WO2018032798A1 - 一种里氏木霉突变菌株及其应用 - Google Patents

一种里氏木霉突变菌株及其应用 Download PDF

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WO2018032798A1
WO2018032798A1 PCT/CN2017/081986 CN2017081986W WO2018032798A1 WO 2018032798 A1 WO2018032798 A1 WO 2018032798A1 CN 2017081986 W CN2017081986 W CN 2017081986W WO 2018032798 A1 WO2018032798 A1 WO 2018032798A1
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fermentation
mass
parts
activity
enzyme
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吴佳鹏
周利伟
王华明
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青岛蔚蓝生物集团有限公司
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase

Definitions

  • the invention relates to the field of microorganisms, in particular to a mutant strain of Trichoderma reesei and application thereof.
  • Non-starch polysaccharides are plant tissues that are composed of a variety of monosaccharides and uronic acids linked by glycosidic bonds. Most of them have a branched chain structure, often combined with inorganic ions and proteins. The main component of the cell wall is generally difficult to be hydrolyzed by digestive enzymes secreted by monogastric animals.
  • the non-starch polysaccharides in common feeds are mainly arabinoxylan, beta-glucan and cellulose.
  • Corn and sorghum contain a small amount of non-starch polysaccharides, and the water-soluble non-starch polysaccharides in oats and barley are mainly ⁇ -glucans.
  • the grain contains a small amount of pectin polysaccharide, which is not found in other plants except rice.
  • Cereal by-products contain large amounts of cell wall components, such as rice bran containing about 20% to 25% of non-starch polysaccharides, primarily equal amounts of arabinoxylan and cellulose.
  • Non-starch polysaccharides are mainly composed of various glycosidases, which can improve the utilization of feed nutrients by eliminating the anti-nutritional effects of non-starch polysaccharides in feeds. When the suitable ratio of these enzyme activities is consistent with the composition of non-starch polysaccharides in feeds When you get the best results.
  • Non-starch polysaccharide enzymes include cellulase, xylanase, ⁇ -glucanase, ⁇ -mannanase, pectinase and the like.
  • Cellulase can break the cell wall rich in fiber, release the nutrients such as protein and starch contained and use it, and at the same time degrade the fiber into reducing sugar which can be digested and absorbed by livestock and poultry, thereby improving feed utilization.
  • Microorganisms that produce cellulase are mostly fungi, and there are few studies on bacteria and actinomycetes. The microorganisms currently used to produce cellulase are mainly Trichoderma, Aspergillus niger, Penicillium and Rhizopus.
  • the Phytophthora, anti-animal rumen, fibrin, yellow fiber, sclerotium, Myxobacteria, Clostridium faecalis, etc. can also produce cellulase.
  • Xylanase is a specific degrading enzyme of xylan, belonging to hydrolase, including endo-xylanase, exo-xylanase and xylosidase.
  • hydrolase including endo-xylanase, exo-xylanase and xylosidase.
  • ⁇ -glucanase can degrade ⁇ -1,3 and ⁇ -1,4 glycoside chains in ⁇ -glucan molecules, degrade them into small molecules, lose hydrophilicity and viscosity, and change the gut of monogastric animals. Characteristics of the contents, activity of digestive enzymes, environment of action of intestinal microorganisms, and the like. Microorganisms secreting ⁇ -glucanase, one is bacteria, the other is fungi, and the fungi are mainly molds, mainly Trichoderma koningii, Trichoderma reesei, Trichoderma virens, Trichoderma viride, Aspergillus oryzae , Mucor, and Aspergillus niger.
  • Pectinase is a generic term for enzymes that decompose pectin. It is also a multi-enzyme complex, which usually includes three enzymes: pectinase, pectin hydrolase, and pectic acidase. The combined action of these three enzymes allows the pectin to be completely decomposed.
  • the strains for industrial production of pectinase are mainly molds, and the commonly used strains are Aspergillus variabilis, Penicillium citrinum, Aspergillus niger, Rhizoctonia solani, Aspergillus oryzae, yeast and the like.
  • Mannanase is a hemicellulose hydrolase that degrades ⁇ -1,4 glycosidic bonds in an endogenous manner.
  • the non-reducing end of the degradation product is mannose, and its substrate includes glucomannan and galactomannan. And ⁇ -mannan and the like. It can not only reduce the viscosity of the intestine, promote the digestion and absorption of nutrients, but also eliminate the interference of ⁇ -mannose rich in beans on glucose absorption, and greatly improve the energy digestibility of cakes, especially soybean meal; At the same time, the animal's resistance and uniformity were improved after the addition of mannanase.
  • non-starch polysaccharides are mainly through biological fermentation.
  • the strains that can be used to produce various non-starch polysaccharide enzymes are Trichoderma viride, Trichoderma viride, Aspergillus niger and Penicillium funiculosum.
  • the microorganisms that produce cellulase most of them are fungi, and there are few studies on bacteria and actinomycetes.
  • the microorganisms currently used to produce cellulase are mainly Trichoderma, Aspergillus niger, Penicillium and Rhizopus. There are many studies on the ability of Trichoderma, Aspergillus and Bacterium to produce xylanase at home and abroad.
  • the commercial xylanase-producing strains are mainly Trichoderma and Aspergillus.
  • Microorganisms secreting ⁇ -glucanase one type is bacteria, mainly Bacillus, mainly Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis; the other is fungi, mainly mold, mainly Trichoderma koningii, Trichoderma reesei, Trichoderma pseudomonas, Trichoderma viride, Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger.
  • the strains of industrial production of pectinase are mainly molds, and the commonly used strains are Aspergillus wenii, Penicillium apple, Aspergillus niger, white rot fungus, Aspergillus oryzae, yeast, etc.
  • the present invention provides a mutant strain of Trichoderma reesei having high yield of non-starch polysaccharide enzyme and application thereof.
  • the invention obtains a mutant strain of Trichoderma reesei with high yield non-starch polysaccharide enzyme by ultraviolet mutagenesis, can greatly increase the expression level of non-starch polysaccharide enzyme, and can be widely applied to the production of non-starch polysaccharide enzyme.
  • the present invention provides the following technical solutions:
  • the present invention provides a strain of Trichoderma reesei, the preservation number is CCTCC NO: M2016363.
  • the xylanase activity of the mutant strain shake flask fermentation supernatant was 102u/ml, which was increased by 126.6% compared with the starting bacteria; the cellulase activity was 95u/ml, which was 66.6% higher than that of the starting bacteria; ⁇ -glucanase enzyme The activity was 229u/ml, which was 54.7% higher than that of the starting bacteria; the mannanase activity was 25u/ml, which was 257.1% higher than that of the starting bacteria. Lactose was used as the inducer.
  • Fermentation tank fermentation showed that the xylanase activity in the fermentation supernatant of Trichoderma reesei NSP-51 was 1497u/ml, which was 305.6% higher than that of the starting bacteria; the cellulase activity was 935u/ml. Compared with the starting bacteria, the activity was increased by 54%; the ⁇ -glucanase activity was 1288u/ml, which was 56.5% higher than that of the starting bacteria; the mannanase activity was 89u/ml, which was 323.8% higher than that of the starting bacteria. Using liquid sugar as the inducer, the fermenter fermentation results showed that the xylanase activity of the mutant strain T.
  • reesei NSP-51 was 206u/ml, which was 1187% higher than that of the starting bacteria; cellulase activity 894u/ Ml, increased by 97.7% compared with the starting bacteria; ⁇ -glucanase activity 925u/ml, 54.4% higher than the starting bacteria; mannanase activity 34u/ml, increased by 161.5% compared with the starting bacteria.
