KR20160000987A - Method for Producing Cellulase with Improved Activity - Google Patents

Abstract

The present invention relates to a method for producing a cellulase by using Trichoderma reesei DSCE24 (deposit number KCTC 18256P) strains, Penicillium verruculosum COKE4E (KCTC 11897BP) strains, or β-glucosidase. According to the present invention, the cellulase produced by the method of the present invention has a markedly higher activity compared with a cellulase produced by using the Trichoderma reesei DSCE24 (deposit number KCTC 18256P) strains, and also has the same or more excellent activity compared with cellulases on the market, thereby being able to replace the conventional cellulases.

Description

[0001] The present invention relates to a method for producing a highly active cellulase,

The invention tricot der town reseyi (Trichoderma reesei and penicillium verruculosum . The present invention also relates to a method for producing high activity cellulase using Penicillium verruculosum .

Cellulose is the most abundant renewable resource in the world and can be biodegraded by cellulase produced by microorganisms. However, most of the cellulose resources are left as agricultural and forest waste products, which is a problem of environmental pollution. Development of an efficient saccharification process technology to solve this problem can be very helpful for the production of bio-based products and energy, as well as for solving environmental problems through reduction of carbon dioxide emission. The sugar conversion of cellulose has physical and chemical treatment methods and enzymatic conversion methods. In the case of high-temperature and high-pressure decomposition methods using strong acids and strong bases, the production equipment and associated waste disposal costs are expensive and non-environmentally problematic. Therefore, it is a recent trend to carry out a chemical pretreatment and a conversion method to a monosaccharide using a cellulase concentrated at a high concentration. In addition, high-activity cellulase can actively decompose cellulose, which is an ovoidable carbohydrate, to improve the utilization of livestock feed, which is actively undergoing research in the feed industry.

Currently produced strains of cellulase are industrially deficient in enzyme concentration, and low enzyme concentrations may affect yield of bioethanol production. Therefore, it is required to develop a cellulase producing strain of high activity and high concentration through genetic mutation. In the present invention, a strain of Trichoderma maresia, a strain of Penicillium verruculum or a strain of Trichoderma maresia and a strain of β-glucosidase To provide bioethanol and a cellulase as a feed additive.

Korean Patent Laid-Open Publication No. 2013-000628 discloses a strain of Penicillium GDX01 which produces a cellulase. However, it does not disclose the strain of Tricoderma maresii, and a strain of Penicillium verruculus or a strain of β-glucosidase A method for enhancing the cellulase activity of the above-mentioned Trichoderma maresia strain has not been disclosed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have intensively studied to develop a highly active cellulase used in the step of converting cellulose and hemicellulose into glucose for bioethanol production. As a result, Trichoderma reesei) mutation treatment to a strain KCCM11770 mutants to high than moju produce cellulase activity tricot der town reseyi DSCE24 (Accession No. KCTC 18256P) for, as a cellulase to the penny silryum beru Cool in which the mutants produced were separated syum (Penicillium verruculosum COKE4E (KCTC 11897BP) or β-glucosidase, the cellulase activity is further increased, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method of producing a cellulase using a strain of Tricoderma maresa DSCE24 (Accession No. KCTC 18256P) and a strain of Penicillium verulchulosum COKE4E (KCTC 11897BP).

It is another object of the present invention to provide a method for producing a cellulase using a strain of Tricoderma marcesca DSCE24 (Accession No. KCTC 18256P) and? -Glucosidase.

It is another object of the present invention to provide a cellulase produced by the method of the present invention.

Another object of the present invention is to provide a composition for producing a cellulase comprising a strain of Tricoderma maresis DSCE24 (accession number KCTC 18256P), and a strain penicillium verruchulosum COKE4E (KCTC 11897BP) or? -Glucosidase.

Yet another object of the present invention is to provide a feed additive for cellulose degradation comprising a strain of Tricoderma maresis DSCE24 (accession number KCTC 18256P), and a strain penicillium beruchulosum COKE4E (KCTC 11897BP) or? -Glucosidase .

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) Trichoderma reesei) DSCE24 (Accession No. KCTC 18256P) strain and a penny silryum beru cool syum (Penicillium verruculosum COKE4E (KCTC 11897BP) in a medium; And (b) culturing the strain of step (a) to produce a cellulase.

