CN104031860A - Bacillus sphaericus for high-yield high-temperature-resistant xylanase and application thereof - Google Patents
Bacillus sphaericus for high-yield high-temperature-resistant xylanase and application thereof Download PDFInfo
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- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 21
- 241000193386 Lysinibacillus sphaericus Species 0.000 title claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 34
- 230000004151 fermentation Effects 0.000 claims abstract description 34
- 229920002488 Hemicellulose Polymers 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 16
- 108010080698 Peptones Proteins 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- 235000019319 peptone Nutrition 0.000 claims description 16
- 239000012153 distilled water Substances 0.000 claims description 15
- 229920001221 xylan Polymers 0.000 claims description 14
- 150000004823 xylans Chemical class 0.000 claims description 14
- 230000009970 fire resistant effect Effects 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000010902 straw Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 8
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 229930182817 methionine Natural products 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 230000000593 degrading effect Effects 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 230000002255 enzymatic effect Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 27
- 108090000790 Enzymes Proteins 0.000 abstract description 20
- 102000004190 Enzymes Human genes 0.000 abstract description 20
- 238000013461 design Methods 0.000 abstract description 6
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- 238000004321 preservation Methods 0.000 abstract description 4
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 238000005211 surface analysis Methods 0.000 abstract description 2
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 abstract 1
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 abstract 1
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 abstract 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract 1
- 239000004472 Lysine Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 32
- 239000002609 medium Substances 0.000 description 13
- 230000008569 process Effects 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000012216 screening Methods 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 8
- 239000000725 suspension Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000009973 maize Nutrition 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 235000007686 potassium Nutrition 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229960003487 xylose Drugs 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 239000002361 compost Substances 0.000 description 2
- 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 2
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- 238000011081 inoculation Methods 0.000 description 2
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- 239000000047 product Substances 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
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- 239000000758 substrate Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical compound O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
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- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
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- 230000036425 denaturation Effects 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
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- 235000013312 flour Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- WVTHODDBDXSFGC-UHFFFAOYSA-K magnesium sodium chloride sulfate Chemical compound [Na+].[Mg+2].[Cl-].[O-]S([O-])(=O)=O WVTHODDBDXSFGC-UHFFFAOYSA-K 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
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- 239000002994 raw material Substances 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
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- 239000002023 wood Substances 0.000 description 1
- 150000003740 xylobioses Chemical class 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The bacillus sphaericus for high yield of high-temperature resistant xylanase and the application thereof are classified and named as bacillus sphaericus (lysine bacillus sphaericus) Xyn-1, are preserved in China Center for Type Culture Collection (CCTCC) at 5 months and 5 days in 2014, and have the preservation number of CCTCCM 2014183. The optimal fermentation enzyme production condition of the shake flask of the screened strain is determined by a Plackett-Burman design and a response surface analysis method, and the fermentation level under the condition can reach 5678.5 IU/mL. The enzyme produced by the fermentation of the strain is applied to the degradation of plant hemicellulose, and the yield of xylobiose obtained by enzymolysis of the hemicellulose can reach more than 60%.
Description
Technical field
The present invention relates to a kind of spherical bacillus and application thereof of high-yield thermostable zytase, belong to using microbe technical field.
Background technology
Zytase (Xylanase E.C3.2.1.8) is a kind of main xylan degrading enzyme, and it is by the β-Isosorbide-5-Nitrae wood sugar glycosidic bond in internal-cutting way degradation of xylan, and hydrolysate is mostly xylo-oligosaccharide, and with a small amount of wood sugar and pectinose.Zytase is with a wide range of applications, aspect paper industry: zytase can be used as bio-bleaching agent and substitutes traditional chemical bleaching, thereby reduce in a large number the consumption of chlorine, alleviate the pollution of papermaking to environment, zytase has been widely used in western developed country as " slurrying enzyme ", has brought good economic and social benefit; The preparation that zytase is widely used in the making food such as flour products, fruit juice and wood oligose in food service industry is produced; In fermentation industry, zytase is widely used in the saccharification of the fermentation raw materials such as wheat and processes; Feedstuff industry adds enzyme as a class feed, can effectively improve feed nutrition.Zytase has become a staple product in zymin production industry, and market also presents the trend growing with each passing day for its demand.