  • the applicant has deposited at the China Center for Type Culture Collection of Wuhan University, Wuhan, China on July 4, 2016, with the accession number CCTCC NO: M2016363.
  • the invention also provides the use of the T. reesei for the fermentation production of non-starch polysaccharide enzymes.
  • the non-starch polysaccharide enzyme is a mixture of one or more of a xylanase, a cellulase, a beta-glucanase, and a mannanase.
  • the present invention also provides a fermentation method for producing a non-starch polysaccharide enzyme, wherein the T. reesei is a fermentation strain.
  • the fermentation process comprises shake flask fermentation and fermentor fermentation
  • the fermentation medium of the fermentation method comprises: 2 parts by mass of glucose; 1.5 parts by mass of corn syrup; 0.9 parts by mass of ammonium sulfate; 2 parts by mass of potassium dihydrogen phosphate; 0.4 parts by mass of diammonium hydrogen phosphate; 0.15 parts by mass of magnesium sulfate heptahydrate ; 0.073 parts by mass of citric acid; 0.12 parts by mass of calcium chloride; 0.075 parts by mass of ferrous sulfate heptahydrate; 0.006 parts by mass of zinc sulfate heptahydrate; 0.0012 parts by mass of copper sulfate pentahydrate; 0.00053 parts by mass of manganese sulfate monohydrate; Share.
  • the fermentation temperature of the shake flask fermentation in the fermentation method is 30 ° C, and the fermentation time is 5-7 days.
  • the fermentor fermentation in the fermentation method comprises shake flask culture and fermentor culture
  • the shake flask culture medium comprises glucose 10-30 g / L, potatoes 100-200 g / L;
  • the shake flask culture conditions were as follows: shaking at 30 ° C, 200 rpm shaker for 48 h.
  • the fermentation tank is cultured under the conditions of: inoculation of the fermentation strain at 30 ⁇ 1° C., pH 5.0 ⁇ 0.1, and stirring speed of 600 rpm. After culturing for 24 hours in the fermentation medium, the inducer was added to control the dissolved oxygen to 30-40% and ferment for 170 hours.
  • the inducer in the fermentation process is lactose or liquid sugar.
  • the present invention also provides a non-starch polysaccharide enzyme obtained by fermentation of the fermentation method; the non-starch polysaccharide enzyme is one of xylanase, cellulase, ⁇ -glucanase and mannanase or In a mixture of two or more, the ratio of the xylanase to the cellulase is from 1:2 to 3:2.
  • the results of shake flask fermentation showed that the xylanase activity in the supernatant of Trichoderma reesei U1 fermentation was 45 u/ml, the cellulase activity was 57 u/ml, and the ⁇ -glucanase activity was 148 u/ml.
  • the mannanase enzyme activity was 7 u/ml.
  • the xylanase activity in the supernatant of the mutant strain was 102u/ml, which was increased by 126.6% compared with the starting bacteria; the cellulase activity was 95u/ml, which was 66.6% higher than that of the starting bacteria; ⁇ -glucanase activity 229u /ml, which is 54.7% higher than the starting bacteria; the mannanase activity is 25u/ml, It is 257.1% higher than the original bacteria and has achieved unexpected technical effects.
  • Lactose was used as the inducer. Fermentation tank fermentation showed that the xylanase activity in the supernatant of Trichoderma reesei U1 fermentation was 369 u/ml, and the cellulase activity was 607 u/ml, ⁇ -glucan. The enzyme activity was 823 u/ml and the mannanase activity was 21 u/ml.
  • the xylanase activity in the fermentation supernatant of Trichoderma reesei NSP-51 was 1497u/ml, which was 305.6% higher than that of the starting bacteria; the cellulase activity was 935u/ml, which was 54% higher than that of the starting bacteria; ⁇ - The glucanase activity was 1288u/ml, which was 56.5% higher than that of the starting bacteria; the mannanase activity was 89u/ml, which was 323.8% higher than that of the starting bacteria, and unexpected technical effects were obtained.
  • the fermenter fermentation results showed that the xylanase activity in the supernatant of Trichoderma reesei U1 fermentation was 16 u/ml, the cellulase activity was 452 u/ml, and ⁇ -glucan The carbohydrase activity was 599 u/ml and the mannanase activity was 13 u/ml.
  • the xylanase activity of the mutant strain T. reesei NSP-51 was 206u/ml, which was 1187% higher than that of the starting bacteria.
  • the cellulase activity was 894u/ml, which was 97.7% higher than that of the starting bacteria.
  • the glucanase activity was 925u/ml, which was 54.4% higher than that of the starting bacteria; the mannanase activity was 34u/ml, which was 161.5% higher than that of the starting bacteria, and unexpected technical effects were obtained.
  • the biomaterial T. reesei NSP-51 classified as: Trichoderma reesei NSP-51, deposited on July 4, 2016 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.
  • the center address is: Wushan Laoshan Wuhan University, Wuchang, Hubei province; the deposit number is CCTCC NO: M2016363.
  • the invention discloses a mutant strain of Trichoderma reesei and application thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • One aspect of the present invention relates to a mutant strain of Trichoderma reesei NSP-51, which was deposited on July 4, 2016 at the China Center for Type Culture Collection of Wuhan University, Wuhan, China, under the accession number CCTCC. NO: M2016363.
  • the present invention also relates to a novel non-starch polysaccharide enzyme mixture produced by Trichoderma reesei of the CCTCC NO: M2016363.
  • the invention provides a non-starch polysaccharide enzyme mixture produced by Trichoderma reesei of CCTCC NO: M2016363, containing at least xylanase, cellulase, ⁇ -glucanase and mannan Enzyme.
  • the ratio of xylanase to cellulase is 1:2 to 3:2.
  • a method for producing a non-starch polysaccharide enzyme is prepared by fermenting the above-mentioned mutant strain of Trichoderma reesei.
  • the inducer used in the fermentation process is lactose or liquid sugar, and further preferably lactose.
  • the components of the medium used in the fermentation process and the mass ratio thereof are lactose 2%; glucose 1%; corn syrup 1.5%; ammonium sulfate 0.9%; potassium dihydrogen phosphate 2%; diammonium phosphate 0.4%; Magnesium sulfate 0.15%; citric acid 0.073%; calcium chloride 0.12%; ferrous sulfate heptahydrate 0.075%; zinc sulfate heptahydrate 0.006%; copper sulfate pentahydrate 0.0012%; manganese sulfate monohydrate 0.00053%; boric acid 0.0003%.
  • the mutant strain T. reesei NSP-51 obtained by the screening of the invention has a xylanase activity of 102 u/ml in the fermentation supernatant after 5 days of shake flask fermentation, which is increased by 126.6% compared with the starting bacteria; cellulase activity 95u/ml, which was 66.6% higher than the starting bacteria; ⁇ -glucanase activity was 229u/ml, which was 54.7% higher than that of the starting bacteria; the mannanase activity was 25u/ml, which was 257.1% higher than that of the starting bacteria. Further, the applicant verified by fermentation in a 20L fermenter.
  • the xylanase activity in the fermentation supernatant was 1497u/ml after fermentation for 160h, which was 305.6% higher than that of the starting bacteria; Enzyme activity 935u/ml, 54% higher than the original bacteria; ⁇ -glucanase activity 1288u/ml, 56.5% higher than the starting bacteria; mannanase activity 89u/ml, improved than the starting bacteria 323%; by comparing the activity data of liquid sugar as an inducer, the mutant strain T.
