CN108220170B - Schizochytrium limacinum and application thereof in producing cellulase - Google Patents
Schizochytrium limacinum and application thereof in producing cellulase Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 54
- 229940106157 cellulase Drugs 0.000 title claims abstract description 53
- 241000003595 Aurantiochytrium limacinum Species 0.000 title abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 51
- 102000004190 Enzymes Human genes 0.000 claims abstract description 51
- 229940088598 enzyme Drugs 0.000 claims abstract description 51
- 241000233671 Schizochytrium Species 0.000 claims abstract description 37
- 239000001963 growth medium Substances 0.000 claims abstract description 31
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 25
- 239000001888 Peptone Substances 0.000 claims abstract description 25
- 108010080698 Peptones Proteins 0.000 claims abstract description 25
- 239000008103 glucose Substances 0.000 claims abstract description 25
- 235000019319 peptone Nutrition 0.000 claims abstract description 25
- 239000000843 powder Substances 0.000 claims abstract description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 23
- 235000002639 sodium chloride Nutrition 0.000 claims abstract description 23
- 239000011780 sodium chloride Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims description 48
- 230000004151 fermentation Effects 0.000 claims description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 239000002904 solvent Substances 0.000 claims description 22
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 15
- 238000011218 seed culture Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 229920001817 Agar Polymers 0.000 claims description 9
- 239000008272 agar Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 7
- 241000598397 Schizochytrium sp. Species 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000009423 ventilation Methods 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000009776 industrial production Methods 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 5
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- 230000000694 effects Effects 0.000 description 33
- 239000000243 solution Substances 0.000 description 28
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- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 14
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 14
- 230000001954 sterilising effect Effects 0.000 description 12
- 239000000872 buffer Substances 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 10
- 239000000758 substrate Substances 0.000 description 7
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 5
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 5
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 5
- 241001052560 Thallis Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
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- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000004753 textile Substances 0.000 description 3
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- 108010084185 Cellulases Proteins 0.000 description 2
- 102000005575 Cellulases Human genes 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 101710166469 Endoglucanase Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241001306132 Aurantiochytrium Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101710112457 Exoglucanase Proteins 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- 108010022901 Heparin Lyase Proteins 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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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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- 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)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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Abstract
The invention discloses Schizochytrium WZYU059 and application thereof in producing cellulase. The method uses schizochytrium limacinum to rapidly produce the cellulase in a culture medium containing cheap raw materials such as glucose, peptone, yeast powder and sea salt for the first time. The cellulase prepared by the preparation method has good pH stability, and can solve the problem of poor pH stability of the enzyme in the acid-base change of the environment in industrial production. Meanwhile, the method has the advantages of low cost, simple process flow, low equipment requirement and strong controllability, and is suitable for industrial production.
Description
(I) technical field
The invention relates to preparation of cellulase, in particular to a method for producing cellulase by utilizing schizochytrium limacinum.
(II) background of the invention
Schizochytrium sp is heterotrophic microorganism of marine fungi, is mostly distributed in marine habitat with variable environment, can fully utilize own multienzyme system to efficiently decompose various matrixes in the environment, and is suitable for rapid growth of environmental factors such as temperature, pH and the like. However, no enzyme preparation derived from Schizochytrium and having industrial value has been reported.
Cellulases are complex enzyme systems consisting of a variety of hydrolases, which are generally divided into three classes: i.e., endoglucanases, exoglucanases, and glucosidases. Since cellulose is the most abundant renewable resource in nature, cellulase is one of the most widely used enzymes in industrial production, and can be used for treating, processing and biotransformation of lignocellulose; in addition, cellulases are widely used in the textile, paper, feed, food and detergent industries.
Microorganisms are important sources of cellulase, and the microorganisms such as bacteria, fungi, yeast and the like all report the production of cellulase at present. The cellulase can be divided into acidic, neutral and alkaline cellulases according to the pH value of the cellulase, wherein the acidic cellulase is mainly used in the fields of biological energy, textile washing and the like; the neutral cellulase is used in the textile washing industry; and alkaline cellulase is mainly used in detergents. Although the cellulase has different action pH, the problem of insufficient pH stability exists generally at present, and the cellulase is difficult to adapt to acid-base change in industrial production.