In addition, can find by the integrated environment analysis to applying in the market zytase, the applied environment of zytase agent is mostly in hot conditions, and for example paper-making process, needs high-temperature operation to remove delignification etc.; Food production generally needs the operations such as high-temperature sterilization.Research discovery, the optimal reactive temperature of zytase is generally lower than 45 DEG C, and therefore zytase needs to lower the temperature in advance to improve the catalytic activity of enzyme before coming into operation in said process, and these operations are not only consumed energy and are also greatly hindered the application development of zytase.Therefore, obtain high yield heat resistant xylanase bacterial strain and devoted real attenuation and produce and there is wide research and application prospect.
Summary of the invention
The object of this invention is to provide a kind of spherical bacillus of high-yield thermostable zytase and produce the application in fire resistant xylanase in fermentation, there is very large promotional value.
The spherical bacillus of high-yield thermostable zytase, Classification And Nomenclature is spherical Methionin genus bacillus Xyn-1 (Lysinibacillus sphaericus Xyn-1), in the center preservation of Chinese Typical Representative culture collection, deposit number is: CCTCC M2014183, preservation date is on May 5th, 2014, address: Wuhan, China Wuhan University.
The screening method of bacterial strain of the present invention is as follows:
Screening and culturing based formulas: peptone 10g, yeast extract paste 3g, NaCl5g, xylan 4g, agar powder 15g, distilled water 1000mL, pH8.0,121 DEG C of sterilizings, 20min.
Collection is arranged in Nanjing University of Technology's biological methane production plant some duplicate samples of agricultural straw compost different positions around and is inoculated into above-mentioned screening culture medium, cultivate 24～48h for 55 DEG C, with 0.1% Congo red solution-treated 30min, again with the decolouring of 1mol/L NaCl solution, then choose on substratum the large and obvious bacterial strain of hydrolysis circle of transparent circle as primary dcreening operation bacterial strain; Subsequently, the enzymatic production ability of primary dcreening operation bacterial strain is verified, taking xylan as substrate, measured enzyme live based on DNS method, choose the strain strain X yn-1 that output is the highest, its initial fermentation level is 3245.5IU/mL.
The growth of physiological and biochemical property: strain X yn-1 is with OD
600absorbance represent, growth Dai Shiwei 3.0h is calculated in the growth by quantity in bacterium logarithm period unit.The molecular biology identification of bacterial strain shows, the 16S rRNA gene order of strain X yn-1 is KJ755851 at the number of registration of GenBank, the similarity of kind Lysinibacillus sphaericus the most close to it is 91%, be a new kind bacterium, the spherical Methionin genus bacillus of called after Xyn-1 (Lysinibacillus sphaericus Xyn-1).This bacterial strain is preserved in Chinese Typical Representative culture collection center on May 5th, 2013, and deposit number is respectively: CCTCC M2014183.
The spherical bacillus of described high-yield thermostable zytase produces the application of zytase in fermentation.
The application of the spherical bacillus of described high-yield thermostable zytase in degrading straw hemicellulose.
The concrete steps of application are as follows:
1) bacterial strain is carried out to slant culture, be placed at 37 DEG C, cultivate 48～72h, adopt beef-protein medium, specifically formula: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 20g, is dissolved in 1000mL distilled water pH7.4-7.6.By (peptone 10g in the LB substratum of the bacterial strain streak inoculation sterilizing of above-mentioned slant culture, yeast extract 5g, NaCl10g, be dissolved in 1000mL distilled water, pH7.0～7.2), under 37 DEG C of conditions, 200～250r/min cultivates 12h, utilizes cell counting count board to measure bacterial concentration, and is diluted to bacteria suspension concentration 5 × 10
6individual/mL.
2) by step 1) the bacterium liquid that obtains is seeded in fermention medium, and inoculum size is 4% (v/v), and substratum consumption is 25mL/250mL Erlenmeyer flask, 45 DEG C of culture temperature, shaking flask rotating speed 200r/min, fermentation time is 72h.Described fermention medium is: xylan 10.7g, yeast powder 5.6g, peptone 2g, NH
4nO
34g, (NH
4)
2sO
46g, KH
2pO
40.9g, MgSO
47H
2o1g, NaCl5g, distilled water 1000mL.