The present inventors have intensively studied to develop a highly active cellulase used in the step of converting cellulose and hemicellulose into glucose for bioethanol production. As a result, Trichoderma reesei) mutation treatment to a strain KCCM11770 mutants to high than moju produce cellulase activity tricot der town reseyi DSCE24 (Accession No. KCTC 18256P) for, as a cellulase to the penny silryum beru Cool in which the mutants produced were separated syum (Penicillium verruculosum ) COKE4E (KCTC 11897BP) or β-glucosidase, the activity of the cellulase is further increased.

Accordingly, the present invention relates to a method for producing a cellulase using a strain of Tricoderma maresis DSCE24 (Accession No. KCTC 18256P) and a strain of Penicillium verruchulosum COKE4E (KCTC 11897BP) or? -Glucosidase.

The strain of T. korea DSCE24 of the present invention is a strain prepared by treating NTG (MNNG: N-methyl-N'-nitro-N-nitrosoguanidine) with Trichoderma maresii KCCM11770.

NTG, a substance for producing the strain used in the present invention, is known as a substance causing DNA mutation to general bacteria (Pleven, C. et al, Glioblastomas and chemical mutagenesis in biology laboratories. Report of 3 deaths in the same institute .) Arch Mal Prof, 44: ..... 411-418 (1983); Martin, MS et al, Susceptibility of inbredrats to gastric and duodenal carcinomas induced by N-methyl-N'-nitro-Nnitrosoguanidine J. natl Cancer Inst . , 53: 837-840 (1974); IARC Monographs, Suppl. 6: 394-398 (1987)).

The chemical structure of NTG is shown in the following Chemical Formula 1.

Formula 1

As a result of analyzing the nucleotide sequence of the ITS (internal space region) region of the strain of the present invention, the strains DSCE24 of the present invention were found to contain a strain of Hypocrea jecorina GTXKohli-1, which is a conventional Trichoderma maresia strain, And the strain Tricoderma marcesa DSCE24 of the present invention was deposited on August 27, 2013 with the deposit number KCTC 18256P at the Korea Research Institute of Bioscience and Biotechnology.

As used herein, the term " cellulase " refers to an enzyme that hydrolyzes cellulose and produces glucose, cellobiose, and cellooligosaccharide as the primary products. The term " cellulose "in the present specification means a polymer compound in which D-glucose is linked in a straight chain form with a (1? 4) -? - type glycoside bond.

According to one embodiment of the present invention, the cellulase of the present invention is endo-β-1,4-glucanase (endo-glucanase or carboxymethylcellulase, EG, EC 3.2.1.4) (Exo-β-1,4-glucan cellobiohydrolase or exocellobiohydrolase, CBH, EC 3.2.1.91), filter paperase (Fpase) and β- And at least one enzyme selected from the group consisting of glucosidase (cellobiase, BG, EC 3.2.1.21).

The method of the present invention will be described in detail in each step as follows:

Step (a): Inoculating the strain into the medium

According to the present invention, the culture medium is first inoculated with a strain of Trichoderma marese DSCE24 (accession number KCTC 18256P) and a strain of penicillium verruchulosum COKE4E (KCTC 11897BP).

The medium of the present invention includes any medium in the art used for fungal culture, including, but not limited to, YM medium and modified Mandel's medium.

According to one embodiment of the present invention, the inoculation of the present invention is carried out in a separate culture medium, each strain of Tricoderma marcesca DSCE24 (Accession No. KCTC 18256P) and Penicillium verruculosum COKE4E (KCTC 11897BP).

According to one embodiment of the present invention, the medium of the present invention comprises a carbon source. According to another embodiment of the present invention, the carbon source of the medium of the present invention is at least one carbon source selected from the group consisting of barley straw, wheat straw, wheat bran and palm by-products. According to another embodiment of the present invention, the carbon source of the medium of the present invention is a mixture of palm by-product and wheat bran, straw straw, or bran. According to some embodiments of the present invention, the carbon source of the medium of the present invention is barley straw or wheat straw or palm by-product. According to a specific embodiment of the present invention, when the carbon source of the medium of the present invention is a mixture of bran and palm by-products, the content of the palm by-product is 0.1-5, 0.1-3, 0.5-3, 0.5-2.5, 1-2.5, 1.5-2.5 or 1.7-2.3 (weight / volume), and the bran content is 0.1-5, 0.5-5, 1-5, 1-4, 1.5-4, 1.5-3.5, 2-3.5, 2.5- 3.5 or 2.7-3.3 (weight / volume). As demonstrated in the following examples, saccharification experiments were carried out using barn and straw, and as a result, the result of cultivation of a strain of Trichoderma maresei DSCE24 (Accession No. KCTC 18256P) (for example, Trichoderma maresei DSCE24 (Accession No. KCTC 18256P) (Fig. 4a and 4b), which is higher than that of Cellic Ctec_II, a commercially available cellulase, as compared with the case where the enzyme of the strain was used alone. In addition, even when the by-product of the palm was used, the saccharifying power was significantly increased as compared with the result of culturing the strain of Tricoderder maizei DSCE24 (accession number KCTC 18256P) (for example, enzyme concentrate of strain Tricoderder maizei DSCE24 (accession number KCTC 18256P)) , And it was confirmed that the cellulolytic activity was similar to that of Cellic Ctec_II, a commercially available cellulase (FIG. 4C).