Disclosure of the invention
The invention aims to provide a new strain-schizochytrium sp WZYU059 and a method for efficiently producing pH stable cellulase by using the schizochytrium sp WZYU 059. The invention uses the marine microorganism schizochytrium who passes food safety certification for the first time, adopts cheap raw materials to produce the cellulase efficiently, effectively reduces the production cost of the cellulase, has higher activity in a wide pH range, solves the problem of insufficient stability of industrial cellulase, has simple whole process flow, low equipment requirement and strong controllability, and is suitable for industrial production.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain-schizochytrium sp WZYU059, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: M2016620, preservation date is: 2016, 11/7/2016, and the preservation address is Wuhan, Wuhan university, Wuhan, Japan, and the zip code 430072.
The invention also provides a method for producing cellulase by using schizochytrium WZYU059, which comprises the following steps: inoculating schizochytrium WZYU059 into a fermentation culture medium, performing shaking culture at the temperature of 25-35 ℃ and the rpm of 50-600 to obtain a fermentation broth, centrifuging the fermentation broth, removing cell precipitates to obtain a crude enzyme solution containing cellulase, and separating and purifying the crude enzyme solution to obtain the cellulase; the fermentation medium comprises the following components: 20-120g/L of glucose, 10-80g/L of yeast powder, 0-40g/L of peptone (0 refers to the condition that no peptone is contained, and the content is 5-20 g/L when peptone is contained), 20g/L of sea salt, water as a solvent and natural pH value.
Further, before inoculation of the schizochytrium WZYU059, slant activation and seed expansion culture are carried out: (1) slant culture: inoculating Schizochytrium WZYU059 into a plate culture medium, and culturing at 28-30 deg.C for 2-3 days; the slant culture medium comprises the following components: 10-40g/L of glucose, 10g/L of yeast powder, 5g/L of peptone, 20g/L of sea salt, 20g/L of agar and water as a solvent, wherein the pH value is natural; (2) seed culture: inoculating the slant thallus into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 180rpm at 28-30 ℃ for 1-2 days to obtain a seed solution; the seed culture medium comprises the following components: 60g/L glucose, 10g/L yeast powder, 5g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
Further, the fermentation culture method of the schizochytrium WZYU059 comprises the following steps: inoculating the seed solution into a fermentation tank containing fermentation medium, and culturing at 25-35 deg.C at rotation speed of 50-600rpm, ventilation amount of 10-200L/min and dissolved oxygen amount of 10-60% for 1-6 days to obtain fermentation liquid, preferably under the following conditions: at 28-32 deg.C, rotation speed of 100-.
Further, the seed solution is inoculated to a fermentation medium in an inoculation amount of 1-20% of the volume concentration.
Further, the fermentation liquor centrifugation conditions are as follows: centrifuging at 8000rpm and 4 deg.C for 5 min.
Further, the fermentation medium consists of: 100g/L glucose, 20g/L yeast powder, 10g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
The cellulase produced by the schizochytrium WZYU059 has the following characteristics: the enzyme has the optimum action pH of 5.0, and still retains more than 80% of activity of the optimum activity within the range of pH3.0-8.0; the enzyme has good stability in the pH range of 3.0-9.0, and the relative enzyme activity can be kept above 70%.
Compared with the prior art, the invention has the following beneficial effects:
schizochytrium has many advantages over other cellulase producing strains: 1) the culture medium has simple formula and low cost, and is suitable for large-scale production; 2) the mode of producing the cellulase is extracellular secretion, and the extraction and separation processes are simplified; 3) the produced cellulase has higher pH stability, and the problem of insufficient pH stability of the cellulase in actual production is solved; 4) the schizochytrium limacinum passes authoritative safety certification and can be applied to the fields of food, medicine and the like.
