WO2018032797A1 - 一种黑曲霉突变菌株及其应用 - Google Patents

一种黑曲霉突变菌株及其应用 Download PDF

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WO2018032797A1
WO2018032797A1 PCT/CN2017/081985 CN2017081985W WO2018032797A1 WO 2018032797 A1 WO2018032797 A1 WO 2018032797A1 CN 2017081985 W CN2017081985 W CN 2017081985W WO 2018032797 A1 WO2018032797 A1 WO 2018032797A1
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fermentation
enzyme
aspergillus niger
mass
activity
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PCT/CN2017/081985
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French (fr)
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徐晓东
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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Publication of WO2018032797A1 publication Critical patent/WO2018032797A1/zh

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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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    • C12N9/2402—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)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N9/248—Xylanases
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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
    • C12N9/248—Xylanases
    • C12N9/2485—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1,3-beta-xylanase
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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
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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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)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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    • 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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    • C12Y302/00—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)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
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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)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
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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)
    • C12Y302/01072—Xylan 1,3-beta-xylosidase (3.2.1.72)
    • C—CHEMISTRY; METALLURGY
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

Definitions

  • the invention relates to the field of microorganisms, in particular to a mutant strain of Aspergillus niger 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 Aspergillus niger with high yield of non-starch polysaccharide enzyme and application thereof.
  • the invention obtains a strain of Aspergillus niger which is 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 Aspergillus niger, the preservation number of which is CCTCC NO: M2016362.
  • the invention obtains a mutant strain with the highest NSP enzyme yield by ultraviolet mutagenesis, and is named as Aspergillus niger NSP-5.
  • the xylanase activity of the strain in the shake flask fermentation supernatant is 106u. /ml, 96% higher than the starting bacteria; cellulase activity 4.4u / ml, 91% higher than the starting bacteria; ⁇ -glucanase enzyme activity 41.4u / ml, 55% higher than the starting bacteria;
  • the mannanase activity was 1.9 u/ml, which was 175% higher than that of the starting bacteria.
  • Fermentation tank fermentation experiment showed that the xylanase activity of the fermentation supernatant of the mutant Aspergillus niger NSP-5 was 205u/ml, which was 107% higher than that of the starting bacteria; the cellulase activity was 17.6u/ml, which was better than the starting bacteria. Increased by 69%; ⁇ -glucanase activity 67.7u/ml, 78% higher than the original bacteria; mannanase activity 3.9u/ml, 160% higher than the starting bacteria, the expression of each component Compared with the shake flask, the amplification effect is obvious.
  • the applicant of the present invention deposited the above-mentioned mutant strain Aspergillus niger NSP-5 on the Chinese typical culture collection center of Wuhan University, Wuhan, China on July 4, 2016, and the deposit number is CCTCC NO: M2016362.
  • the invention also provides the use of the Aspergillus niger in 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 invention also provides a fermentation method for producing non-starch polysaccharide enzyme, the Aspergillus niger For the 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 wheat bran; 0.3 parts by mass of lactose; 2 parts by mass of corn syrup; 0.5 parts by mass of ammonium sulfate; 0.35 parts by mass of potassium dihydrogen phosphate; 0.75 parts by mass of dipotassium hydrogen phosphate; Water magnesium sulfate 0.03 parts by mass.
  • the fermentation temperature of the shake flask fermentation in the fermentation process is 30 ° C, and the fermentation time is 5 d.
  • 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 cultured at 30 ° C, 200 rpm shaker for 20 h.
  • the fermentation tank is cultured under the conditions of: inoculation of the fermentation strain at 30 ⁇ 1° C., pH 5.0 ⁇ 0.2, and stirring speed of 600 rpm. After culturing for 24 hours in the fermentation medium according to claim 5, xylose + glucose reverse syrup is added to control dissolved oxygen to 30-40%, and fermentation is performed for 160 hours.
  • the method for preparing the xylose+glucose reverse syrup in the fermentation method is: taking xylose 25%, glucose 25%, 85% phosphoric acid 5% v/v, 121 ° C Processed for 30 min.