  • the mutant strain of Trichoderma reesei can be widely applied to the production of non-starch polysaccharide enzyme, thereby reducing the production cost of non-starch polysaccharide enzyme and promoting the promotion and application of non-starch polysaccharide enzyme in the feed field.
  • the present invention employs conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). . These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can use other conventional methods, experimental solutions and reagents in the art based on the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention.
  • T. reesei mutant strains provided by the present invention and the materials and reagents used in the application thereof are commercially available.
  • the starting strain Trichoderma reesei U1 (this strain was inoculated by the inventor Wu Jiapeng in February 2015 from the soil of Laoshan District, Qingdao) was inoculated into fresh PDA plates and cultured at 30 ° C for 5-7 days. .
  • the fermentation broth was centrifuged, and the obtained supernatant was a crude enzyme solution.
  • the fermentation supernatant was assayed for cellulase, glucanase, xylanase, and mannanase activity, respectively.
  • the results showed that the xylanase activity in the supernatant of Trichoderma reesei U1 fermentation was 45 u/ml, the cellulase activity was 57 u/ml, and the ⁇ -glucanase activity was 148 u/ml.
  • Mannan The enzyme activity was 7 u/ml.
  • 0.5 ml of CMC substrate was added to each of the four tubes, and preheated for 5 min in a 50 ° C water bath with the enzyme solution to be tested.
  • the first three tubes were sample tubes and the fourth tube was blank tubes.
  • 0.5 ml of the test solution was added to each of the first three tubes, and timed, and reacted in a 50 ° C water bath for 15 minutes.
  • the amount of enzyme required to degrade and release 1 ⁇ mol of reducing sugar per minute from a ⁇ -glucan solution having a concentration of 4 mg/ml at 37 ° C and a pH of 5.5 was an enzyme activity unit U.
  • Each of the above-mentioned concentration series of glucose standard solutions was separately taken up to 2.00 ml (two parallels), respectively, and added to a graduated test tube, and then 2.0 ml of an acetic acid-sodium acetate buffer solution and 5.0 ml of a DNS reagent were separately added. Electromagnetic oscillation for 3s ⁇ 5s, heated in boiling water bath for 5min. It was then cooled to room temperature with tap water and made up to 25 ml with water. The standard blank was used as a control to zero, and the absorbance OD value was measured at 540 nm.
  • a standard curve is drawn with the glucose concentration as the Y-axis and the absorbance OD as the X-axis. A standard curve needs to be redrawn each time a new DNS reagent is prepared.
  • X D is the activity of xylanase in the diluted enzyme solution, U/ml
  • a E is the absorbance of the enzyme reaction solution
  • a B is the absorbance of the enzyme blank
  • K is the slope of the standard curve
  • C 0 is the standard The intercept of the curve
  • M is the molar mass of xylose, 150.2 g/mol
  • t is the enzymatic reaction time, min
  • N is the dilution factor of the enzyme solution
  • the amount of enzyme required to release 1 ⁇ mol of reducing sugar per minute from a xylan solution having a concentration of 5 mg/ml at 37 ° C and a pH of 5.5 is an enzyme activity unit U.
  • X D is the activity of xylanase in the diluted enzyme solution, U/ml
  • a E is the absorbance of the enzyme reaction solution
  • a B is the absorbance of the enzyme blank
  • K is the slope of the standard curve
  • C 0 is the standard The intercept of the curve
  • M is the molar mass of xylose, 150.2 g/mol
  • t is the enzymatic reaction time, min
  • N is the dilution factor of the enzyme solution
  • the amount of enzyme required to degrade and release 1 umol of reducing sugar per minute from a 3 mg/ml mannan solution at 37 ° C and a pH of 5.5 is an enzyme activity unit U.
  • the mannose solution (5.5) 1.00, 2.00, 3.00, 4.00, 5.00, 6.00 and 7.00 ml were respectively taken up, and the volume was adjusted to 100 ml with acetic acid-sodium acetate buffer solution to prepare a concentration of 0.10-0.70 mg/ml D-mannose. standard solution.
  • Each of the above concentration series of mannose standard solutions was separately taken up to 2.00 ml (two parallels), and added to a graduated test tube, respectively, and then 2 ml of an acetic acid-sodium acetate buffer solution and 5 ml of DNS reagent were separately added. Electromagnetic oscillation for 3s, heated in boiling water bath for 5min. It was then cooled to room temperature with tap water and made up to 25 ml with water. The standard blank was used as a control to zero, and the absorbance OD value was measured at 540 nm.
  • a standard curve was prepared with the mannose concentration as the Y-axis and the absorbance OD as the X-axis. A standard curve needs to be redrawn each time a new DNS reagent is prepared.
  • X D is the activity of xylanase in the diluted enzyme solution, U/ml
  • a E is the absorbance of the enzyme reaction solution
  • a B is the absorbance of the enzyme blank
  • K is the slope of the standard curve
  • C 0 is the standard The intercept of the curve
  • M is the molar mass of xylose, 150.2 g/mol
  • t is the enzymatic reaction time, min
  • N is the dilution factor of the enzyme solution
  • the starting strain Trichoderma reesei U1 was inoculated on a PDA plate and cultured at 30 ° C for 5-7 days. When the surface of the colony becomes white and a large number of spores are produced, 5 ml of sterile water is aspirated to obtain a spore solution. After centrifugation, it is resuspended in sterile water and counted with a hemocytometer to make the spore concentration about 5 ⁇ 10 7 /ml. .
  • Mutagenesis screening Take a 90 mm sterile Petri dish placed in the rotor, add 10 ml of the diluted spore suspension (concentration: 5 ⁇ 10 7 ), and stir on a magnetic stirrer to keep the spore solution in a uniform state.
  • a sterile ultra-clean workbench use a UV lamp with a power of 9w to illuminate above a vertical distance of 20cm, irradiate for 120s, then place it in the dark for 30min, dilute 10000 times, take 100ul coated PDA plate, and incubate at 30°C for 2-3d. .
  • the fermentation results are shown in Table 1.
  • the xylanase activity in the supernatant of the mutant strain was 102u/ml, which was increased by 126.6% compared with the starting bacteria; the cellulase activity was 95u/ml, which was 66.6% higher than that of the starting bacteria; ⁇ -glucanase activity 229u /ml, which is 54.7% higher than the starting bacteria; the mannanase activity is 25u/ml, which is 257.1% higher than the starting bacteria, and an unexpected technical effect is obtained.
  • the starting strain T. reesei U1 and the mutant T. reesei NSP-51 were inoculated separately into the same shake flask seed medium (glucose 10-30 g/L, potato 100-200 g/L), shaken at 30 ° C, 200 rpm.
  • the fermentation broth was then transferred to a 20 L fermenter (formulation: glucose 2%; corn syrup 1.5%; ammonium sulfate 0.9%; potassium dihydrogen phosphate 2%; diammonium phosphate 0.4%; heptahydrate sulfuric acid) Magnesium 0.15%; citric acid 0.073%; calcium chloride 0.12%; ferrous sulfate heptahydrate 0.075%; zinc sulfate heptahydrate 0.006%; copper sulfate pentahydrate 0.0012%; manganese sulfate monohydrate 0.00053%; boric acid 0.0003%), temperature Both are controlled at 30 ⁇ 1°C, the pH value is controlled at 5.0 ⁇ 0.1, and the stirring speed is 600 rpm. After 24 hours of fermentation in the fermenter, the addition of lactose induces the production of enzymes, and the dissolved oxygen is controlled at 30-40%. About 170 h, a fermentation broth was prepared.