According to one embodiment of the present invention, the medium of the present invention may further comprise a nitrogen source or inorganic salts. According to another embodiment of the present invention, the nitrogen source of the medium of the present invention is at least one selected from the group consisting of peptone, meat extract, yeast extract, dried yeast, soybean cake, urea, thiourea, ammonium salt, nitrate and other organic or inorganic nitrogen- But it is not limited to these components. According to another embodiment of the present invention, the inorganic salts of the medium of the present invention may include phosphates such as magnesium, manganese, potassium, calcium, iron, zinc and cobalt, nitrate, carbonate and chloride. It is not. Amino acids, vitamins, nucleic acids and related compounds in addition to the carbon source, the nitrogen source and the components of the inorganic salts may be additionally added to the medium of the present invention.

According to one embodiment of the present invention, the medium of the present invention is pH 4-6, pH 4.5-6.0, pH 4.5-5.5, pH 5.0-5.5.

Step (b): Cellulase production by culturing the strain

After step (a), the strain of step (a) is cultured to produce a cellulase.

The present invention can be cultured through a known culture method for pre-culturing or seed culture of Trichoderma maresei DSCE24 (Accession No. KCTC 18256P) strain and Penicillium verruchulosum COKE4E (KCTC 11897BP). The resulting enzyme solution of the culture contains cellulase.

According to one embodiment of the present invention, the cultivation of the present invention is carried out at 20-35 占 폚, 25-35 占 폚, 25-33 占 폚 or 27-33 占 폚.

According to one embodiment of the present invention, the cultivation of the present invention is carried out at a stirring speed of 100-300 rpm, 150-300 rpm, 150-250 rpm or 150-200 rpm.

According to one embodiment of the present invention, the incubation of the present invention is 120-168 hours, 132-156 hours, 138-150 hours or 141-147 hours.

According to another embodiment of the present invention, when each strain of the present invention is inoculated into a separate medium, the step of culturing each strain and then mixing the resultant thereof is further included. According to some embodiments of the present invention, the cellulase of the present invention is a mixture of the cultured product of a strain of Trichoderma maresca DSCE24 (Accession No. KCTC 18256P) and the result of culturing of Penicillium verulchulosum COKE4E (KCTC 11897BP). According to some embodiments of the present invention, the result of culturing the penicillium beruchullochium COKE4E (KCTC 11897BP) of the present invention is such that the volume of the resultant culture of the strain Tricoderma maresis DSCE24 (accession number KCTC 18256P) is 100 parts skin 0.5-30, 0.5-25, 0.5-20, 0.5-15, 0.5-10, 0.7-10, 1-10, 1-8, 3-8, 3-6 or 4-6 parts of skin. According to a specific embodiment of the present invention, the activity of the cultured product of the strain of Trichoderma maresae DSCE24 (Accession No. KCTC 18256P) of the present invention is 1-2.3, 1.5-2.3, 1.7-2.3, 1.7-2.1, 1.9-2.1 or 2 FPU / ml. According to another specific embodiment of the present invention, the activity of the culture product of the penicillium veruculum bercoleum COKE4E (KCTC 11897BP) strain of the present invention is 100-200, 100-180, 100-160, 120-160, 130-160 , 130-150 or 140 U / ml.