  • 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 a mixture of more than two.
  • the results of shake flask experiments showed that the xylanase activity in the fermentation supernatant of Aspergillus niger NSP-1 was 54 u/ml, the cellulase activity was 2.3 u/ml, and the ⁇ -glucanase activity was 26.6 u/ The enzyme activity of ml, mannanase was 0.69 u/ml.
  • the invention obtains a mutant strain with the highest NSP enzyme yield, named as Aspergillus niger NSP-5, and the xylanase activity of the strain in the fermentation supernatant is 106 u/ml, which is higher than that of the starting bacteria.
  • the applicant of the present invention deposited the above-mentioned mutant strain Aspergillus niger NSP-5 on the Chinese typical culture collection center of Wuhan University, Wuhan, China on July 4, 2016, and the deposit number is CCTCC NO: M2016362.
  • the fermentation experiment results of the fermenter showed that the xylanase activity of the fermentation supernatant of the starting bacteria was 99 u/ml, the cellulase activity was 10.4 u/ml, and the ⁇ -glucanase activity was 37.9 u/ml.
  • the mannanase enzyme activity was 1.5 u/ml.
  • the xylanase activity of the fermentation supernatant of the mutant Aspergillus niger NSP-5 was 205u/ml, which was 107% higher than that of the starting bacteria; the cellulase activity was 17.6u/ml, which was 69% higher than that of the starting bacteria; ⁇ - The glucanase activity was 67.7u/ml, which was 78% higher than that of the original bacteria; the mannanase activity was 3.9u/ml, which was 160% higher than the starting bacteria.
  • the expression of each component was amplified compared with the shake flask. Obviously, unexpected technical effects have been achieved.
  • the biomaterial Aspergillus niger NSP-5 named as follows: Aspergillus niger NSP-5, deposited on July 4, 2016 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China.
  • the deposit center is: Within the Wuhan University of Wushan, Wuchang, Hubei province; the deposit number is CCTCC NO: M2016362.
  • the invention discloses a mutant strain of Aspergillus niger 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 novel mutant strain Aspergillus niger NSP-5, 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: M2016362.
  • the components of the medium used in the fermentation process and the mass ratio thereof are wheat bran 2%; lactose 0.3%; corn syrup 2%; ammonium sulfate 0.5%; potassium dihydrogen phosphate 0.35%; dipotassium hydrogen phosphate 0.75% ; Magnesium sulfate heptahydrate 0.03%.
  • the mutant strain Aspergillus niger NSP-5 obtained by the present invention was cultured for 5 days in a shake flask, and the xylanase activity in the fermentation supernatant was 106 u/ml, which was 96% higher than that of the starting bacteria; the cellulase activity was 4.4. u/ml, which is 91% higher than the starting bacteria; ⁇ -glucanase activity is 41.4u/ml, which is 55% higher than the starting bacteria; the mannanase activity is 1.9u/ml, which is higher than the starting bacteria. 175%, and achieved unexpected technical results.
  • the fermentation experiment results of the fermenter showed that the xylanase activity of the fermentation supernatant of the starting bacteria was 99 u/ml, the cellulase activity was 10.4 u/ml, and the ⁇ -glucanase activity was 37.9 u/ml.
  • the mannanase enzyme activity was 1.5 u/ml.
  • the xylanase activity of the fermentation supernatant of the mutant Aspergillus niger NSP-5 was 205u/ml, which was 107% higher than that of the starting bacteria; the cellulase activity was 17.6u/ml, which was 69% higher than that of the starting bacteria; ⁇ - The glucanase activity was 67.7u/ml, which was 78% higher than that of the original bacteria; the mannanase activity was 3.9u/ml, which was 160% higher than the starting bacteria.
  • the expression of each component was amplified compared with the shake flask. Obviously, unexpected technical effects have been achieved.
  • the Aspergillus niger mutant strain can be widely applied to the production of non-starch polysaccharide enzyme, thereby contributing to 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.
  • the Aspergillus niger mutant strain provided by the present invention and the materials and reagents used in the application thereof are commercially available.
