glutathione peroxidase (M _r -45 kDa) and differential expression is observed on a number of carbon inducers (numbers 1-18 represent different inducers). In contrast, other T.P. The extracellular activity for any of the Emersony strains is
It was not observed even in the concentrated sample. Strain IMI393751 also produces significant levels of extracellular glutathione peroxidase during the growth of TL / PPL.

Catalase culture filtrate samples (non-concentrated and concentrated samples) were run on a 7.5% native PAGE gel followed by incubation with 3% H ₂ O ₂ . The gel was then stained with 1% ferric chloride / 1% potassium ferricyanide (the catalase band appears as a deep yellow band). Enzymatic electrophoretic staining was also supplemented by enzymatic assays on the culture filtrate using standard published methods.

IMI393751 produces extracellular catalase (M _r -230 kDa) and differential expression is observed on a number of carbon inducers (numbers 1-18 represent different inducers).
In contrast, other T.P. No extracellular activity was observed in any of the concentrated samples for any of the Emersonii strains.

The presence of these enzyme activities in the culture filtrate of IMI393751 is potentially important in affecting the structure of the substrate, but eliminates any compounds that can oxidize key hydrolase activity, such as xylanase or glucanase. Is also potentially important to do.

Example 20: Ten T. cerevisiae on various solid (agar) media. Comparison of phenotypes of Emersony strains Agar medium
1. Sabouraud Glucose Agar (Oxoid Ltd., UK)
2. Potato maltose agar (Oxoid Ltd., UK)
3. Czapek Doc (Oxoid Ltd., UK; pH not adjusted)
4). Cornmeal Agar (Oxoid Ltd., UK)
5. Malt extract agar (Oxoid Ltd., UK)
6). Nutrient Agar (Difco Ltd., UK)
7). Emerson agar (yeast potassium soluble starch; YpSs)
The following ingredients were added to 1 L distilled H ₂ O:
15.0 g soluble starch (Sigma-Aldrich, Dublin, Irel
and)
4.0 g yeast extract (Oxoid Ltd., UK)
1.0 g potassium dihydrogen phosphate (KH ₂ PO ₄ )
0.5 g of magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O)
Agar No. 20.0 g 1 (Oxoid Ltd., UK)
8). Yeast glucose agar (YGA)
The following ingredients were added to 1 L distilled H ₂ O:
20.0 g of glucose (Sigma-Aldrich, Dublin, Irelan
d)
10.0 g yeast extract (Oxoid Ltd., UK)
Agar No. 15.0 g 1 (Oxoid Ltd., UK)

The agar plate (each type of agar) has 10 different T. agar plates. Emersony strain was inoculated. One batch of plates was incubated at 45 ° C and the second at 55 ° C (water content was ~
20-30%). The culture was confirmed daily and the following results were recorded:
(A) Measurement of culture diameter (using caliper)
(B) Photographic recording using a 7.0 M pixel digital camera (c) Record visible phenotypic changes / differences (d) Display of sporulation (microscopic analysis) or otherwise

result:
T. on various agar media. The culture of the Emersonii strain is 393751 with respect to:
Revealed clear differences between the strain and the other nine strains:
Tables 43 and 44: Summary of growth rate and range at 45 ° C.-Measurement of culture diameter performed on days 2 and 5 Table 45: Growth rate at 55 ° C.-culture performed on day 5 Object Diameter Measurements Table 46: Summary of evidence for visual phenotypic changes and sporulation.
Important observation 1: Many IMI / CBS strains resulted in multicolor cultures. However, the purity of these cultures was confirmed. A color / pigment is produced and the production pattern is consistent with many species of penicillium, e.g. It is peculiar to pinofilm.
Important observation 2: As shown in Table 20, there is a clear difference in sporulation between 393751 strain and others.