According to another embodiment of the present invention, the cultured product of the strain of Trichoderma maresca DSCE24 (Accession No. KCTC 18256P) of the present invention includes endo-glucanase, cellobiohydrolase and? -Glucosidase . According to yet another embodiment of the invention, the result of the culture of Penicillium verruchulosum COKE4E (KCTC 11897BP) of the present invention comprises? -Glucosidase. According to a specific embodiment of the present invention, the cellulase of the present invention is an endo-glucanase, cellobiohydrolase and? -Glucosidase of a strain of Tricoderma maresis DSCE24 (accession number KCTC 18256P) Penicillium verruculosum COKE4E (KCTC 11897BP).

According to another embodiment of the present invention, the result of culturing the penicillium beruchulosum COKE4E (KCTC 11897BP) of the present invention contains a protein of 30-150, 30-75 or 30-70 kDa.

According to one embodiment of the present invention, the cellulase of the present invention has an activity of 2.3-5.0 FPU / ml. According to another embodiment of the present invention, the cellulase has an activity of 2.3-4.0, 2.3-3.5, 2.3-3.0 or 2.5-3.0 FPU / ml. According to some embodiments of the present invention, the cellulase has an activity of 2.5-5.0, 2.5-4.0 or 2.5-3.5 FPU / ml. Such activity of the cellulase of the present invention can be confirmed by the results of culturing the product of Tricoderma lysine DSCE24 (for example, using a solution of penicillium verulchulosum COKE4E enzyme alone in an amount of 0% Compared to the control group) (Table 5 and Fig. 3).

According to another aspect of the present invention, the present invention provides a method of producing a microorganism, comprising: a) inoculating a culture medium with a strain of Tricoderma maresis DSCE24 (accession number KCTC 18256P); (b) culturing the strain of step (a); And (c) mixing the result of step (b) with? -Glucosidase to produce a cellulase.

The method of the present invention will be described in detail in each step as follows:

Step (a): Inoculating the strain into the medium

According to the present invention, the strain is first inoculated with a strain of Trichoderma marese DSCE24 (accession number KCTC 18256P).

Step (b): Culture of the strain

After step (a) is carried out, the strain of step (a) is cultured.

Step (c): Production of cellulase by mixing ? -Glucosidase

After step (b) is carried out, the result of step (b) is mixed with? -Glucosidase.

According to some embodiments of the present invention, the β-glucosidase of the present invention can be used in the form of a mixture of 1-30, 1-25, and 10-21 when the volume of the resultant culture of the strain Tricoderma maresca DSCE24 (accession number KCTC 18256P) 1-20, 3-20, 3-15, 5-15, 7-15, 7-13, 9-13 or 9-11 parts.

According to one embodiment of the present invention, the result of step (b) of the present invention is an endo-glucanase, cellobiohydrolase and? -Glucosidase of the strain of Tricoderma maresis DSCE24 (accession number KCTC 18256P) .

According to one embodiment of the present invention, the cellulase of the present invention has an activity of 2.3-5.0 FPU / ml. According to another embodiment of the present invention, the cellulase has an activity of 2.5-5.0, 3.0-5.0, 3.0-4.5, 3.5-4.5 or 3.5-4.0 FPU / ml. According to some embodiments of the present invention, the cellulase has an activity of 2.3-4.5 or 2.5-4.5 FPU / ml. This activity of the cellulase of the present invention is superior to that of the active control of the cellulase (a control group with 0% substitution of? -Glucosidase substitution) only in the strain of Tricoderder marese DSCE24 (accession number KCTC 18256P) as demonstrated in the following examples It was confirmed that the total cellulase activity increased 1.6-2 times (Table 4 and Fig. 2).

Since the method for producing a cellulase of the present invention uses the same strain, culture medium and culture method of Trichoderma maresia DSCE24 (accession number KCTC 18256P) which is the same as the method for producing a cellulase of the above embodiment, The description is omitted in order to avoid undue complexity of the present specification.

According to another aspect of the present invention, the present invention provides a cellulase produced by the method of the present invention.

According to another aspect of the present invention, the present invention provides a method for producing a cellulase comprising a strain of Tricoderma maresis DSCE24 (Accession No. KCTC 18256P) and a strain of Penicillium verruculum COKE4E (KCTC 11897BP) or? -Glucosidase Lt; / RTI >

The composition for producing a cellulase of the present invention may contain the medium component of the present invention.

According to one embodiment of the present invention, the composition for producing a cellulase of the present invention decomposes a biomass selected from the group consisting of barnyardgrass, wheat straw, and palm by-product into glucose.

According to another aspect of the present invention, the present invention provides a method for degrading cellulose comprising Tricoderma maresis DSCE24 (Accession No. KCTC 18256P) strain and Penicillium verruchulosum COKE4E (KCTC 11897BP) strain or? -Glucosidase Feed additives.