  • Example 1 Aspergillus niger shake flask fermentation and enzyme activity detection
  • the starting strain Aspergillus niger NSP-1 (the strain is invented by the invention)
  • One of the people, Xu Xiaodong was screened from the soil of Laoshan District, Qingdao in January 2015, and inoculated into fresh PDA plates and cultured at 30 °C for 5-7 days.
  • 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.
  • Each Newly formulated DNS reagents require a redrawing of the standard curve.
  • 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), respectively, and added to a graduated test tube, and then 2 ml of an acetic acid-sodium acetate buffer solution and 5 ml of a 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 Aspergillus niger NSP-1 was inoculated on a PDA plate and cultured at 30 ° C for 5-7 days. When the surface of the colony turns black and a large amount of spores are produced, 5 ml of sterile water is aspirated to obtain a spore solution, which is centrifuged, resuspended in sterile water, and counted using a hemocytometer. A 90 mm culture dish was taken, 5 ml of the diluted spore suspension (concentration of about 1 ⁇ 10 7 /mL) was added, and the rotor was added and stirred on a magnetic stirrer to make the spore liquid in a uniform state.
  • a UV lamp with a power of 9w to illuminate above a vertical distance of 20cm, respectively, for 30s, 45s, 60s, 75s, 90s, 105s, 120s, and dilute the spore solution after irradiation for 10, 100, 1000 times, 100 ul coated PDA plates were taken, and cultured at 30 ° C for 2-3 days, and counted, and the undetected spore solution was used as a control to calculate the lethality.
  • the mortality rate was 95%, and the irradiation time was selected for subsequent mutagenesis experiments.
  • Mutagenesis screening Take a 90 mm culture dish, add 5 ml of diluted spore suspension (concentration of 1 ⁇ 10 7 ), add the rotor and stir on a magnetic stirrer to make the spore liquid 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 20 cm, dilute 1000 times after 90 s, take 100 ul of coated PDA plates, and incubate at 30 ° C for 2-3 d.
  • Each transformant was cut into 2cm ⁇ 2cm size pieces and inoculated separately into 50ml liquid shake flask medium (wheat bran 2%; lactose 0.3%; corn syrup 2%; ammonium sulfate 0.5%; potassium dihydrogen phosphate 0.35%) ; Dipotassium hydrogen phosphate 0.75%; magnesium sulfate heptahydrate 0.03%) Fermentation, cultured at 30 ° C for 5 days, the supernatant was centrifuged to obtain the crude enzyme solution. The NSP enzyme activity was detected by the obtained crude enzyme solution.
  • the applicant finally screened a mutant strain with the highest NSP enzyme yield, named Aspergillus niger NSP-5, and the xylanase activity of the strain in the fermentation supernatant (see Table 1) 106u/ Ml, 96% higher than the starting bacteria; cellulase activity 4.4u / ml, 91% higher than the starting bacteria; ⁇ -glucanase enzyme activity 41.4u / ml, 55% higher than the starting bacteria; The activity of glycanase was 1.9 u/ml, which was 175% higher than that of the original bacteria. Compared with the original strain, the mutant strain was extremely significant (P ⁇ 0.01), which increased the NSP enzyme production and achieved unexpected technical effects.
  • the starting strain NSP-1 and the mutant Aspergillus niger NSP-5 were respectively inoculated into the same shake flask seed medium (glucose 10-30 g/L, potato 100-200 g/L), and cultured at 30 ° C for 20 h on a shaker at 200 rpm.
  • the xylose+glucose reverse syrup is added (the formula is: Xylose 25%, glucose 25%, 85% phosphoric acid 5% v / v, treated at 121 ° C for 30 min) induced enzyme production, dissolved oxygen control at 30-40%, fermentation time is about 160h, made into fermentation bacteria liquid.
  • the fermentation broth was centrifuged, and the supernatant was taken for detection of NSP enzyme activity.
  • the results are shown in Table 2.
  • the xylanase activity of the fermentation supernatant of the starting bacteria was 99 u/ml
  • the cellulase activity was 10.4 u/ml
  • the ⁇ -glucanase activity was 37.9 u/ml.