(IV) description of the drawings
FIG. 1 shows the colony morphology of Schizochytrium WZYU059 strain.
FIG. 2 is a transparent circle formed by the Schizochytrium WZYU059 strain on a safranin plate.
FIG. 3 is a graph of the effect of relative cellulase activity of Schizochytrium WZYU 059.
FIG. 4 is the pH stability of temperature versus Schizochytrium WZYU059 cellulase.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 Schizochytrium WZYU059 screening and identification
(1) Screening of strain WZYU059
A leaf mold sample is collected at seaside of Leqing gulf of Wenzhou, Zhejiang, cleaned, placed in YP culture solution (1g/L yeast powder, 1g/L peptone, 50mg/L ampicillin, 50mg/L streptomycin, water preparation, pH 6.0) with 0.1g of pollen pini suspended on the surface, cultured in the dark at 30 ℃ for 1 week, 50 microliter of culture is taken and spread on YP plates (1g/L yeast powder, 1g/L peptone, 50mg/L ampicillin, 50mg/L streptomycin, agar 20g/L, water preparation, pH 6.0), cultured at 28 ℃ for 3 days until a single colony grows out, and streaking GYP plate purification culture is repeated for three times to obtain a pure strain. Pure colonies were spotted on CMC-Congo red plates, cultured for 3 days at 30 ℃ in an inverted manner, and the clear circles were observed to mark the strain WZYU 059.
GYP plate culture medium comprises (g/L) glucose 20, yeast powder 10, dried egg white 5, sea salt 20, agar 20, and solvent water, and has natural pH value, sterilized at 115 deg.C for 30min, and poured into plate.
The CMC-Congo red flat plate component is (g/L): 5 parts of sodium carboxymethylcellulose (CMC), 10 parts of yeast powder, 5 parts of egg white, 0.2 part of Congo red, 20 parts of sea salt, 20 parts of agar and water as a solvent, wherein the pH value is 7.0, and the mixture is sterilized at 115 ℃ for 30min and poured into a flat plate.
(2) Identification of strain WZYU059
The morphological characteristics of the cells are as follows:
the strain WZYU059 is inoculated in GYP culture medium, cultured at 28 ℃ for 2 days, and microscopically, the cells are round or oval, the diameter is 5-20 μm, the cell surface is bright, the cells have a scaly structure, the edges are clear, the cells are divided and proliferated, and an even number of cells are gathered together (figure 1).
The 18SrDNA sequence of the strain WZYU059 is shown in SEQ ID No. 1. The similarity of the 18SrDNA sequence of the strain and strains such as Aurantiochytrium sp, KRS101 and ST-2012 can reach 99 percent, so the strain WZYU059 is identified as schizochytrium sp, named as schizochytrium sp WZYU059, deposited in the China center for type culture Collection with the deposit number: CCTCC NO: M2016620, preservation date is: 2016, 11/7/2016, and the preservation address is Wuhan, Wuhan university, Wuhan, Japan, and the zip code 430072.
Example 2 production of cellulase by liquid fermentation of Schizochytrium WZYU059
Transferring the Schizochytrium WZYU059 strain stored at-80 deg.C to slant culture medium, and static culturing at 30 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 30 ℃ and 180rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 10% of volume concentration, culturing for 3 days at 30 deg.C, 300rpm, 80L/min of ventilation capacity and 30% of dissolved oxygen to obtain fermentation broth; centrifuging the fermentation liquid at 8000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 128.4U/mL.
The activity of the cellulase is determined by taking sodium carboxymethylcellulose (CMC) as a substrate and combining a DNS (3, 5-dinitrosalicylic acid) method. Definition of enzyme activity: the amount of enzyme required to release 1. mu. mol of reducing sugar (calculated as glucose) per minute from 5mg/mL of CMC at 50 ℃ and pH4.8 was one enzyme activity unit (U). Mixing 0.5mL and 5mg/mL CMC water solution with 0.5mL enzyme solution, reacting for 15min at 50 ℃ and pH4.8, adding 1.5mL LDNS reagent, boiling in water bath for 5min, cooling to room temperature, diluting to 5mL, taking the enzyme solution inactivated in advance as a reference, measuring absorbance at 540nm, and calculating enzyme activity.