In order to produce the feed additive of the present invention, the mutant of the present invention or a culture solution thereof may be directly formulated or diluted with a diluent such as wheat flour, starch, and texturin and a seed cake rich in bran oil and fat such as grains, rice hull and degreased rice bran Can be formulated together with feedstuff raw materials. The cellulase contained in the obtained mutant of the present invention is administered to an animal in the form of a feed additive containing it or in the form of a feed in an appropriate amount to give rise to weight gain and accelerated growth.

In addition, the feed additive of the present invention may be in the form of a dry or liquid preparation, and may further comprise one or more enzyme preparations. The added enzyme preparation can be either dry or liquid. It can be used as a lipase, a lipase, a lipase, a phytase that decomposes phytic acid to form phosphate and inositol phosphate, a starch and a glycogen, Amylase, which is an enzyme that hydrolyzes an alpha -1,4-glycoside bond, phosphatase, which is an enzyme that hydrolyzes organic phosphate ester, xylanase which degrades xylose, maltase and saccharose which hydrolyze maltose into two molecules of glucose to hydrolyze glucose-fructose (glucose) and a sugar-producing enzyme such as an invertase for producing a mixture of a sugar and a fructose.

The feed additives of the present invention may include excipients for feed addition. The excipient for feed addition includes, but is not limited to, zeolite, corn or rice bran.

According to another aspect of the present invention, there is provided a feed for livestock growing comprising the feed additive of the present invention.

The feed of the present invention can be suitably used for feed materials such as peanuts, peas, beet pulp, pulp, grain by-products, animal powder, fish meal powder and the like including various grains and soybean protein. The processing is preferably a process in which the feed material is filled with a feedstock under a pressure and is compressed to a constant outlet. In the case of a protein, extrusion is preferably used in which denaturation and utilization are increased, but the present invention is not limited thereto. Extrusion molding has the advantages of denaturing proteins through heat treatment, destroying antioxidant factors, and increasing the activity of proteolytic enzymes by enzymatic exposure to new sites due to molecular structural changes. In the case of the soy protein, the protein digestibility is improved by extrusion. Inactivation of anti-nutrients such as trypsin inhibitor, which is one of protease inhibitors in soybean, And the nutritional value of soy protein can be increased.

Representative examples of animals in which the feed of the present invention can be used are: domestic animals such as edible wool, cows, calves, pigs, sheep, sheep, goats, horses, rabbits, dogs, cats and the like; Poultry such as chickens, chickens, domestic chickens, roosters, ducks, geese, turkeys, quail, small birds,

In addition, the dosage may be suitably used depending on the kind of animal to be administered, age and other kinds of feed ingredients.

According to another aspect of the present invention, the present invention provides a detergent composition comprising a strain of Trichoderma marese DSCE24 (accession number KCTC 18256P), and a strain penicillium verruchulosum COKE4E (KCTC 11897BP) or? -Glucosidase to provide.

The detergent compositions of the present invention may be in the form of part 1 and part 2 aqueous detergent compositions, non-aqueous liquid detergent compositions, cast solids, granular forms, particulate forms, compressed tablets, gels and / or pastes and slurries. Such detergent compositions can be used to quickly remove stubborn foodstuffs, residual foodstuffs, and other small amounts of food compositions. The detergent compositions of the present invention may be effective for catalyzed hydrolysis of starch polysaccharides to remove dried stains.

The detergent composition of the present invention can provide a detergent composition including a detergent composition for cleaning hard surfaces, a detergent composition for cleaning fabrics, a detergent composition for dishwashing, a detergent composition for oral cleaning, a detergent for denture cleaning and a contact lens cleaning solution .

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a cellulase using a strain of Trichoderma marese DSCE24 (Accession No. KCTC 18256P), and a strain of Penicillium verruculum COKE4E (KCTC 11897BP) or β-glucosidase.

(b) The present invention provides a cellulase produced by the method of the present invention.

(c) The present invention relates to a composition for producing a cellulase comprising a strain of Tricoderma maresis DSCE24 (accession number KCTC 18256P) of the present invention, a strain of Pennicillium beruchulosum COKE4E (KCTC 11897BP) or β-glucosidase, Compositions and detergent compositions.