3) fermentation prepare the enzymolysis of zytase for wheat stalk hemicellulose: by step 2) obtain fermented liquid at 5500r/min, centrifugal 5min under 4 DEG C of conditions.Collect supernatant liquor and obtain the fermentation fire resistant xylanase liquid that produces, and be applied to the enzymolysis of Technique of Hemicellulose from Cornstalk.Enzymolysis process adds zytase liquid with the ratio of 100～150IU/g hemicellulose, under 55 DEG C of conditions, reacts 72h.After reaction finishes, filter and collect enzymolysis solution, utilize HPLC method to measure each reducing sugar content in enzymolysis solution, and calculate the enzymolysis efficiency of this process with this.
Beneficial effect
The invention provides a plant height and produce fire resistant xylanase strain X yn-1, its culture condition is relatively gentle, bacterium is tested and appraised and belongs to spherical Methionin genus bacillus, not yet carried out report for the zytase research of this bacterial strain before this, the present invention has further enriched the correlative study content of this kind fungus strain.
The fermentation level that the strain X yn-1 that screening obtains produces fire resistant xylanase reaches 5678.5IU/mL, the zytase throughput of relatively finding this bacterial strain is higher, the fire resistant xylanase that fermentation obtains simultaneously has good thermostability, can be widely used in the industry such as papermaking, food.
Adopt statistical method to be optimized research to the production fermenting process of strain X yn-1, obtained best culture scheme, the zytase fermentation level of bacterial strain has had significant raising, brings up to 5678.5IU/mL by the 3245.5IU/mL that initially ferments.
Brief description of the drawings
The spherical Methionin genus bacillus of Fig. 1 Xyn-1 flat-plate bacterial colony aspect graph.
Embodiment
Screening and the cultivation of embodiment 1 high-yield thermostable zytase bacterial strain
Enrichment medium: NaNO
30.5g, K
2hPO
4l g, MgSO
47H
2o0.5g, KCl0.5g, FeSO
47H
2o0.005g, distilled water 1000mL, pH nature, 121 DEG C of sterilizings, 20min.
Screening culture medium: peptone 10g, yeast extract paste 3g, NaCl5g, xylan 4g, agar powder 15g,
Distilled water 1000mL, pH8.0,121 DEG C of sterilizings, 20min.
Fermention medium: peptone 10g, yeast extract paste 3g, NaCl5g, xylan 4g, distilled water 1000mL, pH nature, 121 DEG C of sterilizings, 20min.
LB substratum: peptone 10g, yeast extract 5g, NaCl10g, agar 20g, is dissolved in 1000mL distilled water, pH7.0～7.2,121 DEG C of sterilizings, 20min.
Collection is positioned at Nanjing University of Technology's biological methane production plant some duplicate samples 5g of straw compost different positions around, it is placed in respectively to 250mL Erlenmeyer flask, add 50mL sterilized water, 37 DEG C, 180r/min are cultivated 1h and are prepared suspension, getting 5mL suspension joins in the triangular flask that fills 50mL enrichment medium, after shaking culture 5～7d, get nutrient solution and dilute respectively 10
-1～10
-6, and respectively it is evenly applied in screening culture medium, cultivate 24～48h for 55 DEG C, with 0.1% Congo red solution-treated 30min, then decolour with 1mol/L NaCl solution.Observe the transparent circle situation of periphery of bacterial colonies, can compare bacterial strain according to the size of transparent circle and bacterium colony size and produce the height of Xylanase activity, and carry out the primary dcreening operation of bacterial strain using this as index.After primary dcreening operation completes, to the bacterium liquid dilution 10 of some primary dcreening operation bacterial strains
-1～10
-6, be inoculated into respectively in fresh fermention medium 35 DEG C and cultivate 72h, carry out bacterial strain using zytase output in fermented liquid as direct standard and sieve again.So succeeding transfer culture, the culture that superseded fermentation level is weak, finally obtain a strain and keep higher xylanase ability and stable bacterial strain, utilize LB substratum to carry out the preservation of bacterial strain purifying to it, utilize glycerine training method to carry out cold storage processing to purifying bacterial strain simultaneously.