  • the above fermentation broth was centrifuged, and the supernatant was taken. The results are shown in Table 2.
  • the xylanase activity in the supernatant of Trichoderma reesei U1 fermentation was 369 u/ml
  • the cellulase activity was 607 u/ml
  • the ⁇ -glucanase activity was 823 u/ml
  • the mannanase activity was It is 21u/ml.
  • the xylanase activity in the fermentation supernatant of Trichoderma reesei NSP-51 was 1497u/ml, which was 30.6% higher than that of the starting bacteria; the cellulase activity was 935u/ml, which was 54% higher than that of the starting bacteria; ⁇ -Port The glycanase activity was 1288u/ml, which was 56.5% higher than that of the starting bacteria; the mannanase activity was 89u/ml, which was 323.8% higher than that of the starting bacteria, and unexpected technical effects were obtained.
  • the starting strain T. reesei U1 and the mutant T. reesei NSP-51 were inoculated separately into the same shake flask seed medium (glucose 10-30 g/L, potato 100-200 g/L), 30 ° C, 200 rpm shaker
  • the fermentation broth was separately transferred to a 20 L fermentor (formulation: glucose 2%; corn syrup 1.5%; ammonium sulfate 0.9%; potassium dihydrogen phosphate 2%; diammonium phosphate 0.4%; magnesium sulfate heptahydrate) 0.15%; citric acid 0.073%; calcium chloride 0.12%; ferrous sulfate heptahydrate 0.075%; Zinc sulfate heptahydrate 0.006%; copper sulfate pentahydrate 0.0012%; manganese sulfate monohydrate 0.00053%; boric acid 0.0003%), the temperature is controlled at 30 ⁇ 1 ° C, the pH value is controlled at 5.0
  • the above fermentation broth was centrifuged, and the supernatant was taken. The results are shown in Table 3.
  • the xylanase activity in the supernatant of Trichoderma reesei U1 fermentation was 16 u/ml, the cellulase activity was 452 u/ml, and the ⁇ -glucanase activity was 599 u/ml. Mannanase The enzyme activity was 13 u/ml.
  • the xylanase activity of the mutant strain T. reesei NSP-51 was 206u/ml, which was 1187% higher than that of the starting bacteria.
  • the cellulase activity was 894u/ml, which was 97.7% higher than that of the starting bacteria.
  • the glucanase activity was 925u/ml, which was 54.4% higher than that of the starting bacteria; the mannanase activity was 34u/ml, which was 161.5% higher than that of the starting bacteria, and unexpected technical effects were obtained.
  • the expression level of non-starch polysaccharide enzyme components in the mutant strain T. reesei NSP-51 was increased by more than 50% compared with the starting strain U1. Moreover, the inhibitory effect of glucose on xylanase expression is mainly alleviated, and the proportion of xylanase is greatly increased. Under the induction of lactose, the enzyme activity ratio of xylanase to cellulase in the fermentation broth is about 1:2. Raised to about 3:2, and achieved unexpected results.
  • the present invention utilizes mutagenesis technology combined with shake flask screening, and the obtained mutant Trichoderma reesei NSP-51 can greatly increase the yield of non-starch polysaccharide enzyme, and can significantly increase the expression ratio of xylanase, which is beneficial to reduce Production costs and application prospects are extensive.

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Abstract