The slant culture medium comprises (g/L) glucose 40, yeast powder 10, peptone 5, sea salt 20, agar 20, and solvent water, with natural pH, and sterilizing at 115 deg.C for 30 min; the seed culture medium (g/L) comprises glucose 60, yeast powder 10, peptone 5, sea salt 20, and solvent water, with natural pH, and sterilizing at 115 deg.C for 30 min; the fermentation medium (g/L) comprises glucose 60, yeast powder 10, peptone 5, sea salt 20, and solvent water, with natural pH value, and sterilizing at 115 deg.C for 30 min.
Example 3 production of cellulase by liquid fermentation of Schizochytrium WZYU059
Transferring the Schizochytrium WZYU059 strain stored at-80 deg.C to slant culture medium, and standing at 28 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 28 ℃ and 180rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 20% of volume concentration, culturing for 2 days at 25 deg.C, rotation speed of 600rpm, ventilation of 100L/min, and dissolved oxygen of 50% to obtain fermentation broth; centrifuging the fermentation liquid at 8000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 92.4U/mL.
The activity of the cellulase is determined by taking sodium carboxymethylcellulose (CMC) as a substrate and combining a DNS (3, 5-dinitrosalicylic acid) method. Definition of enzyme activity: the amount of enzyme required to release 1. mu. mol of reducing sugar (calculated as glucose) per minute from 5mg/mL of CMC at 50 ℃ and pH4.8 was one enzyme activity unit (U). Mixing 0.5mL and 5mg/mL CMC water solution with 0.5mL enzyme solution, reacting for 15min at 50 ℃ and pH4.8, adding 1.5mL LDNS reagent, boiling in water bath for 5min, cooling to room temperature, diluting to 5mL, taking the enzyme solution inactivated in advance as a reference, measuring absorbance at 540nm, and calculating enzyme activity.
The slant culture medium comprises the following components in percentage by weight (g/L): 40 parts of glucose, 10 parts of yeast powder, 5 parts of peptone, 20 parts of sea salt, 20 parts of agar and a solvent of water, wherein the pH value is natural, and the sterilization is carried out for 30min at 115 ℃; the seed culture medium (g/L) comprises glucose 60, yeast powder 10, peptone 5, sea salt 20, and solvent water, with natural pH, and sterilizing at 115 deg.C for 30 min; the fermentation medium (g/L) comprises glucose 20, yeast powder 10, peptone 0, sea salt 20, and solvent water, with natural pH value, and sterilizing at 115 deg.C for 30 min.
Example 4 production of cellulase by liquid fermentation of Schizochytrium WZYU059
Transferring the Schizochytrium WZYU059 strain stored at-80 deg.C to slant culture medium, and standing at 28 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 28 ℃ and 180rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 1% of volume concentration, culturing for 6 days at 35 deg.C, 500rpm, 200L/min of ventilation capacity and 60% of dissolved oxygen to obtain fermentation broth; centrifuging the fermentation liquid at 8000rpm and 4 deg.C for 5min, removing cell precipitate to obtain crude enzyme solution with enzyme activity of 48.2U/mL.
The activity of the cellulase is determined by taking sodium carboxymethylcellulose (CMC) as a substrate and combining a DNS (3, 5-dinitrosalicylic acid) method. Definition of enzyme activity: the amount of enzyme required to release 1. mu. mol of reducing sugar (calculated as glucose) per minute from 5mg/mL of CMC at 50 ℃ and pH4.8 was one enzyme activity unit (U). Mixing 0.5mL of LCMC solution with 0.5mL of enzyme solution, reacting for 15min at 50 ℃ and pH4.8, adding 1.5mL of LDNS reagent, boiling in a water bath for 5min, cooling to room temperature, diluting to 5mL, taking the enzyme solution inactivated in advance as a control, measuring absorbance at 540nm, and calculating enzyme activity.