(d) The cellulase produced by the method of the present invention is significantly higher in activity than the cellulase produced using the strain of Tricoderma maresis DSCE24 (Accession No. KCTC 18256P), and has the same or superior activity as the commercially available cellulase And has an advantage of being able to replace conventional cellulases.

Fig. 1 shows the total cellulase activity when commercially available? -Glucosidase was added to an enzyme solution of Tricoderder marese DSCE24.
Fig. 2 shows the total cellulase activity in the case of adding an enzyme solution produced by penicillium berukulosum COKE4E to an enzyme solution of tricorder marese DSCE24.
Fig. 3 shows the total cellulase activity of the enzyme Tricoderma marcesca DSCE24 and the solution of penicillium verrucoulosum COKE4E.
Fig. 4 shows the results of measurement of the saccharification rate depending on the kinds of cow and biomass. Fig. 4A shows the results when straw straw is used as the biomass, Fig. 4B shows the results when the wheat straw is used, and Fig. 4C shows the results when using the palm by-products.
FIG. 5 shows the result after saccharification according to the type of biomass (the biomass is a straw straw in the case of FIG. 5A, a whitish in the case of FIG. 5B, and a farm by-product in the case of FIG. 5C).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Example  1: Enzyme profile search for highly active mutants of cellulase

High activity of cellulase, which was treated with NTG (MNNG: N-methyl-N'-nitro-N-nitrosoguanidine) on Trichoderma maresei KCCM11770 (total mosquito), increased total cellulase activity by 48% The activity of glucosidase was increased by about 54%.) The strain Tricoderdermarease DSCE24 (Accession No. KCTC 18256P) was cultured to produce an enzyme solution, which was compared with Cellic Ctec II (Novozymes, Denmark), which was evaluated as highly active cellulase To search for deficient enzymes. The Cellic Ctec II enzyme is an enzyme prepared for commercial use and enriched in high concentration (120-140 FPU / ml). Therefore, Cellic Ctec II was diluted 60-70 times with 50 mM citrate buffer (pH 4.8), adjusted to 2 FPU / ml, and compared with the enzyme solution of Tricoderder marcy DSCE24 in order to compare the exact enzyme profile. The preparation method of the above-mentioned Tricoderdermarease DSCE24 (Accession No. KCTC 18256P) strain is described in detail in Korean Patent Application No. 2013-0104329 of the present inventors, which is incorporated herein by reference. The above-mentioned Trichoderma maresei DSCE24 (Accession No. KCTC 18256P) strain was deposited on August 27, 2013 by the depository institution Korea Biotechnology Research Institute, and received the deposit number KCTC 18256P.

The flask culture of Trichoderma maresca DSCE24 (accession number KCTC 18256P) was performed by streaking on a PDA plate, and spores of the cellulase highly active strain were suspended using SDW. The spore suspension was firstly cultured in YM medium and then secondary cultured in modified Mandel's medium containing glucose. Then, glucose was replaced with palm by-product and wheat bran and the pH was adjusted to 5.2 with NaOH The transformed Mandel medium (Table 1) was subjected to the main culture (30 ° C, 170 rpm, 6 days). Then, the culture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used as a reference enzyme solution.

Total cellulase ( cellulase )

Total cellulase activity was measured using the filter paper assay method (Technical Report NREL / TP-510-42628; www.nrel.gov) presented by National Renewable Energy Laboratory (NREL).

1 ml of 0.05 M citrate buffer was added to a test tube (Φ18 × h180 mm), 50 mg (1 × 6 cm) of whatman NO.1 filter paper (Whatman) was added as a substrate, 0.5 ml of the enzyme solution of Lysey DSCE24 (Accession No. KCTC 18256P) strain was added. After reaction at 50 ° C for 60 minutes, 3 ml of 3,5-dinitrosalicylic acid (DNS: SIGMA, USA) was added, and the reaction was stopped by boiling at 100 ° C for 5 minutes. Respectively. After cooling in cold water, the absorbance was measured at a wavelength of 540 nm. The amount of reducing sugar produced by substitution into a previously prepared glucose standard curve was calculated. The enzyme activity unit used was filter paper unit (FPU), which was defined as the amount of enzyme used to produce 1 μmol of glucose per minute at the above reaction conditions.

Endo- Glucanase ( endo - glucanase ) And Cellrobiohydrolase ( cellobiohydrolase ) Activity analysis

The activity of endo-glucanase and cellobiohydrolase was determined by reducing endogenous (Ghose TK (1987) Pure & Appl . Chem ., Vol.59, NO.2, pp. 257-268 was measured using a)).