提供了一种里氏木霉突变菌株及其应用，相比于出发菌株，该里氏木霉突变菌株显著提高了非淀粉多糖酶的产量，可广泛应用于非淀粉多糖酶的生产。

Description

一种里氏木霉突变菌株及其应用
本申请要求于2016年8月18日提交中国专利局、申请号为201610688711.1、发明名称为“一种里氏木霉突变菌株及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及微生物领域，特别涉及一种里氏木霉突变菌株及其应用。
背景技术
非淀粉多糖(non-starchpolysaccharides，NSP)是植物组织中由多种单糖和糖醛酸经糖苷键连接而成，大多为有分支的链状结构，常与无机离子和蛋白质结合在一起，是细胞壁的主要成分，一般难于被单胃动物分泌的消化酶所水解。常见饲料中的非淀粉多糖主要是阿拉伯木聚糖、β-葡聚糖和纤维素。玉米和高粱中含有少量的非淀粉多糖，燕麦和大麦中的水溶性非淀粉多糖主要是β-葡聚糖。谷物中含有少量果胶多糖，除大米外，其他植物中均未发现。谷物副产品含有大量的细胞壁成分，如米糠含有大约20％-25％的非淀粉多糖，主要是等量的阿拉伯木聚糖和纤维素。
非淀粉多糖酶则以多种糖苷酶为主体，通过消除饲料中的非淀粉多糖的抗营养作用，提高动物对饲料养分的利用率，当这些酶活性的适合比例与饲料中非淀粉多糖组成一致时，可获得最佳的使用效果。非淀粉多糖酶包括纤维素酶、木聚糖酶、β-葡聚糖酶、β-甘露聚糖酶、果胶酶等。
纤维素酶可破解富含纤维的细胞壁，使其包含的蛋白质、淀粉等营养物质释放出来并加以利用，同时又可将纤维降解为可被畜禽机体消化吸收的还原糖，从而提高饲料利用率。产生纤维素酶的微生物研究较多的是真菌，对细菌和放线菌研究很少。当前用来生产纤维素酶的微生物主要是木霉、黑曲霉、青霉和根霉，此外，漆斑霉、反当动物瘤胃菌、嗜纤维菌、产黄纤维单抱菌、侧抱菌、粘细菌、梭状芽抱杆菌等也能产生纤维素酶。
木聚糖酶是木聚糖的专一降解酶，属于水解酶类，包括内切木聚糖酶、外切木聚糖酶和木糖苷酶三种。国内外关于木霉、曲霉、细菌产木聚糖酶能力的研究较多，现在商业化的产木聚糖酶的菌株主要是木霉和曲霉属。
β-葡聚糖酶能降解β-葡聚糖分子中的β-1，3和β-1，4糖苷链，使之降解为小分子，失去亲水性和粘性，改变单胃动物肠道内容物的特性、消化酶的活性、肠道微生物的作用环境等。分泌β-葡聚糖酶的微生物，一类是细菌，另一类是真菌，真菌以霉菌为主，主要有康氏木霉、里氏木霉、拟氏木霉、绿色木霉、米曲霉、冻土毛霉、黑曲霉等。
果胶酶是分解果胶的酶的通称，也是一个多酶复合物，它通常包括原果胶酶、果胶甲酷水解酶、果胶酸酶三种酶。这三种酶的联合作用使果胶质得以完全分解。工业生产果胶酶的菌种主要是霉菌，常用菌种有文氏曲霉、苹果青霉、黑曲霉、白腐核菌、米曲霉、酵母等。
甘露聚糖酶是一种半纤维素水解酶,以内切方式降解β-1,4糖苷键,降解产物的非还原末端为甘露糖,其作用底物包括葡萄甘露聚糖、半乳甘露聚糖及β-甘露聚糖等。它不仅能够降低肠道粘度,促进营养物质的消化和吸收,而且还可消除豆类中富含的β-露聚糖对葡萄糖吸收的干扰,极大提高饼粕尤其是豆粕的能量消化率；同时,添加了甘露聚糖酶后动物的抵抗力及整齐度都有所提高。
非淀粉多糖酶的生产主要通过生物发酵法，目前人们寻找的能产生各种非淀粉多糖酶的菌株有绿色木霉、红色木霉、黑曲霉和索状青霉等。其中针对产生纤维素酶的微生物研究较多的是真菌，对细菌和放线菌研究很少，当前用来生产纤维素酶的微生物主要是木霉、黑曲霉、青霉和根霉。国内外关于木霉、曲霉、细菌产木聚糖酶能力的研究较多，现在商业化的产木聚糖酶的菌株主要是木霉和曲霉属。分泌β-葡聚糖酶的微生物，一类是细菌，以芽孢杆菌为主，主要有枯草芽孢杆菌、解淀粉芽孢杆菌、地衣芽孢杆菌等；另一类是真菌，以霉菌为主，主要有康氏木霉、里氏木霉、拟氏木霉、绿色木霉、米曲霉、冻土毛霉、黑曲霉等。工业生产果胶酶的菌种主要是霉菌，常用菌种有文氏曲霉、苹果青霉、 黑曲霉、白腐核菌、米曲霉、酵母等。
我国主要能量饲料资源短缺，而非淀粉多糖极大的限制了谷物及其副产品在饲料中的应用，因此急需针对不同饲粮背景开发高产的非淀粉多糖酶菌株，降低非淀粉多糖酶应用于饲料工业的成本，有效缓解资源短缺问题。
发明内容
有鉴于此，本发明提供一种高产非淀粉多糖酶的里氏木霉突变菌株及其应用。本发明通过紫外诱变的方法获得一株高产非淀粉多糖酶的里氏木霉突变菌株，能大幅度提高非淀粉多糖酶的表达量，可广泛应用于非淀粉多糖酶的生产。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一株里氏木霉，其保藏编号为CCTCC NO:M2016363。
该突变菌株摇瓶发酵上清液中木聚糖酶活102u/ml，比出发菌提高了126.6％；纤维素酶活95u/ml，比出发菌提高了66.6％；β-葡聚糖酶酶活229u/ml，比出发菌提高了54.7％；甘露聚糖酶酶活25u/ml，比出发菌提高了257.1％。以乳糖为诱导物，发酵罐发酵结果显示，突变菌里氏木霉NSP-51发酵上清液中木聚糖酶活1497u/ml，比出发菌提高了305.6％；纤维素酶活935u/ml，比出发菌提高了54％；β-葡聚糖酶酶活1288u/ml，比出发菌提高了56.5％；甘露聚糖酶酶活89u/ml，比出发菌提高了323.8％。以液糖为诱导物，发酵罐发酵结果显示，突变菌里氏木霉NSP-51发酵上清液中木聚糖酶活206u/ml，比出发菌提高了1187％；纤维素酶活894u/ml，比出发菌提高了97.7％；β-葡聚糖酶酶活925u/ml，比出发菌提高了54.4％；甘露聚糖酶酶活34u/ml，比出发菌提高了161.5％。本申请人已于2016年7月4日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M2016363。
本发明还提供了所述里氏木霉在发酵生产非淀粉多糖酶中的应用。
在本发明的一些具体实施方案中，所述非淀粉多糖酶为木聚糖酶，纤维素酶，β-葡聚糖酶和甘露聚糖酶中的一种或两者以上的混合物。
本发明还提供了一种生产非淀粉多糖酶的发酵方法，以所述里氏木霉为发酵菌株。
在本发明的一些具体实施方案中，所述发酵方法包括摇瓶发酵和发酵罐发酵；
所述发酵方法的发酵培养基包括：葡萄糖2质量份；玉米浆1.5质量份；硫酸铵0.9质量份；磷酸二氢钾2质量份；磷酸氢二铵0.4质量份；七水硫酸镁0.15质量份；柠檬酸0.073质量份；氯化钙0.12质量份；七水硫酸亚铁0.075质量份；七水硫酸锌0.006质量份；五水硫酸铜0.0012质量份；一水硫酸锰0.00053质量份；硼酸0.0003质量份。
在本发明的一些具体实施方案中，所述发酵方法中所述摇瓶发酵的发酵温度为30℃，发酵时间为5～7d。
在本发明的一些具体实施方案中，所述发酵方法中所述发酵罐发酵包括摇瓶培养和发酵罐培养；
所述摇瓶培养的培养基包括葡萄糖10-30g/L，土豆100-200g/L；
所述摇瓶培养的条件为于30℃、200rpm摇床培养48h。
在本发明的一些具体实施方案中，所述发酵方法中所述发酵罐培养的条件为：在30±1℃，pH值为5.0±0.1，搅拌速度为600rpm的条件下，将发酵菌株接种于所述的发酵培养基中培养24h后，补加诱导物，控制溶氧为30-40％，发酵170h。
在本发明的一些具体实施方案中，所述发酵方法中所述诱导物为乳糖或液糖。
本发明还提供了所述的发酵方法发酵获得的非淀粉多糖酶；所述非淀粉多糖酶为木聚糖酶，纤维素酶，β-葡聚糖酶和甘露聚糖酶中的一种或两者以上的混合物，所述木聚糖酶与所述纤维素酶的比例为1:2～3:2。
摇瓶发酵培养结果显示，出发菌里氏木霉U1发酵上清液中木聚糖酶活为45u/ml，纤维素酶活为57u/ml，β-葡聚糖酶酶活为148u/ml，甘露聚糖酶酶活为7u/ml。该突变菌株发酵上清液中木聚糖酶活102u/ml，比出发菌提高了126.6％；纤维素酶活95u/ml，比出发菌提高了66.6％；β-葡聚糖酶酶活229u/ml，比出发菌提高了54.7％；甘露聚糖酶酶活25u/ml， 比出发菌提高了257.1％，取得了意料不到的技术效果。