The slant culture medium comprises the following components in percentage by weight (g/L): 40 parts of glucose, 10 parts of yeast powder, 5 parts of peptone, 20 parts of sea salt, 20 parts of agar and a solvent of water, wherein the pH value is natural, and the sterilization is carried out for 30min at 115 ℃; the seed culture medium (g/L) comprises glucose 60, yeast powder 10, peptone 5, sea salt 20, and solvent water, with natural pH, and sterilizing at 115 deg.C for 30 min; the fermentation medium (g/L) comprises glucose 120, yeast powder 80, peptone 20, sea salt 20, and solvent water, with natural pH value, and sterilizing at 115 deg.C for 30 min.
Example 5 production of cellulase by liquid fermentation of Schizochytrium WZYU059
Transferring the Schizochytrium WZYU059 strain stored at-80 deg.C to slant culture medium, and standing at 28 deg.C for 2 days; inoculating the thalli into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 28 ℃ and 180rpm for 1 day to obtain seed liquid; transferring the seed solution into a fermentation tank containing 3L fermentation medium according to the inoculum size of 15% of volume concentration, culturing for 4 days at 28 deg.C, 50rpm, 10L/min of ventilation capacity and 10% of dissolved oxygen to obtain fermentation broth; and centrifuging the fermentation liquor at 8000rpm and 4 ℃ for 5min, removing cell precipitates to obtain a crude enzyme solution, wherein the enzyme activity is 53.9U/mL.
The activity of the cellulase is determined by taking sodium carboxymethylcellulose (CMC) as a substrate and combining a DNS (3, 5-dinitrosalicylic acid) method. Definition of enzyme activity: the amount of enzyme required to release 1. mu. mol of reducing sugar (calculated as glucose) per minute from 5mg/mL of CMC at 50 ℃ and pH4.8 was one enzyme activity unit (U). Mixing 0.5mL and 5mg/mL CMC water solution with 0.5mL enzyme solution, reacting for 15min at 50 ℃ and pH4.8, adding 1.5mL LDNS reagent, boiling in water bath for 5min, cooling to room temperature, diluting to 5mL, taking the enzyme solution inactivated in advance as a reference, measuring absorbance at 540nm, and calculating enzyme activity.
The slant culture medium comprises the following components in percentage by weight (g/L): 10 parts of glucose, 10 parts of yeast powder, 5 parts of peptone, 20 parts of sea salt, 20 parts of agar and a solvent of water, wherein the pH value is natural, and the sterilization is carried out for 30min at 115 ℃; the seed culture medium (g/L) comprises glucose 60, yeast powder 10, peptone 5, sea salt 20, and solvent water, with natural pH, and sterilizing at 115 deg.C for 30 min; the fermentation medium (g/L) comprises glucose 100, yeast powder 20, peptone 40, sea salt 20, and solvent water, with natural pH value, and sterilizing at 115 deg.C for 30 min.
Example 6 Schizochytrium WZYU059 cellulase optimum pH and stability
The cellulase activity of the crude enzyme solution prepared by fermenting the schizochytrium WZYU059 strain of example 2 was measured by combining the DNS standard method using buffers of different pH values (0.1M glycine-hydrochloric acid buffer at pH 2.0; 0.1M citric acid-sodium citrate buffer at pH 3.0-6.0; 0.1M barbiturate-hydrochloric acid buffer at pH 7.0-9.0; glycine-sodium hydroxide buffer at pH 10.0; 0.0-6.0; 0.1M barbiturate-hydrochloric acid buffer at pH 7.0-9.0; 5.0; 5g/L CMC as substrate) and the optimum pH of the cellulase was 5.0, which was taken as 100%, and the enzyme activities corresponding to substrates of other pH values under the same conditions were compared to obtain the relative cellulase activity (see fig. 3). The results showed that the enzyme had a high enzymatic activity (greater than 80% of the optimal activity) in the pH range 3.0-8.0.
