CN117568317A - Complex enzyme for efficiently degrading cereal fibers and application thereof - Google Patents

Complex enzyme for efficiently degrading cereal fibers and application thereof Download PDF

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Publication number
CN117568317A
CN117568317A CN202311611528.8A CN202311611528A CN117568317A CN 117568317 A CN117568317 A CN 117568317A CN 202311611528 A CN202311611528 A CN 202311611528A CN 117568317 A CN117568317 A CN 117568317A
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enzyme
beta
xylanase
complex enzyme
glucanase
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屈汶辉
谢春元
崔然宰
金根佑
周黔光
刘阳
张民
刘睿
徐晓明
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Xijieuter Shandong Biotechnology Co ltd
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Xijieuter Shandong Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of enzyme preparations, and particularly relates to a composite enzyme for efficiently degrading cereal fibers and application thereof. In order to efficiently degrade a large amount of cellulose and hemicellulose in the cereal fiber, the invention selects Trichoderma reesei enzyme system as a main enzyme system, and further strengthens or supplements xylanase and beta-glucanase on the basis. The composite enzyme for efficiently degrading the cereal fibers has universal applicability to corn, byproducts, bran, barley and other grains, can obviously improve the dry matter degradation rate of common grains and byproducts, and simultaneously provides an application method of the composite enzyme.

Description

Complex enzyme for efficiently degrading cereal fibers and application thereof
Technical Field
The invention belongs to the technical field of enzyme preparations, and particularly relates to a composite enzyme for efficiently degrading cereal fibers and application thereof.
Background
The grains are main energy raw materials in livestock and poultry feeds, and have huge demands, but the grain fibers contain a large amount of Non-starch polysaccharides (Non-starch polysaccharides, NSPs), mainly cellulose, xylan, beta-glucan (accounting for more than 70% of the total NSPs) and the like, and the Non-starch polysaccharides are connected in a staggered manner (Choct et al, 1997) to form a complex space structure. In the production of livestock and poultry, the soluble part and the insoluble part respectively reduce the digestion and absorption of nutrient substances by animals by increasing the viscosity of chyme and the barrier effect, so that the enzymolysis of the crude fiber of the grains requires specific complex enzyme for high-efficiency enzymolysis. Patent CN105462948A discloses a wheat ration specific xylan degrading complex enzyme preparation, the active ingredients of which comprise xylanase, arabinofuranosidase and/or feruloyl esterase; patent CN109679934a discloses a complex enzyme composition for efficiently degrading corn xylan, the main active components of which are xylanase a, xylanase b, alpha-L-arabinofuranosidase and/or feruloyl esterase; patent CN1392256A discloses an early indica rice feeding enzyme preparation, which comprises xylanase, beta-glucanase, cellulose CMC enzyme, acid proteinase and the like as main components. Patent CN104630182A discloses a compound enzyme for growing pigs and a preparation method thereof, wherein the compound enzyme comprises xylanase, cellulase, beta-glucanase, moderate temperature alpha-amylase, galactosidase, pectinase and the like as main components. Said invention has the advantages of simple compatibility and strong pertinence; however, the enzyme types adopted by the complex enzyme compatibility scheme are limited to a plurality of enzymes which are compounded, and most of the complex enzyme compatibility scheme is endo-enzyme, and is lack of exoenzyme, side chain enzyme, auxiliary protein and other auxiliary factors, so that the fiber (especially the fiber with complex structure or insoluble fiber) has lower degradation efficiency and narrower application range, and the requirement of degrading complex feed components can not be completely met.