References
1. Raeder U. and Broda P. (1985), Lett. Appl. Microbiol. Vol. 1 pgs 17-20.
2. P. Chomozynski and N. Sacchi (1987),, Anal. Biochem Vol. 162, 156-159.
3. J. Sambrook, EF Fritsch and T. Maniatis (1989), "Cold Spring Harbor Labora
tory Press, New York.
4. G. Zaide, D. Shallom, S. Shulami, G. Zolotnitsky, G. Golan, T. Baasov, G. Sho
ham and Y. Shoham (2001) Eur. J. Biochem. Vol. 268, 3006-1016.
5. PG Murray, CM Collins, A. Grassick and MG Tuohy (2003), Biochem. Biophy
s. Res. Commun. Vol. 301, 280-286.
6. AJ Harvey, M. Hrmova, R. De Gori, JN Varghese and GB Fincher, (2000) Pr
oteins Vol. 41, 257-269.
7. AP Moloney, TJ Hackett, PJ Considine, MP Coughlan, (1983) Enzyme Micr
ob. Technol. Vol. 5, 260-264.
8. GL Miller (1951), Anal. Chem. Vol. 31, 426-428.
9. M. Dubois, K. Gilles, PA Hamillton, PA Rebers, F. Smith, (1956) Anal. Che
m. 28, 350-356.
10. UR Laemmli, (1970), Nature (London), Vol. 227, 680-685.
11. PG Murray, A-Grassick, CD Laffey, MM Cuffe, T. Higgins, AV Savage, A
Planas, MG Tuohy, (2001), Enzyme Microbial Technol. Vol. 29, 90-98.
12. OH Lowry, NJ Rosebrough, AL Farr, RJ Randall (1951), J. Biol. Chem.
193: 265-275.
13. L. Falquet, M. Pagni, P. Bucher, N. HuIo, CJ Sigrist, K. Hofmann, A. Bairo
ch, (2002). Nucleic Acids Research, Vol. 30, 235-238.
14. H. Nielsen, J. Engelbrecht, S. Brunak, G. on Heigne, (1997), Int. J. Neural
Syst, Vol. 8, 581- 599.
15. D. Higgins, J. Thompson, T. Gibson (1994): Nucleic Acids Res, Vo. 22. 4673-
4680.
16. AC Stolk, RA Samson (1972) Studies in Mycology No.2, pp. 1-65.
17. Tuohy MG, Coughlan, MP (1992) Bioresource Technol. 39, 131-137
18. Tuohy, MG, Walsh, DJ, Murray, PG, Claeyssens, M., Cuffe, MM, Savage,
AV, Coughlan, MP (2002). Biochim. Biophys. Acta, 1596, 336-380.
19. Reynolds, PJ (1986) Ph.D. Thesis, National University of Ireland, Galway.
20. APHA, A., WEF. (1992) Standard methods for the examination of wastewater.
21. Walsh, DJ (1997) Ph.D. Thesis, National University of Ireland, Galway.
22. Gilleran, CT (2004) Ph.D. Thesis, National University of Ireland, Galway.
23. Maloney, AP, Callan, SM, Murray, PG, Tuohy, MG (2004) Eur. J. Bioche
m. 271, 3115-3126.
24. Braet, C. (2005) Ph.D. Thesis, National University of Ireland, Galway. Bensa
doun, A. and Weinstein, D. (1976). Anal. Biochem. 70: 241-250.
25. Tuohy, MG, Laffey, CD & Coughlan. MP (1994) Bioresource Technol. 50, 3
7-42. Tuohy, MG, Puls, J., Claeyssens, M. Vrsanska, M. & Coughlan, MP (1993)
Biochem. J. 290, 515-523.
26. Murray, PG, Aro, N., Collins, C. Grassick, A., Penttila, M., Saloheimo, M.
& Tuohy, M. 2004. Prot. Expr. Purif. Vol. 38, pp. 248-257.
27. McCarthy, T., Hanniffy, O., Lalor, E., Savage, AV & Tuohy, MG 2005. Proc
ess Biochem. Vol. 40, pp. 1741-1748.
28. McCarthy, T., Hanniffy, O., Savage, AV & Tuohy, MG (2003) Int. J. Biol.
Macrmol. 33: 141-148.
29. Colleran, E. & Pistilli, A. (1994) Ann. Microbiol. Enzimol. 44: 1-18.
30. Coates, JD, Coughlan, MF & Colleran, E. (1996) J. Microbiol Meth. 26: 23
7-246.

Claims (3)

  It has a molecular weight of 17.5 kDa and a pH optimum of 4 to 4.5, retains 91% activity at pH 3.0, and has a degradation activity for both mixed-bonded D-xylan and mixed-bonded D-glucan A xylanase.   The xylanase according to claim 1, which also has activity against aryl-β-xyloside.   The xylanase according to claim 1 or 2, derived from a Tallalomyces emersonii strain having the deposit number IMI393751 or a strain substantially similar thereto or a mutant thereof.