The pH values of the crude enzyme solutions prepared by fermentation of the schizochytrium wZYU059 strain of example 2 were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively using buffers of different pH values (0.1M glycine-hydrochloric acid buffer at pH 2.0; 0.1M citric acid-sodium citrate buffer at pH 3.0-6.0; 0.1M barbiturate-hydrochloric acid buffer at pH 7.0-9.0; glycine-sodium hydroxide buffer at pH 10.0) and stored at 4 ℃ for 60min, the activity of the schizochytrium wZYU059 cellulase was determined using CMC as a substrate in combination with the DNS standard method, and the relative enzyme activities under different pH conditions were calculated using the stock solution of natural pH as 100% to evaluate the pH stability of the enzyme (see FIG. 4). The result shows that the enzyme has better pH stability in the pH range of 3.0-9.0, and the relative enzyme activity can be kept above 70%.
Sequence listing
<110> Zhejiang industrial university
<120> Schizochytrium limacinum and application thereof in producing cellulase
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1770
<212>DNA
<213> Schizochytrium sp (2 AmbystAlternatiochytrium sp.)
<400>1
tcctgccagt agtcatatgc tcgtctcaaa gattaagcca tgcatgtgta agtataagcg 60
attgtactgt gagactgcga acggctcatt atatcagtaa taatttcttc ggtagtttct 120
tttatatgga tacctgcagt aattctggaa ataatacatg ctgtaagagc cctgtatggg 180
gctgcactta ttagattgaa gccgatttta ttggtgaatc atgataattg agcagattga 240
ctatttttgt cgatgaatcg tttgagtttc tgccccatca gttgtcgacg gtagtgtatt 300
ggactacggt gactataacg ggtgacggag ggttagggct cgactccgga gagggagcct 360
gagagacggc taccatatcc aaggatagca gcaggcgcgt aaattaccca ctgtggactc 420
cacgaggtag tgacgagaaa tatcgatgcg aagcgtgtat gcgttttgct atcggaatga 480
gagcaatgta aaaccctcat cgaggatcaa ctggagggca agtctggtgc cagcagccgc 540
ggtaattcca gctccagaag catatgctaa agttgttgca gttaaaaagc tcgtagttga 600
atttctggca tgggcgaccg gtgctttccc tgaatgggga ttgattgtct gtgttgcctt 660
ggccatcttt ctcatgctat tttggtatga gatctttcac tgtaatcaaa gcagagtgtt 720
ccaagcaggt cgtatgaccg gtatgtttat tatgggatga taagatagga cttgggtgct 780
attttgttgg tttgcacgcc tgagtaatgg ttaataggaa cagttggggg tattcgtatt 840
taggagctag aggtgaaatt cttggatttc cgaaagacga actaaagcga aggcatttac 900
caagcatgtt ttcattaatc aagaacgaaa gtctggggat cgaagatgat tagataccat 960
cgtagtctag accgtaaacg atgccgactt gcgattgttg ggtgcttttt tatatgggcc 1020
tcagcagcag cacatgagaa atcaaagtct ttgggttccg gggggagtat ggtcgcaagg 1080
ctgaaactta aaggaattga cggaagggca ccaccaggag tggagcctgc ggcttaattt 1140
gactcaacac gggaaaactt accaggtcca gacataggta ggattgacag attgagagct 1200
ctttcatgat tctatgggtg gtggtgcatg gccgttctta gttggtggag tgatttgtct 1260
ggttaattcc gttaacgaac gagacctcgg cctactaaat agtgcgtggt atggcaacat 1320
agtgcgtttt tacttcttag agggacatgt ccggtttacg ggcaggaagt tcgaggcaat 1380
aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact gatgggttca 1440
tcgggtttta atttcatttt tggaattgag tgcttggtcg gaaggcctgg ctaatccttg 1500
gaacgctcat cgtgctgggg ctagattttt gcaattatta atctccaacg aggaattcct 1560
agtaaacgca agtcatcagc ttgcattgaa tacgtccctg ccctttgtac acaccgcccg 1620
tcgcacctac cgattgaacg gtccgatgaa accatgggat gtttgtgttt ggattaattt 1680
ttggacatag gcagaactcg ggtgaatctt attgtttaga ggaaggtgaa gtcgtaacaa 1740
ggtttccgta ggtgaacctg cggaggatca 1770
Claims (8)
1. Schizochytrium sp (WZYU 059) deposited in the China center for type culture Collection with the deposit number: CCTCC NO: M2016620, preservation date is: 2016, 11/7/2016, and the preservation address is Wuhan, Wuhan university, Wuhan, Japan, and the zip code 430072.