以乳糖为诱导物，发酵罐发酵结果显示，出发菌里氏木霉U1发酵上清液中木聚糖酶酶活为369u/ml，纤维素酶酶活为607u/ml，β-葡聚糖酶酶活为823u/ml，甘露聚糖酶酶活为21u/ml。突变菌里氏木霉NSP-51发酵上清液中木聚糖酶酶活1497u/ml，比出发菌提高了305.6％；纤维素酶酶活935u/ml，比出发菌提高了54％；β-葡聚糖酶酶活1288u/ml，比出发菌提高了56.5％；甘露聚糖酶酶活89u/ml，比出发菌提高了323.8％，取得了意料不到的技术效果。
以液糖为诱导物，发酵罐发酵结果显示，出发菌里氏木霉U1发酵上清液中木聚糖酶酶活为16u/ml，纤维素酶酶活为452u/ml，β-葡聚糖酶酶活为599u/ml，甘露聚糖酶酶活为13u/ml。突变菌里氏木霉NSP-51发酵上清液中木聚糖酶酶活206u/ml，比出发菌提高了1187％；纤维素酶酶活894u/ml，比出发菌提高了97.7％；β-葡聚糖酶酶活925u/ml，比出发菌提高了54.4％；甘露聚糖酶酶活34u/ml，比出发菌提高了161.5％，取得了意料不到的技术效果。
生物保藏说明
生物材料里氏木霉NSP-51，分类命名：里氏木霉NSP-51(Trichoderma reesei NSP-51)，于2016年7月4日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏中心地址为：湖北省武昌珞珈山武汉大学校内；保藏编号为CCTCC NO:M2016363。
具体实施方式
本发明公开了一种里氏木霉突变菌株及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明一方面涉及一种突变菌株里氏木霉NSP-51(Trichoderma reesei NSP-51)，已于2016年7月4日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M2016363。
本发明还涉及一种新的非淀粉多糖酶混合物，是由保藏号为CCTCC NO:M2016363的里氏木霉菌产生的。
本发明提供的一种非淀粉多糖酶混合物，是由保藏号为CCTCC NO:M2016363的里氏木霉菌产生的，至少含有木聚糖酶，纤维素酶，β-葡聚糖酶和甘露聚糖酶。
所述非淀粉多糖酶混合物中，木聚糖酶与纤维素酶的比例为1:2～3:2。
一种生产非淀粉多糖酶的方法，是通过上述里氏木霉突变菌株发酵制备得到的。
所述发酵过程中使用的诱导物为乳糖或液糖，进一步优选乳糖。
所述发酵过程中使用的培养基各组分及其质量比为乳糖2％；葡萄糖1％；玉米浆1.5％；硫酸铵0.9％；磷酸二氢钾2％；磷酸氢二铵0.4％；七水硫酸镁0.15％；柠檬酸0.073％；氯化钙0.12％；七水硫酸亚铁0.075％；七水硫酸锌0.006％；五水硫酸铜0.0012％；一水硫酸锰0.00053％；硼酸0.0003％。
本发明筛选获得的突变菌株里氏木霉NSP-51，其摇瓶发酵5d后，发酵上清液中木聚糖酶酶活102u/ml，比出发菌提高了126.6％；纤维素酶酶活95u/ml，比出发菌提高了66.6％；β-葡聚糖酶酶活229u/ml，比出发菌提高了54.7％；甘露聚糖酶酶活25u/ml，比出发菌提高了257.1％。进一步地，申请人通过20L发酵罐发酵进行验证，当以乳糖为诱导物时，发酵160h后，发酵上清液中木聚糖酶酶活1497u/ml，比出发菌提高了305.6％；纤维素酶酶活935u/ml，比出发菌提高了54％；β-葡聚糖酶酶活1288u/ml，比出发菌提高了56.5％；甘露聚糖酶酶活89u/ml，比出发菌提高了323％；通过对比以液糖为诱导物的酶活数据可知，突变菌株里氏木霉NSP-51，相对出发菌株U1，不仅普遍提高了木聚糖酶、纤维素酶、β-葡聚糖酶和甘露聚糖酶的表达量，并且减轻了葡萄糖对木聚糖酶表达的 抑制作用，大大提高了木聚糖酶的比例，使发酵液中木聚糖酶与纤维素酶的酶活比例从约1:2提高到了约3:2，取得了意料不到的技术效果。所述里氏木霉突变菌株可广泛应用于非淀粉多糖酶的生产，从而有利于降低非淀粉多糖酶的生产成本，促进非淀粉多糖酶在饲料领域中的推广与应用。
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING:A LABORATORY MANUAL,3nd Ed.(Sambrook,2001)和CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Ausubel,2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。
本发明提供的里氏木霉突变菌株及其应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1出发菌里氏木霉摇瓶发酵及酶活检测
将出发菌里氏木霉U1(Trichoderma reesei U1)(该菌株是由发明人之一吴佳鹏于2015年2月筛选自青岛市崂山区土壤中)接种至新鲜的PDA平板，30℃培养5-7d。
割取2cm×2cm大小的菌块，接种到50ml液体摇瓶培养基(乳糖2％；葡萄糖1％；玉米浆1.5％；硫酸铵0.9％；磷酸二氢钾2％；磷酸氢二铵0.4％；七水硫酸镁0.15％；柠檬酸0.073％；氯化钙0.12％；七水硫酸亚铁0.075％；七水硫酸锌0.006％；五水硫酸铜0.0012％；一水硫酸锰0.00053％；硼酸0.0003％)中发酵，30℃培养2天，然后25℃培养3天。培养5天后，离心发酵液，获得的上清液即为粗酶液。将发酵上清液分别进行纤维素酶、葡聚糖酶、木聚糖酶酶、甘露聚糖酶活力测定。结果显示，出发菌里氏木霉U1发酵上清液中木聚糖酶活为45u/ml，纤维素酶活为57u/ml，β-葡聚糖酶酶活为148u/ml，甘露聚糖酶酶活为7u/ml。
1.纤维素酶活力检测
(1)纤维素酶酶活单位的定义
在50℃，pH为4.80条件下(中性为pH6.0)，每分钟从浓度为5mg/ml的羧甲基纤维素钠溶液中降解释放1umol还原糖所需要的酶量为一个活力单位(IU)，还原糖以葡萄糖等量。
(2)酶活测定方法
(2.1)标准曲线的绘制：
取8支试管按下表加入相关试液后再加入1.5ml DNS试剂，充分摇匀，置沸水浴中反应5min。迅速冷却至室温，用水定容至5.0ml，用0号试管试液作为对照，在540nm波长下测其它各试管试液的吸光度。以吸光度为纵坐标，以(葡萄糖含量/100)为横坐标绘制标准曲线。
试管号 0 1 2 3 4 5 6 7
缓冲液加入量(ul) 1000 990 985 980 975 970 965 960
葡萄糖标准液加入量(ul) 0 10 15 20 25 30 35 40
葡萄糖含量(ug) 0 100 150 200 250 300 350 400
(2.2)酶活力测定：
取四支试管各加入0.5ml CMC底物，与待测酶液一起50℃水浴预热5min，前三支为样品试管，第四支为空白管。在前三支试管中各加入0.5ml待测液，并计时，50℃水浴中反应15min。
反应完后在前三支试管中各加入1.5ml的DNS试剂。然后依次向各空白管加入1.5mLDNS，最后依次向空白管中补加0.5ml的待测酶液。
取出并摇匀三支试管后，在沸水浴中反应5min。迅速冷却至室温，用水定到5.0ml。以空白试管试液为对照在540nm波长条件下测定样品管试液的吸光度，吸光度在0.25～0.30之间为宜。若不在此范围，需改变稀释倍数重测。
酶活计算公式：
酶活力(IU/ml或IU/g)＝(葡萄糖等量值/180/15/0.5)×n