Trichoderma reesei (Trichoderma reesei) is a multicellular eukaryotic microorganism, an asexual form of Brevibacterium roseum (Hypocrea jecorina), belonging to the genus Trichoderma (Trichoderma) of the order Trichoderma reesei (Moniliales). After being induced as industrial strain, the secretory group of the strain is identified by protein, and the secretory group comprises 31 enzymes and auxiliary proteins (Pedersen, N.R. and the like, 2021), wherein cellulose degradation related proteins (cellulase systems) account for more than 85% of total proteins (Berrin, J.G. and the like, 2014), and the secretory group is widely used for processing plant cellulose (such as textile, papermaking and the like). However, enzyme systems for hemicellulose-related degradation, including xylanase and glucanase, are relatively low. Therefore, the patent CN 113174382A uses trichoderma reesei enzyme system as main enzyme system, and is a complex cellulase system prepared by adding xylanase and arabinofuranosidase; or one or two of acetylxylan esterase and feruloyl esterase are further added on the basis of the above, so that the conversion rate of corn fiber sugar and the ethanol yield of unit corn are improved. The invention is only directed to saccharification of corn fiber, and thus only allows for degradation of two types of polysaccharides, cellulose (the corresponding degrading enzyme is the Trichoderma reesei enzyme system) and xylan (the corresponding degrading enzyme includes endo-xylanase, arabinofuranosidase, acetylxylanase esterase and arabinofuranosidase). However, in livestock production, glucan is a third polysaccharide content (especially in wheat diets) in addition to cellulose and xylan. Therefore, the invention is based on Trichoderma reesei enzyme system, and supplements beta-1, 3-1, 4-glucanase besides strengthening xylanase. On the other hand, unlike the CN 113174382A invention, the main purpose is to obtain monosaccharides, thereby further obtaining ethanol. The invention is mainly used for livestock production, and aims to open a structure, reduce the viscosity of chyme and release nutrient substances, degrade the chyme into oligosaccharide and be more beneficial to the utilization of intestinal microorganisms of livestock. Therefore, only endoxylanase with higher oligosaccharide production efficiency is enhanced in the xylanolytic enzyme, and other auxiliary enzymes are derived from Trichoderma reesei enzyme systems.
Disclosure of Invention
In order to efficiently degrade a large amount of cellulose and hemicellulose in the cereal fiber, the invention selects Trichoderma reesei enzyme system as a main enzyme system, and further strengthens or supplements xylanase and beta-glucanase on the basis. The composite enzyme for efficiently degrading the cereal fibers has universal applicability to corn, byproducts, bran, barley and other grains, can obviously improve the dry matter degradation rate of common grains and byproducts, and simultaneously provides an application method of the composite enzyme.
The invention is realized by the following technical scheme:
the composite enzyme for efficiently degrading cereal fibers is prepared from Trichoderma reesei enzyme system as a main enzyme system, and also comprises beta-1, 4-xylanase and beta-1, 3-1, 4-glucanase, wherein active ingredients of the composite enzyme mainly comprise cellulase (EC 3.2.1.4), beta-1, 4-xylanase (EC 3.2.1.8) and beta-1, 3-1, 4-glucanase (EC 3.2.1.73).
The Trichoderma reesei enzyme system is obtained by the method of U.S. patent No. 6190189B 1. The CMCase activity of the cellulase is 1500-3000U/g, and the FPase activity is 10-20U/g.
The Trichoderma reesei enzyme system mainly comprises a cellulase enzyme system, a xylanase enzyme system and other enzymes or auxiliary proteins;
the cellulase enzyme system comprises one or more of cellulase and a cleavable polysaccharide monooxygenase, wherein the cellulase comprises one or more of endo beta-1, 4-glucanase, exo beta-1, 4-glucanase and beta-glucosidase;
the xylanase enzyme system comprises one or more of beta-1, 4-xylanase, beta-xylanase, alpha-L-arabinosidase, alpha-D-glucuronidase, acetyl xylanase and phenolic acid esterase.