2. A method for producing cellulase by using the Schizochytrium WZYU059 of claim 1, which is characterized in that the method comprises the following steps: inoculating schizochytrium WZYU059 into a fermentation culture medium, performing shaking culture at the temperature of 25-35 ℃ and the rpm of 50-600 to obtain a fermentation broth, centrifuging the fermentation broth, removing cell precipitates to obtain a crude enzyme solution containing cellulase, and separating and purifying the crude enzyme solution to obtain the cellulase; the fermentation medium comprises the following components: 20-120g/L of glucose, 10-80g/L of yeast powder, 0-40g/L of peptone, 20g/L of sea salt and water as a solvent, and the pH value is natural.
3. The method for producing cellulase by using schizochytrium WZYU059 as claimed in claim 2, wherein the schizochytrium WZYU059 is inoculated with slant activation and seed expansion culture: (1) bevel activation: inoculating Schizochytrium WZYU059 into slant culture medium, and culturing at 28-30 deg.C for 2-3 days to obtain slant thallus; the slant culture medium comprises the following components: 10-40g/L of glucose, 10g/L of yeast powder, 5g/L of peptone, 20g/L of sea salt, 20g/L of agar and water as a solvent, wherein the pH value is natural; (2) seed culture: inoculating the slant thallus into a 500mL triangular flask containing 100mL seed culture medium, and performing shake culture at 180rpm at 28-30 ℃ for 1-2 days to obtain a seed solution; the seed culture medium comprises the following components: 60g/L glucose, 10g/L yeast powder, 5g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
4. The method for producing cellulase by using schizochytrium WZYU059 as claimed in claim 3, wherein the fermentation broth is prepared by the following method: inoculating the seed solution into a fermentation tank containing fermentation culture medium, and culturing at 25-35 deg.C at rotation speed of 50-600rpm, ventilation amount of 10-200L/min, and dissolved oxygen amount of 10-60% for 1-6 days to obtain fermentation liquid.
5. The method for producing cellulase by using schizochytrium WZYU059 as claimed in claim 3, wherein the seed solution is inoculated to the fermentation medium in an inoculum size of 1% -20% by volume concentration.
6. The method for producing cellulase by using schizochytrium WZYU059 as claimed in claim 4, wherein the fermentation culture conditions are: at 28-32 deg.C, rotation speed of 100-.
7. The method for producing cellulase by using schizochytrium WZYU059 as claimed in claim 2, wherein the fermentation broth centrifugation conditions are: centrifuging at 8000rpm and 4 deg.C for 5 min.
8. The method for producing cellulase using schizochytrium WZYU059 of claim 2, wherein the fermentation medium consists of: 100g/L glucose, 20g/L yeast powder, 10g/L peptone, 20g/L sea salt and water as solvent, and the pH value is natural.
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| CN104498371A (en) * | 2014-12-31 | 2015-04-08 | 浙江工业大学 | Aurantiochytrium sp. YLH70 and application of Aurantiochytrium sp. YLH70 for synthesizing DHA |
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