式中：180――葡萄糖从微克换算成微摩尔
15――待测液与底物的反应时间
0.5――加入反应的待测酶液量
n――酶样的稀释倍数
2.β－葡聚糖酶活力检测
(1)β－葡聚糖酶酶活单位的定义
在37℃、pH值为5.5的条件下，每分钟从浓度为4mg/ml的β－葡聚糖溶液中降解释放1μmol还原糖所需要的酶量为一个酶活力单位U。
(2)酶活测定方法
(2.1)标准曲线的绘制：
吸取乙酸-乙酸钠缓冲溶液4.0ml，加入DNS试剂5.0ml，沸水浴加热5min。用自来水冷却至室温，用水定容至25.0ml，制成标准空白样。分别吸取葡萄糖溶液1.00ml、2.00ml、3.00ml、4.00ml、5.00ml、6.00ml和7.00ml，分别用乙酸-乙酸钠缓冲溶液定容至100ml，配制成浓度为0.10mg/ml、0.20mg/ml、0.30mg/ml、0.40mg/ml、0.50mg/ml、0.60mg/ml、0.70mg/ml葡萄糖标准溶液。
分别吸取上述浓度系列的葡萄糖标准溶液各2.00ml(做二个平行)，分别加入到刻度试管中，再分别加入2.0ml乙酸—乙酸钠缓冲溶液和5.0ml DNS试剂。电磁振荡3s～5s，沸水浴加热5min。然后用自来水冷却到室温，再用水定容至25ml。以标准空白样为对照调零，在540nm处测定吸光度OD值。
以葡萄糖浓度为Y轴、吸光度OD值为X轴，绘制标准曲线。每次新配制DNS试剂均需要重新绘制标准曲线。
(2.2)酶活力测定：
吸取10.0mlβ－葡聚糖溶液，37℃平衡20min。
吸取10.0ml经过适当稀释的酶液，37℃平衡10min。
吸取2.00ml经过适当稀释的酶液(已经过37℃平衡)，加入到刻度试管中，再加入5ml DNS试剂，电磁振荡3s。然后加入2.0mlβ－葡聚糖溶液，37℃平衡30min，沸水浴加热5min。用自来水冷却至室温，加水定容至25ml，电磁振荡3s～5s。以标准空白样为空白对照，在540nm处测定吸光度AB。
吸取2.00ml经过适当稀释的酶液(已经过37℃平衡)，加入到刻度试管中，再加入2.0mlβ－葡聚糖溶液(已经过37℃平衡)，电磁振荡3s，37℃精确保温30min。加入5.0ml DNS试剂，电磁振荡3s，以终止酶解反应。沸水浴加热5min，用自来水冷却至室温，加水定容至25ml，电磁振荡3s。以标准空白样为空白对照，在540nm处测定吸光度AE。
酶活计算公式：
Figure PCTCN2017081986-appb-000001
式中：XD为稀释酶液中木聚糖酶的活力，U/ml；AE为酶反应液的吸光度；AB为酶空白液的吸光度；K为标准曲线的斜率；C0为标准曲线的截距；M为木糖的摩尔质量，150.2g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子，1mmol＝1000μmol。
3.木聚糖酶活力检测
(1)木聚糖酶酶活单位的定义
在37℃、pH值为5.5的条件下，每分钟从浓度为5mg/ml的木聚糖溶液中释放1μmol还原糖所需要的酶量即为一个酶活力单位U。
(2)酶活测定方法
取2ml浓度为1％的木聚糖底物(pH5.5乙酸-乙酸钠缓冲液配制)，加入到比色管中，37℃平衡10min，再加入2ml经pH5.5乙酸-乙酸钠缓冲液适当稀释并经37℃平衡好的酸性木聚糖酶酶液，混匀于37℃精确保温反应30min。反应结束后，加入5ml DNS试剂，混匀以终止反应。然后沸水浴煮沸5min，用自来水冷却至室温，加蒸馏水定容至25ml，混匀后，以标准空白样为空白对照，在540nm处测定吸光值AE。
酶活计算公式：
Figure PCTCN2017081986-appb-000002
式中：XD为稀释酶液中木聚糖酶的活力，U/ml；AE为酶反应液的吸光度；AB为酶空白液的吸光度；K为标准曲线的斜率；C0为标准曲线的截距；M为木糖的摩尔质量，150.2g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子，1mmol＝1000μmol。
4.甘露聚糖酶活力检测
(1)甘露聚糖酶酶活单位的定义
在37℃、pH值为5.5的条件下，每分钟从浓度为3mg/ml的甘露聚糖溶液中降解释放1umol还原糖所需要的酶量为一个酶活力单位U。
(2)酶活测定方法
(2.1)标准曲线的绘制：
吸取乙酸-乙酸钠缓冲溶液4.0ml，加入DNS试剂5.0ml，沸水浴加热5min。用自来水冷却至室温，用水定容至25.0ml，制成标准空白样。
分别吸取甘露糖溶液(5.5)1.00、2.00、3.00、4.00、5.00、6.00和7.00ml，分别用乙酸-乙酸钠缓冲溶液定容至100ml，配制成浓度为0.10—0.70mg/ml D-甘露糖标准溶液。
分别吸取上述浓度系列的甘露糖标准溶液各2.00ml(做二个平行)，分别加入到刻度试管中，再分别加入2ml乙酸-乙酸钠缓冲溶液和5mlDNS试剂。电磁振荡3s，沸水浴加热5min。然后用自来水冷却到室温，再用水定容至25ml。以标准空白样为对照调零，在540nm处测定吸光度OD值。
以甘露糖浓度为Y轴、吸光度OD值为X轴，绘制标准曲线。每次新配制DNS试剂均需要重新绘制标准曲线。
(2.2)酶活力测定：
吸取10.0ml甘露聚糖溶液，37℃平衡10min。
吸取10.0ml经过适当稀释的酶液，37℃平衡10min。
吸取2.00ml经过适当稀释的酶液(已经过37℃平衡)，加入到刻度试管中，再加入5mlDNS试剂，电磁振荡3s。然后加入2.0ml甘露聚糖溶液，37℃保温30min，沸水浴加热5min。用自来水冷却至室温，加水定容至25ml，电磁振荡3s。以标准空白样为空白对照，在540nm处测定吸光度AB。
吸取2.0ml经过适当稀释的酶液(已经过37℃平衡)，加入到刻度试管中，再加入2.0ml甘露聚糖溶液(已经过37℃平衡)，电磁振荡3s，37℃精确保温30min。加入5.0mlDNS试剂，电磁振荡3s，酶解反应。沸水浴加热5min，用自来水冷却至室温，加水定容至25ml，电磁振荡3s。以标 准空白样为空白对照，在540nm处测定吸光度AE。
酶活计算公式：
Figure PCTCN2017081986-appb-000003
式中：XD为稀释酶液中木聚糖酶的活力，U/ml；AE为酶反应液的吸光度；AB为酶空白液的吸光度；K为标准曲线的斜率；C0为标准曲线的截距；M为木糖的摩尔质量，150.2g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子，1mmol＝1000μmol。
5液糖的制备方法
称取500g葡萄糖到1L烧杯中，加蒸馏水到约900mL，全部溶解后，加入25mL85％磷酸，然后定容到1L，在灭菌锅中121℃处理30min。得到的棕色溶液即为液糖。
实施例2紫外诱变与突变菌筛选
确定致死率：将出发菌株里氏木霉U1接种于PDA平板，30℃培养5-7d。待菌落表面变白，产生大量孢子时，吸取5ml无菌水洗脱，获得孢子液，离心后用无菌水重悬，用血球计数板计数，使孢子浓度约为5×107个/ml。取一个放入转子的90mm无菌培养皿，加入10ml稀释好的孢子悬液，在磁力搅拌器上搅拌使孢子液处于均匀状态。在无菌超净工作台中，用功率为9w的紫外灯于垂直距离20cm的上方照射，分别照射60s、90s、120s、150s、180s，取照射后的孢子液稀释10000倍，取100ul涂布PDA平板，30℃培养2-3d后计数，以未照射的孢子液为对照，计算致死率。其中照射120s时，致死率为90％，选取该照射时间进行后续诱变实验。
诱变筛选：取一个放入转子的90mm无菌培养皿，加入10ml稀释好的孢子悬液(浓度为5×107)，在磁力搅拌器上搅拌使孢子液处于均匀状态。在无菌超净工作台中，用功率为9w的紫外灯于垂直距离20cm的上方照射，照射120s，然后黑暗条件下放置30min，稀释10000倍，取100ul涂布PDA平板，30℃培养2-3d。