  • the glycanase enzyme activity was 1.5 u/ml.
  • the xylanase activity of the fermentation supernatant of the mutant Aspergillus niger NSP-5 was 205u/ml, which was 107% higher than that of the starting bacteria; the cellulase activity was 17.6u/ml, which was 69% higher than that of the starting bacteria; ⁇ - The glucanase activity was 67.7u/ml, which was 78% higher than that of the original bacteria; the mannanase activity was 3.9u/ml, which was 160% higher than the starting bacteria.
  • the expression of each component was amplified compared with the shake flask. Obvious (P ⁇ 0.01), unexpected technical effects were achieved.

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Abstract

提供了一种黑曲霉突变菌株及其应用，该黑曲霉突变菌株可用于非淀粉多糖酶的生产，相比于出发菌株，该突变菌株显著提高了非淀粉多糖酶的产量。

Description

一种黑曲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201610701711.0、发明名称为“一种黑曲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，特别涉及一种黑曲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，本发明提供了一种高产非淀粉多糖酶的黑曲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黑曲霉突变菌株，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：
本发明提供了一株黑曲霉，其保藏编号为CCTCC NO:M2016362。
本发明通过紫外诱变的方法获得一株NSP酶产量最高的突变菌株，命名为黑曲霉NSP-5(Aspergillus niger NSP-5)，该菌株摇瓶发酵上清液中木聚糖酶酶活106u/ml，比出发菌提高了96％；纤维素酶酶活4.4u/ml，比出发菌提高了91％；β-葡聚糖酶酶活41.4u/ml，比出发菌提高了55％；甘露聚糖酶酶活1.9u/ml，比出发菌提高了175％。发酵罐发酵实验结果显示：突变菌黑曲霉NSP-5的发酵上清液木聚糖酶酶活205u/ml，比出发菌提高了107％；纤维素酶酶活17.6u/ml，比出发菌提高了69％；β-葡聚糖酶酶活67.7u/ml，比出发菌提高了78％；甘露聚糖酶酶活3.9u/ml，比出发菌提高了160％，各组分表达量与摇瓶相比放大作用明显。本发明申请人已于2016年7月4日将上述突变菌株黑曲霉NSP-5(Aspergillus niger NSP-5)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:M2016362。
本发明还提供了所述黑曲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，以所述黑曲霉 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小麦麸皮2质量份；乳糖0.3质量份；玉米浆2质量份；硫酸铵0.5质量份；磷酸二氢钾0.35质量份；磷酸氢二钾0.75质量份；七水硫酸镁0.03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5d。
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20h。
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±0.2，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，补加木糖+葡萄糖反转糖浆，控制溶氧为30-40％，发酵160h。
在本发明的一些具体实施方案中，所述发酵方法中所述木糖+葡萄糖反转糖浆的制备方法为：取木糖25％，葡萄糖25％，85％磷酸5％v/v，121℃处理30min。
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。