The beta-1, 4-xylanase is insensitive to xylanase inhibitor proteins TAXI-I, TAXI-II and XIP. The beta-1, 4-xylanase expression gene is derived from a bacterium (e.g., bacillus sp., pseudomonas fluorescens Pseudomonas fluorescens, trichoderma Thermobacillus xylaniticus, bacillus agarobacis Bacillus agaradhaerens, bacillus subtilis Bacillus subtilis, cellulars succinate Fibrobacter succinogens, bacillus amyloliquefaciens Bacillus amyloliquefaciens), or a fungus (Penicillium funiculosum Penicillium funiculosum, penicillium purpurogenum Penicillium purpurogenum, penicillium oxalicum penicillium oxalicum, aspergillus aculeatus Aspergillus aculeatus, aspergillus nidulans Aspergillus nidulans, aspergillus niger Aspergillus niger, aspergillus oryzae Aspergillus oryzae, emersen basket Talaromyces emersonii, botrytis cinerea, fusarium Fusarium graminearum, verbena Orpinomyces Strain PC-2, the anaerobic fungus Neocallimastix sp., penicillium griseus Penicillium griseofulvum, trichoderma longibrachiatum Trichoderma longibrachiatum, trichoderma viride Trichoderma viride, trichoderma reesei Trichoderma reesei), or a plant. The fermentation strain comprises bacillus, escherichia coli, trichoderma reesei, aspergillus niger, aspergillus oryzae and yeast.
The beta-1, 3-1, 4-glucanase is different from beta-1, 3 (4) -glucanase and beta-1, 4-glucanase, and the beta-1, 3-1, 4-glucanase specifically hydrolyzes beta-1, 3-1, 4-glucan in grains. The beta-1, 3-1, 4-glucanase expression gene is derived from a bacterium (e.g., bacillus subtilis Bacillus subtilis, bacillus terlazyphi Bacillus tequilensis, bacillus licheniformis Bacillus licheniformis, bacillus amyloliquefaciens Bacillus amyloliquefaciens, bacillus alcalophilus Bacillus halodurans, bacillus circulans Bacillus circulans, paenibacillus Paenibacillus sp., bacillus polymyxa Bacillus polymyxa, bacillus upland Bacillus altitudinis, filamentous succinate producing bacterium Fibrobacter succinogenes, streptococcus bovis Streptococcus bovis, trichosporon Laetiporus sulphureus, and Clostridium thermocellum Clostridium thermocellum), or a fungus (e.g., paecilomyces thermophilus Paecilomyces thermophila, rhizomucor miehei Rhizomucor miehei, mucor tsugae Malbranchea cinnamomea, bacillus rhizopus Orpinomyces Strain PC-2, bispora, emerson basket Talaromyces emersonii, phytophthora infestans Phytophthora infestans, and Mucor garcins) or a plant. The fermentation strain comprises bacillus, escherichia coli, trichoderma reesei, aspergillus niger, aspergillus oryzae and yeast.
The complex enzyme for efficiently degrading the cereal fiber comprises a Trichoderma reesei enzyme system with 1500-3000U/g and activity of 10000-25000U/g of beta-1, 4-xylanase and a beta-1, 3-1, 4-glucanase with activity of 10000-25000U/g according to the CMCase activity;
preferably, the complex enzyme for efficiently degrading the cereal fiber comprises 2000-3000U/g of Trichoderma reesei enzyme system, 15000-25000U/g of beta-1, 4-xylanase and 20000-25000U/g of beta-1, 3-1, 4-glucanase based on the CMCase activity;
further preferably, the complex enzyme for efficiently degrading the cereal fiber comprises a Trichoderma reesei enzyme system with 2500-3000U/g, a beta-1, 4-xylanase with 20000-25000U/g and a beta-1, 3-1, 4-glucanase with 20000-25000U/g, which are calculated according to the cellulase CMCase activity.
The composite enzyme for efficiently degrading the cereal fibers also comprises a carrier, wherein the carrier comprises one or more of corn husks, starch and rice hull powder, and the carrier can be obtained by crushing the raw materials and uniformly mixing the crushed raw materials.
In the composite enzyme for efficiently degrading the cereal fibers, the mass percentage of the added enzyme is 8% -15%.
The application method of the complex enzyme for efficiently degrading the cereal fibers comprises the steps of adding 200-500 g/ton of substrate, and carrying out enzymolysis at 40 ℃ for 14-18 hours, wherein the substrate comprises corn husks, bran and barley.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention aims at the design of the non-starch polysaccharide with complex space structure of the grains and the by-product raw materials (corn, corn husks, DDGS, wheat, bran and barley) thereof, and can eliminate the space obstruction of xylan; the degradation rate of dry matters of the raw materials is improved, the cell walls are fully opened, and more embedded nutrient substances (starch, protein, mineral elements and the like) are released; after the non-starch polysaccharide in the raw materials is degraded, the viscosity of the chyme in the intestinal tracts of animals is reduced, the phenomena of overfeeding and nutritional diarrhea in the feeding process are reduced, the feed cost is saved, meanwhile, the excrement is reduced, and the energy conservation and the emission reduction are facilitated.