共涂布150块PDA平板，30℃培养2-3d后，每个平板长出约50个菌落，先通过观察菌落形态，挑取菌落形态较小、菌丝体致密的突变体 200个接种到PDA平板，30℃培养5-7d。选取生长状态正常的菌落150个，每个突变体菌落割取2cm×2cm大小的菌块，分别接种于50ml液体摇瓶培养基(乳糖2％；葡萄糖1％；玉米浆1.5％；硫酸铵0.9％；磷酸二氢钾2％；磷酸氢二铵0.4％；七水硫酸镁0.15％；柠檬酸0.073％；氯化钙0.12％；七水硫酸亚铁0.075％；七水硫酸锌0.006％；五水硫酸铜0.0012％；一水硫酸锰0.00053％；硼酸0.0003％)中发酵，30℃培养2天，然后25℃培养3天；离心菌体获得上清液即为粗酶液。通过对获得的粗酶液进行NSP酶酶活力检测，最终筛选出一株NSP酶产量最高的突变菌株，命名为里氏木霉NSP-51(Trichoderma reesei NSP-51)。
发酵结果见表1。该突变菌株发酵上清液中木聚糖酶活102u/ml，比出发菌提高了126.6％；纤维素酶活95u/ml，比出发菌提高了66.6％；β-葡聚糖酶酶活229u/ml，比出发菌提高了54.7％；甘露聚糖酶酶活25u/ml，比出发菌提高了257.1％，取得了意料不到的技术效果。
表1
Figure PCTCN2017081986-appb-000004
申请人已于2016年7月4日将上述突变菌株里氏木霉NSP-51(Trichoderma reesei NSP-51)保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M2016363。
实施例3 20L发酵罐发酵验证
1、以乳糖为诱导物
将出发菌里氏木霉U1和突变菌里氏木霉NSP-51分别接种于相同的摇瓶种子培养基(葡萄糖10-30g/L，土豆100-200g/L)，30℃，200rpm摇 床培养48h之后，然后分别将发酵液转入20L发酵罐(配方为：葡萄糖2％；玉米浆1.5％；硫酸铵0.9％；磷酸二氢钾2％；磷酸氢二铵0.4％；七水硫酸镁0.15％；柠檬酸0.073％；氯化钙0.12％；七水硫酸亚铁0.075％；七水硫酸锌0.006％；五水硫酸铜0.0012％；一水硫酸锰0.00053％；硼酸0.0003％)，温度均控制在30±1℃，pH值均控制在5.0±0.1，搅拌速度为600rpm，在发酵罐培养24h之后，开始补加乳糖诱导菌体产酶，溶氧控制在30-40％，发酵时间约为170h，制成发酵菌液。
将上述发酵菌液离心，取上清，结果见表2。出发菌里氏木霉U1发酵上清液中木聚糖酶活为369u/ml，纤维素酶活为607u/ml，β-葡聚糖酶酶活为823u/ml，甘露聚糖酶酶活为21u/ml。突变菌里氏木霉NSP-51发酵上清液中木聚糖酶活1497u/ml，比出发菌提高了305.6％；纤维素酶活935u/ml，比出发菌提高了54％；β-葡聚糖酶酶活1288u/ml，比出发菌提高了56.5％；甘露聚糖酶酶活89u/ml，比出发菌提高了323.8％，取得了意料不到的技术效果。
表2
Figure PCTCN2017081986-appb-000005
2、以液糖为诱导物
将出发菌里氏木霉U1和突变菌里氏木霉NSP-51分别接种于相同的摇瓶种子培养基(葡萄糖10-30g/L，土豆100-200g/L)，30℃，200rpm摇床培养48h之后，然后分别将发酵液转入20L发酵罐(配方为：葡萄糖2％；玉米浆1.5％；硫酸铵0.9％；磷酸二氢钾2％；磷酸氢二铵0.4％；七水硫酸镁0.15％；柠檬酸0.073％；氯化钙0.12％；七水硫酸亚铁0.075％； 七水硫酸锌0.006％；五水硫酸铜0.0012％；一水硫酸锰0.00053％；硼酸0.0003％)，温度均控制在30±1℃，pH值均控制在5.0±0.1，搅拌速度为600rpm，在发酵罐培养24h之后，开始补加液糖诱导菌体产酶，溶氧控制在30-40％，发酵时间约为170h，制成发酵菌液。
将上述发酵菌液离心，取上清，结果见表3。出发菌里氏木霉U1发酵上清液中木聚糖酶酶活为16u/ml，纤维素酶酶活为452u/ml，β-葡聚糖酶酶活为599u/ml，甘露聚糖酶酶活为13u/ml。突变菌里氏木霉NSP-51发酵上清液中木聚糖酶酶活206u/ml，比出发菌提高了1187％；纤维素酶酶活894u/ml，比出发菌提高了97.7％；β-葡聚糖酶酶活925u/ml，比出发菌提高了54.4％；甘露聚糖酶酶活34u/ml，比出发菌提高了161.5％，取得了意料不到的技术效果。
表3
Figure PCTCN2017081986-appb-000006
通过对比乳糖和液糖作为诱导物的发酵酶活数据可知，与出发菌U1相比，突变菌株里氏木霉NSP-51中非淀粉多糖酶各组分的表达量均提高了50％以上，并且主要减轻了葡萄糖对木聚糖酶表达的抑制作用，大大提高了木聚糖酶的比例，在乳糖诱导下，发酵液中木聚糖酶与纤维素酶的酶活比例从约1:2提高到了约3:2，取得了意料不到的效果。
综上，本发明利用诱变技术结合摇瓶筛选，获得的突变菌里氏木霉NSP-51能大幅提高非淀粉多糖酶的产量，并且能显著提高木聚糖酶的表达比例，有利于降低生产成本，应用前景广泛。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普 通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 里氏木霉，其特征在于，其保藏编号为CCTCC NO:M2016363。
  2. 根据权利要求1所述的里氏木霉在发酵生产非淀粉多糖酶中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述非淀粉多糖酶为木聚糖酶，纤维素酶，β-葡聚糖酶和甘露聚糖酶中的一种或两者以上的混合物。
  4. 一种生产非淀粉多糖酶的发酵方法，其特征在于，以如权利要求1所述的里氏木霉为发酵菌株。
  5. 根据权利要求4所述的发酵方法，其特征在于，发酵方法包括摇瓶发酵和发酵罐发酵；
    所述发酵方法的发酵培养基包括：葡萄糖2质量份；玉米浆1.5质量份；硫酸铵0.9质量份；磷酸二氢钾2质量份；磷酸氢二铵0.4质量份；七水硫酸镁0.15质量份；柠檬酸0.073质量份；氯化钙0.12质量份；七水硫酸亚铁0.075质量份；七水硫酸锌0.006质量份；五水硫酸铜0.0012质量份；一水硫酸锰0.00053质量份；硼酸0.0003质量份。
  6. 根据权利要求4或5所述的发酵方法，其特征在于，所述摇瓶发酵的发酵温度为30℃，发酵时间为5～7d。
  7. 根据权利要求4至6任一项所述的发酵方法，其特征在于，所述发酵罐发酵包括摇瓶培养和发酵罐培养；
    所述摇瓶培养的培养基包括葡萄糖10-30g/L，土豆100-200g/L；
    所述摇瓶培养的条件为于30℃、200rpm摇床培养48h。
  8. 根据权利要求7所述的发酵方法，其特征在于，所述发酵罐培养的条件为：在30±1℃，pH值为5.0±0.1，搅拌速度为600rpm的条件下，将发酵菌株接种于如权利要求5所述的发酵培养基中培养24h后，补加诱导物，控制溶氧为30-40％，发酵170h。
  9. 根据权利要求8所述的发酵方法，其特征在于，所述诱导物为乳糖或液糖。
  10. 根据权利要求4至9任一项所述的发酵方法发酵获得的非淀粉多糖酶；所述非淀粉多糖酶为木聚糖酶，纤维素酶，β-葡聚糖酶和甘露聚糖酶中的一种或两者以上的混合物，所述木聚糖酶与所述纤维素酶的比例为1:2～3:2。
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