摇瓶实验结果显示：出发菌黑曲霉NSP-1发酵上清液中木聚糖酶活为54u/ml，纤维素酶活为2.3u/ml，β-葡聚糖酶酶活为26.6u/ml，甘露聚糖酶酶活为0.69u/ml。本发明获得一株NSP酶产量最高的突变菌株，命名为黑曲霉NSP-5(Aspergillus niger NSP-5)，该菌株发酵上清液中木聚糖酶酶活106u/ml，比出发菌提高了96％；纤维素酶酶活4.4u/ml，比出发菌提高了91％；β-葡聚糖酶酶活41.4u/ml，比出发菌提高了55％；甘露聚糖酶酶活1.9u/ml，比出发菌提高了175％，取得了意料不到的技术效果。 本发明申请人已于2016年7月4日将上述突变菌株黑曲霉NSP-5(Aspergillus niger NSP-5)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:M2016362。
发酵罐发酵实验结果显示：出发菌的发酵上清液木聚糖酶酶活为99u/ml，纤维素酶酶活为10.4u/ml，β-葡聚糖酶酶活为37.9u/ml，甘露聚糖酶酶活为1.5u/ml。突变菌黑曲霉NSP-5的发酵上清液木聚糖酶酶活205u/ml，比出发菌提高了107％；纤维素酶酶活17.6u/ml，比出发菌提高了69％；β-葡聚糖酶酶活67.7u/ml，比出发菌提高了78％；甘露聚糖酶酶活3.9u/ml，比出发菌提高了160％，各组分表达量与摇瓶相比放大作用明显，取得了意想不到的技术效果。
生物保藏说明
生物材料黑曲霉NSP-5，分类命名：黑曲霉NSP-5(Aspergillus niger NSP-5)，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:M2016362。
具体实施方式
本发明公开了一种黑曲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新的突变菌株黑曲霉NSP-5(Aspergillus niger NSP-5)，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:M2016362。
所述发酵过程中使用的培养基各组分及其质量比为小麦麸皮2％；乳糖0.3％；玉米浆2％；硫酸铵0.5％；磷酸二氢钾0.35％；磷酸氢二钾0.75％； 七水硫酸镁0.03％。
本发明筛选获得的突变菌株黑曲霉NSP-5，摇瓶培养5天后，发酵上清液中木聚糖酶酶活为106u/ml，比出发菌提高了96％；纤维素酶酶活为4.4u/ml，比出发菌提高了91％；β-葡聚糖酶酶活为41.4u/ml，比出发菌提高了55％；甘露聚糖酶酶活为1.9u/ml，比出发菌提高了175％，取得了意料不到的技术效果。
发酵罐发酵实验结果显示：出发菌的发酵上清液木聚糖酶酶活为99u/ml，纤维素酶酶活为10.4u/ml，β-葡聚糖酶酶活为37.9u/ml，甘露聚糖酶酶活为1.5u/ml。突变菌黑曲霉NSP-5的发酵上清液木聚糖酶酶活205u/ml，比出发菌提高了107％；纤维素酶酶活17.6u/ml，比出发菌提高了69％；β-葡聚糖酶酶活67.7u/ml，比出发菌提高了78％；甘露聚糖酶酶活3.9u/ml，比出发菌提高了160％，各组分表达量与摇瓶相比放大作用明显，取得了意想不到的技术效果。
所述黑曲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。
本发明提供的黑曲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出发菌黑曲霉摇瓶发酵及酶活检测
将出发菌黑曲霉NSP-1(Aspergillus niger NSP-1)(该菌株是由发明 人之一徐晓东于2015年1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，接种至50ml液体摇瓶培养基(小麦麸皮2％；乳糖0.3％；玉米浆2％；硫酸铵0.5％；磷酸二氢钾0.35％；磷酸氢二钾0.75％；七水硫酸镁0.03％)中发酵，30℃培养5天；将发酵液进行离心，获得的上清液即为粗酶液；对上清液分别进行纤维素酶、葡聚糖酶、木聚糖酶酶、甘露聚糖酶的酶活力测定。结果显示，出发菌黑曲霉NSP-1发酵上清液中木聚糖酶活为54u/ml，纤维素酶活为2.3u/ml，β-葡聚糖酶酶活为26.6u/ml，甘露聚糖酶酶活为0.69u/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1.5mlDNS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：
XD＝[(AE-AB)×K+C0]×N×1000÷(M×t)
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：
XD＝[(AE-AB)×K+C0]×N×1000÷(M×t)
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：
XD＝[(AE-AB)×K+C0]×N×1000÷(M×t)
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。
实施例2紫外诱变与突变菌筛选
确定致死率：将上述黑曲霉NSP-1接种于PDA平板，30℃培养5-7d。待菌落表面变黑，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。取一个90mm培养皿，加入5ml稀释好的孢子悬液(浓度约为1×107个/mL)，加入转子并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30s、45s、60s、75s、90s、105s、120s，取照射后的孢子液稀释10、100、1000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95％，选取该照射时间进行后续诱变实验。