β- Glucosidase (β- glucosidase ) Active measurement

β-glucosidase was measured by using para-nitrophenyl-β-D-glucopyranoside (pNPG, SIGMA, USA). The reaction solution was prepared by adding 0.2 ml of 10 mM pNPG as a substrate to 2.7 ml of citrate buffer and then adding 0.1 ml of enzyme solution. After reacting at 50 ° C for 30 minutes, the reaction was stopped by adding 1 ml of sodium carbonate (1M) and the absorbance at 420 nm was measured. The amount of para-nitro-phenol (pNP) produced by substitution in a standard curve prepared in advance was measured to confirm relative glucose production. Activity is expressed as U (unit), where 1 U is defined as the amount of enzyme used to generate 1 μmol of pNP per minute by digesting pNPG under these conditions.

As a result, it was confirmed that the activity of β-glucosidase was significantly lower than that of Cellic Ctec II in the enzyme of Trichoderma maresae DSCE24 (accession number KCTC 18256P), which is a high activity mutant of cellulase, compared with other enzyme groups (Table 2) -Glucosidase activity of the present invention can be further increased by increasing the activity of the total cellulase.

Example  2: Penicillium Beruculo COKE4E Of enzymes

The enzyme produced by Penicillium verruchulosum COKE4E (Accession No. KCTC 11897BP), which has been distributed from the KCTC Microorganism Resource Center, Korea Research Institute of Bioscience and Biotechnology, is insufficient in β-glucosidase The activity of penicillium verruchulosum COKE4E on pNP (pNitrophenyl) type substrate was investigated. The enzyme solution of penicillium verruchulosum COKE4E was prepared by the following method: The culture of the penicillium beruchulosum COKE4E (Deposit No. KCTC 11897BP) strain was performed by streaking on a PDA plate and then cultured using a cutter knife After cutting to 0.5 cm in width and 0.5 cm in size, the cells were firstly cultured in YM medium. Then, the cells were cultured in a modified Mandel's medium containing glucose, glucose was replaced with palm by-products and wheat bran, (30 ° C, 170 rpm, 6 days) was applied to the modified Mandel medium (Table 3) adjusted to 5.2. Then, the culture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used as a reference enzyme solution.

In order to measure the activity of the pNP-based substrate, the reaction solution of penicillium verruchulosum COKE4E was prepared by adding 0.2 ml of 10 mM pNPG as a substrate to 2.7 ml of citrate buffer, followed by addition of 0.1 ml of enzyme solution . After reacting at 50 ° C for 30 minutes, 1 ml of 1 M sodium carbonate was added to stop the reaction, and the absorbance at 420 nm was measured. The amount of para-nitro-phenol (pNP) produced by substitution in a standard curve prepared in advance was measured to confirm relative glucose production. Activity is expressed as U (unit), where 1 U is defined as the amount of enzyme used to generate 1 μmol of pNP per minute by digesting pNPG under these conditions.

As shown in Table 3, the activity of pNP-β-D-glucopyranoside (pNP-β-D-glucopyranoside), which is an index of β-glucosidase activity, The activity against pNP-? -D-cellrobioside and pNP-? -D-xylopyranoside was 100% or higher, and the activity against pNP-? -D-glucopyranoside was 100% (7.1%) and 17.8% (Table 4), respectively. Therefore, the enzyme produced by Penicillium verruculosum COKE4E was found to be capable of enhancing the? -Glucosidase activity including? -Glucosidase.

Example  3: Trichoderma by enzyme reinforcement LESEY DSCE24 Enzyme solution  Ascertained active rise

In order to confirm whether the total cellulase activity of the enzyme solution of Trichoderma maresca DSCE24 was increased when β-glucosidase was added, commercially-available β-glucosidase (600 U / ml, manufactured by SIGMA) The enzymatic activity was confirmed after the enzyme mixture was prepared by adding 0-20% of the volume of the enzyme solution of the tricorder deraceae DSCE24. The activity measurement of the enzyme mixture was carried out by using the same method as that of the cellulase activity assay of Example 1, except that the enzyme solution of Example 1 was replaced with the enzyme mixture.

As a result, as shown in Table 5, it was confirmed that the addition of? -Glucosidase increased the total cellulase activity by 1.6-2 times as compared with the control (Fig. 1).