2. The Trichoderma reesei enzyme system contained in the invention is from deep liquid fermentation of Trichoderma reesei. Trichoderma reesei industrial strains are used for producing enzymes for decomposing different plant raw materials, contain rich cellulases and hemicellulases, and have been used for many years. Trichoderma reesei has many advantages of being suitable for production, has no toxicity to human, and does not produce mycotoxin and antibiotics under enzyme production conditions.
3. The beta-1, 4-xylanase contained in the invention is insensitive to xylanase inhibitor proteins TAXI-I, TAXI-II and XIP in grains, and the combination of the inhibitor proteins and the beta-1, 4-xylanase and a xylan substrate is avoided, so that xylan in feed is better degraded, and the viscosity of chyme is reduced.
4. Beta-1, 3-1, 4-glucanases (EC 3.2.1.73), also known as lichenases, contained in the present invention are widely present in microorganisms and plants and are capable of hydrolysing beta-1, 3 and beta-1, 4 glycosidically linked linear beta-glucans (e.g. cereal beta-glucans and lichenin), degrading only the beta-1, 4 glycoside on the glucose residue substituted at the 3-O position, whereas it is inactive for single beta-1, 4 glycosidically linked cellulose and thus distinguished from endo beta-1, 4-glucanases (EC 3.2.14) in cellulases. Beta-1, 3-1, 4-glucanase has effects in a plurality of degradation pathways of cell wall arabinoxylans, and can be beneficial to degradation of non-starch polysaccharide to generate glucose (figure 1), so that energy supply of raw materials is improved.
Drawings
FIG. 1, role of beta-1, 3-1,4 glucanase in degradation of cereal cell wall arabinoxylans.
Detailed Description
The present invention is further described below with reference to examples, and all the raw materials used in examples and comparative examples, such as Aspergillus niger liquid fermentation complex enzyme and Trichoderma reesei solid fermentation complex enzyme, are commercially available.
Examples
According to the active ingredients in the table 1, adding a carrier, uniformly mixing to enable the mass percentage of the active ingredients to be 10%, wherein the carrier is formed by mixing starch and rice hull powder in a weight ratio of 1:1; the complex enzyme is added into the substrate, the dosage of the complex enzyme is 300 g/ton of the substrate, the enzymolysis temperature is 40 ℃, the enzymolysis time is 16 hours, and the results are shown in Table 1.
TABLE 1
Taking corn husks as an example, the dry matter digestibility assessment method is as follows:
(1) Preparing an enzymolysis substrate sample: after the corn husks are crushed, 95% of the corn husks are sieved by a 40-mesh sieve. Measuring dry matter content (DM) for later use;
(2) And (3) enzyme solution dilution: taking 1.0000g of solid enzyme, dissolving with deionized water for 30min, then fixing the volume to a 50mL volumetric flask, uniformly mixing, taking 10mL, putting into a centrifuge tube, centrifuging at 4000rpm for 5min, and taking supernatant to be diluted to a proper concentration. The enzyme liquid is prepared and used at present;
(3) Preparing filter paper: the filter paper was dried to absolute weight (W1, g) at 105℃for 4 h. The experiment uses whatman inlet filter paper;
(4) pH6.5buffer preparation: 1000mL of the solution contained 0.68g of potassium dihydrogen phosphate and 0.1mol/L of sodium hydroxide 15.2mL. And calculating according to the total volume of the configured Buffer. The concentration of the antibiotic (penicillin potassium) in Buffer is 666IU/mL;
(5) Sample adding: about 2g (accurate to 0.0001 g) of corn husk substrate is weighed by using an electronic balance in a 100mL conical flask, weighing (M, g) of the substrate is recorded, enzyme activities required by 2g of the substrate are diluted into 1mL of enzyme solution, and the enzyme activities are sequentially added according to the following sample adding table, so that the concentration of antibiotics in a system is consistent, and the volumes of buffers added in each group are required to be consistent. In this test, 10mL buffer was added, and the sample had significant flowability. If the substrate is other, the Buffer consumption can be increased or decreased according to the actual situation;
(6) Degradation: and sealing the bottle mouth by using sealing films after each group of samples are added. Putting the mixture into a culture box at 40 ℃ and 150rpm for degradation for 16 hours. The incubator needs to be preheated to a specified temperature in advance;
(7) And (3) cleaning and filtering: after degradation, pouring degradation liquid into absolute dry filter paper, cleaning each sample with 60mL of cleaning liquid in total, and adding the cleaning liquid into the filter paper for a plurality of times according to the requirement, wherein the total volume is kept to be 60 mL;
(8) The residue was oven dried: after the water in the filter paper is completely filtered, drying the filter paper and residues to absolute weight (W2, g) at 105 ℃ for 4 hours;
(9) Dry matter digestibility =；
The above embodiments are not to be taken as limiting the scope of the invention, and any alternatives or modifications to the embodiments of the invention will be apparent to those skilled in the art and fall within the scope of the invention.