诱变筛选：取一个90mm培养皿，加入5ml稀释好的孢子悬液(浓度为1×107)，加入转子并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90s后稀释1000倍，取100ul涂布PDA平板，30℃培养2-3d。
共涂布200块PDA平板，30℃培养2-3d后，每个平板长出30-50个菌落，先通过菌落形态，筛选短分枝的突变体，挑取菌落形态较小、菌丝致密、菌落周围绒毛较短的突变体156个接种到PDA平板，30℃培养5-7d。每个转化子割取2cm×2cm大小的菌块，分别接种于50ml液体摇瓶培养基(小麦麸皮2％；乳糖0.3％；玉米浆2％；硫酸铵0.5％；磷酸二氢钾0.35％；磷酸氢二钾0.75％；七水硫酸镁0.03％)中发酵，30℃培养5d后，离心菌体获得上清液即为粗酶液。通过对获得的粗酶液分别进行NSP酶酶活力检测。
申请人最终筛选出一株NSP酶产量最高的突变菌株，命名为黑曲霉NSP-5(Aspergillus niger NSP-5)，该菌株发酵上清液中(见表1)木聚糖酶酶活106u/ml，比出发菌提高了96％；纤维素酶酶活4.4u/ml，比出发菌提高了91％；β-葡聚糖酶酶活41.4u/ml，比出发菌提高了55％；甘露聚糖酶酶活1.9u/ml，比出发菌提高了175％，突变菌株与出发菌株相比，极显著(P＜0.01)提高了NSP酶产量，取得了意料不到的技术效果。
表1
Figure PCTCN2017081985-appb-000001
申请人已于2016年7月4日将上述突变菌株黑曲霉NSP-5(Aspergillus niger NSP-5)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:M2016362。
实施例3 20L发酵罐发酵验证
将出发菌NSP-1和突变菌黑曲霉NSP-5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20h之后，然后分别将发酵液转入20L发酵罐(配方为：小麦麸皮2％；乳糖0.3％；玉米浆2％；硫酸铵0.5％；磷酸二氢钾0.35％；磷酸氢二钾0.75％；七水硫酸镁0.03％)，温度均控制在30±1℃，pH值均控制在5.0±0.2，搅拌速度为600rpm，在发酵罐培养24h之后，开始补加木糖+葡萄糖反转糖浆(配方为：木糖25％，葡萄糖25％，85％磷酸5％v/v，121℃处理30min)诱导菌体产酶，溶氧控制在30-40％，发酵时间约为160h，制成发酵菌液。
将上述发酵菌液离心，取上清，进行分别进行NSP酶酶活力检测。结果如表2显示，出发菌的发酵上清液木聚糖酶酶活为99u/ml，纤维素酶酶活为10.4u/ml，β-葡聚糖酶酶活为37.9u/ml，甘露聚糖酶酶活为1.5u/ml。突变菌黑曲霉NSP-5的发酵上清液木聚糖酶酶活205u/ml，比出发菌提高了107％；纤维素酶酶活17.6u/ml，比出发菌提高了69％；β-葡聚糖酶酶活67.7u/ml，比出发菌提高了78％；甘露聚糖酶酶活3.9u/ml，比出发菌提高了160％，各组分表达量与摇瓶相比放大作用明显(P＜0.01)，取得了意想不到的技术效果。
表2
Figure PCTCN2017081985-appb-000002
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. 黑曲霉，其特征在于，其保藏编号为CCTCC NO:M2016362。
  2. 根据权利要求1所述的黑曲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所述的黑曲霉为发酵菌株。
  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小麦麸皮2质量份；乳糖0.3质量份；玉米浆2质量份；硫酸铵0.5质量份；磷酸二氢钾0.35质量份；磷酸氢二钾0.75质量份；七水硫酸镁0.03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5d。
  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20h。
  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±0.2，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，补加木糖+葡萄糖反转糖浆，控制溶氧为30-40％，发酵160h。
  9. 根据权利要求8所述的发酵方法，其特征在于，所述木糖+葡萄糖反转糖浆的制备方法为：取木糖25％，葡萄糖25％，85％磷酸5％v/v，121℃处理30min。
  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。
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111778166A (zh) * 2020-08-21 2020-10-16 四川润格生物科技有限公司 一种利用白酒糟生产黑曲霉的技术方法