Example  4: Penicillium Beruculo COKE4E  Trichoderma according to production enzyme reinforcement LESEY DSCE24 Enzyme solution  Ascertained active rise

In order to confirm that the total cellulase activity was increased when the penicillium veruculosum COKE4E enzyme solution of Example 2 was fortified with the enzyme solution of Trichoderma maresia DSCE24 of Example 1, The total cellulase enzyme activity was confirmed after preparing an enzyme mixture by adding 0-20% of the volume of DSCE24 enzyme solution by replacing the penicillium berukulosum COKE4E enrichment enzyme solution. The penicillium beruchulium COKE4E enrichment enzyme solution was used after concentrating the penicillium beruchullozyme COKE4E enzyme of Example 2 to 140 U / ml using an ultrafiltration membrane (Amicon stirred cell, Amicon). The activity measurement of the enzyme mixture was carried out by using the same method as that of the cellulase activity assay of Example 1, except that the enzyme solution of Example 1 was replaced with the enzyme mixture.

As a result, as shown in the following Table 6, when the enzyme mixture was used, the total cellulase activity increased 1.2-1.4 times compared with the control using the enzyme of Example 1, (Fig. 2).

Example  5: Penicillium Berochlorine COKE4E Enzyme solution  Identification of total cellulase activity

In order to confirm whether the result of Example 4 is the effect of the high total cellulase activity of the penicillium berrochium COKE4E enzyme solution, the total cellulase activity of the penicillium berrochium COKE4E enzyme solution was confirmed, and the activity of the enzyme of the tricorder macease DSCE24 enzyme Activity.

As a result, it was confirmed that the total cellulase activity was not substantially exhibited in the case of Penicillium berrochium COKE4E enzyme solution, unlike the enzyme solution of Trichoderma maresia DSCE24 (Fig. 3). Therefore, the result of Example 4 is not considered to be an effect of Penicillium berrochium COKE4E enzyme solution, and it is considered that the use of penicillium berrochium COKE4E enzyme alone would not be suitable for saccharification of biomass.

Example  6: Biomass  Depending on the type Glycation ability  evaluation

The saccharifying ability of each of the enriched solution of the enzyme solution of Trichoderma marinasi DSCE24 of Example 1, the concentrated solution of Cellic Ctec II (Novozymes, Denmark) and the mixture of the enzyme solution of Example 4 was measured by differentiating the biomass as barley straw, wheat straw and palm by-product .

(20 FPU / g biomass) was stirred at 180 rpm at 50 DEG C in a reaction solution (each substrate content 5%, pH 4.8, 1% sodium azide) having the composition shown in Table 7 below The reaction was carried out for 72 hours. The amount of glucose to be converted was determined by taking a sample and centrifuging (10000 rpm x 5 min) and then using supernatant (YSI 2900, YSI life science, USA).

As a result of measuring the sugar content according to the type of enzymes and biomass according to the above method, when using the barley straw and wheat straw as biomass, the sugar content of the enzyme solution of Example 4 was 4-5% higher than that of Cellic Ctec II, It was confirmed that when the by-product of the biomass was used, the glycation rate was similar to that of Cellic Ctec II (FIGS. 4A-4C).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KTCT18256P 20130827

Claims (6)

A method of producing a cellulase comprising the steps of:
(a) Trichoderma reesei) DSCE24 (Accession No. KCTC 18256P) strain and a penny silryum beru cool syum (Penicillium verruculosum COKE4E (KCTC 11897BP) in a medium; And
(b) culturing the strain of step (a) to produce a cellulase.
A method of producing a cellulase comprising the steps of:
(a) inoculating the culture medium with a strain of Trichoderma maresca DSCE24 (Accession No. KCTC 18256P);
(b) culturing the strain of step (a) to produce a cellulase; And
(c) mixing the result of step (b) with? -glucosidase.
The method of producing a cellulase according to claim 1 or 2, wherein the medium of step (a) comprises a carbon source.
4. The method of claim 3, wherein the carbon source is a carbon source selected from the group consisting of barley straw, wheat straw, wheat bran, and palm by-products.
A composition for producing a cellulase comprising a strain of Tricorde maresae DSCE24 (Accession No. KCTC 18256P) and a strain of Penicillium verruchulosum COKE4E (KCTC 11897BP).
A composition for producing a cellulase comprising a strain of Tricoderma marcesca DSCE24 (Accession No. KCTC 18256P) strain and? -Glucosidase.

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