Claims (10)

1. A complex enzyme for efficiently degrading cereal fibers is characterized in that: is mainly composed of Trichoderma reesei enzyme system, beta-1, 4-xylanase and beta-1, 3-1, 4-glucanase.
2. The complex enzyme for efficiently degrading cereal fibers according to claim 1, wherein: the Trichoderma reesei enzyme system mainly comprises a cellulase enzyme system, a xylanase enzyme system and auxiliary proteins.
3. The complex enzyme for efficiently degrading cereal fibers according to claim 2, wherein: the cellulase enzyme system comprises one or more of cellulase and a schizochypolysaccharide monooxygenase; the xylanase enzyme system comprises one or more of beta-1, 4-xylanase, beta-xylanase, alpha-L-arabinosidase, alpha-D-glucuronidase, acetyl xylanase and phenolic acid esterase.
4. A complex enzyme for the efficient degradation of cereal fibres according to claim 3, characterised in that: the cellulase comprises one or more of endo beta-1, 4-glucanase, exo beta-1, 4-glucanase and beta-glucosidase.
5. A complex enzyme for the efficient degradation of cereal fibres according to claim 3, characterised in that: comprises 1500-3000U/g Trichoderma reesei enzyme system, 10000-25000U/g beta-1, 4-xylanase and 10000-25000U/g beta-1, 3-1, 4-glucanase based on cellulase CMCase activity.
6. The complex enzyme for efficiently degrading cereal fibers according to claim 5, wherein: comprises 2000-3000U/g of Trichoderma reesei enzyme system, 15000-25000U/g of beta-1, 4-xylanase and 20000-25000U/g of beta-1, 3-1, 4-glucanase based on cellulase CMCase activity.
7. The complex enzyme for efficiently degrading cereal fibers according to claim 6, wherein: comprises 2500-3000U/g Trichoderma reesei enzyme system with cellulase CMCase activity, 20000-25000U/g beta-1, 4-xylanase with activity 20000-25000U/g beta-1, 3-1, 4-glucanase.
8. The complex enzyme for efficiently degrading cereal fibers according to claim 1, wherein: the beta-1, 4-xylanase is insensitive to xylanase inhibitor proteins TAXI-I, TAXI-II and XIP.
9. The complex enzyme for efficiently degrading cereal fibers according to claim 1, wherein: the complex enzyme also comprises a carrier, and the mass percentage of the added enzyme accounts for 8% -15% of the complex enzyme.
10. Use of a complex enzyme for the efficient degradation of cereal fibres according to any one of claims 1 to 9, characterised in that: the addition amount of the compound enzyme is 200-500 g/ton substrate, the enzymolysis temperature is 40 ℃, and the enzymolysis time is 14-18 hours.
CN202311611528.8A 2023-11-29 2023-11-29 Complex enzyme for efficiently degrading cereal fibers and application thereof Pending CN117568317A (en)

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