CN112111556A (zh) * 2020-09-27 2020-12-22 吉林中粮生化有限公司 一种判断葡萄糖淀粉酶的活力检测是否准确的方法
CN112300945A (zh) * 2020-06-16 2021-02-02 青海民族大学 一种黑曲霉固态发酵产饲用复合酶制剂及其制备工艺
CN114437938A (zh) * 2022-01-14 2022-05-06 山东隆科特酶制剂有限公司 一株高产耐高温酸性β-甘露聚糖酶的菌株及其应用
CN114561418A (zh) * 2020-11-27 2022-05-31 潍坊康地恩生物科技有限公司 一株高产β-1，3-葡聚糖酶的黑曲霉突变菌

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109988717B (zh) * 2019-05-05 2021-09-21 广西壮族自治区畜牧研究所 一株米曲霉菌株及其应用
CN113913305B (zh) * 2021-11-22 2023-10-13 山东隆科特酶制剂有限公司 一株高产酸性木聚糖酶的突变菌株及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103756919A (zh) * 2013-11-28 2014-04-30 青岛蔚蓝生物集团有限公司 一种木聚糖酶的重组菌株及其应用
CN104371934A (zh) * 2014-11-13 2015-02-25 青岛蔚蓝生物集团有限公司 一种里氏木霉突变菌株及其应用
WO2015143960A1 (zh) * 2014-03-28 2015-10-01 中国科学院广州能源研究所 一种高产木聚糖酶的黑曲霉及其应用
CN105505787A (zh) * 2015-12-25 2016-04-20 青岛蔚蓝生物集团有限公司 一种产葡萄糖转苷酶的黑曲霉突变菌株

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100999713A (zh) * 2006-12-20 2007-07-18 浙江省农业科学院 一种黑曲霉菌株及其nsp酶的制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103756919A (zh) * 2013-11-28 2014-04-30 青岛蔚蓝生物集团有限公司 一种木聚糖酶的重组菌株及其应用
WO2015143960A1 (zh) * 2014-03-28 2015-10-01 中国科学院广州能源研究所 一种高产木聚糖酶的黑曲霉及其应用
CN104371934A (zh) * 2014-11-13 2015-02-25 青岛蔚蓝生物集团有限公司 一种里氏木霉突变菌株及其应用
CN105505787A (zh) * 2015-12-25 2016-04-20 青岛蔚蓝生物集团有限公司 一种产葡萄糖转苷酶的黑曲霉突变菌株

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HU , SHIFENG ET AL.: "Solid State Fermentation Conditions for Cellulase Produced by Trichoderma Reesei REMI Mutant Strain", NATURAL SCIENCES, vol. 34, no. 2, 30 April 2008 (2008-04-30), pages 207 - 211, ISSN: 1007-1032 *
JOST WEBER ET AL.: "Microbubble Fermentation of Trichoderma Reesei for Cellulase Production", PROCESS BIOCHEMISTRY, vol. 40, 31 December 2005 (2005-12-31), pages 669 - 676, XP027793878, ISSN: 1359-5113 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112300945A (zh) * 2020-06-16 2021-02-02 青海民族大学 一种黑曲霉固态发酵产饲用复合酶制剂及其制备工艺
CN111778166A (zh) * 2020-08-21 2020-10-16 四川润格生物科技有限公司 一种利用白酒糟生产黑曲霉的技术方法
CN112111556A (zh) * 2020-09-27 2020-12-22 吉林中粮生化有限公司 一种判断葡萄糖淀粉酶的活力检测是否准确的方法
CN112111556B (zh) * 2020-09-27 2023-12-12 吉林中粮生化有限公司 一种判断葡萄糖淀粉酶的活力检测是否准确的方法
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CN114437938A (zh) * 2022-01-14 2022-05-06 山东隆科特酶制剂有限公司 一株高产耐高温酸性β-甘露聚糖酶的菌株及其应用
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