The biological assay of embodiment 2 bacteriums
1. the Morphological Identification of bacterial strain
Separation and purification inoculation, to solid LB substratum, is cultivated to the flat-plate bacterial colony form of 24h observation bacterial classification under 37 DEG C of conditions.As Fig. 1, strain X yn-1, after the growth of solid LB substratum, colony diameter 0.6cm, shape is regular thrust, can form endogenous spore, rounded, the opaque colony that is white in color on substratum, smooth surface, colony edge rule.
2. the molecules of bacterial strain qualification
Screening bacterium is carried out to strain identification, the bacterial 16 S rDNA sequence library of strain identification based on ncbi database.Utilize bacterial genomes to extract the DNA of test kit extraction bacterial strain, and utilizing bacterial 16 S rDNA universal primer to carry out pcr amplification, primer sequence is: 27F (5 '-AGAGTTTGATCMTGGCTCAG-3 '), 1492R (5 '-GGYTACCTTGTTACGACT-3 ').PCR reaction system is (25 μ L): template DNA 10ng, 10 × PCR damping fluid, 2.5 μ L, 25mmol/L MgCl
21.5 μ L, dNTP mix2.5 μ L, the each 0.25 μ L of primer, 5U/ μ L rTaq polysaccharase 0.125 μ L.PCR response procedures is: 94 DEG C of denaturation 5min, 35 circulations (72 DEG C are extended 2min for 94 DEG C of sex change 30s, 55 DEG C of annealing 1min) subsequently, last 72 DEG C of reaction 10min.By the pcr amplified fragment censorship order-checking after purifying, and sequencing result is carried out to similarity comparison by the blast program of Genbank in NCBI.
Molecules qualification result: the 16S rDNA region sequence of bacterium Xyn-1 is as shown in SEQ ID NO:1, this sequence has been committed to Genbank, number of registration is KJ755851, the similarity of kind Lysinibacillus sphaericus the most close to it is 91%, be a new kind bacterium, the spherical Methionin genus bacillus of called after Xyn-1.
The optimization of embodiment 3 fermentation culture conditions
Adopt single-factor analysis therapy to be optimized strain X yn-1 culture condition, determine best culture condition: substratum consumption 25mL/250mL Erlenmeyer flask, 45 DEG C of culture temperature, shaking flask rotating speed 200r/min, bacteria suspension concentration is 5 × 10
6individual/mL, every bottle of inoculum size is 1.0mL, initial pH7.0, fermentation time is 72 hours.The optimization of substratum composition:
Determine relevant 9 factors of substratum of producing enzyme to Xyn-1 by single-factor analysis therapy.Adopt Plackett-Burman design screening to produce the enzyme material impact factor, select the design of test number (TN) N=12,9 factors such as the lactose in substratum, maltose, xylan, yeast powder, peptone, sodium-chlor, magnesium sulfate, ammonium sulfate and potassium primary phosphate (%) are investigated, the results are shown in Table 1～2, on the order of producing enzyme impact be: xylan > yeast powder > potassium primary phosphate > peptone.
Table 1 strain X yn-1 cultivates factor Plackett-Burman design experiment
| Experimental group | A | B | (C) | D | E | F | G | H | I | J | (K) | Enzyme (IU/mL) alive |
| 1 | 1 | -1 | 1 | 1 | -1 | 1 | -1 | -1 | -1 | 1 | 1 | 3279.24 |
| 2 | -1 | 1 | 1 | 1 | -1 | 1 | 1 | -1 | 1 | -1 | -1 | 3391.19 |
| 3 | -1 | 1 | 1 | -1 | 1 | -1 | -1 | -1 | 1 | 1 | 1 | 3267.78 |
| 4 | -1 | -1 | -1 | 1 | 1 | 1 | -1 | 1 | 1 | -1 | 1 | 3011.58 |
| 5 | -1 | 1 | -1 | -1 | -1 | 1 | 1 | 1 | -1 | 1 | 1 | 3311.02 |
| 6 | 1 | -1 | -1 | -1 | 1 | 1 | 1 | -1 | 1 | 1 | -1 | 3138.08 |
| 7 | 1 | 1 | -1 | 1 | -1 | -1 | -1 | 1 | 1 | 1 | -1 | 3395.20 |
| 8 | 1 | -1 | 1 | -1 | -1 | -1 | 1 | 1 | 1 | -1 | 1 | 3084.53 |
| 9 | -1 | -1 | 1 | 1 | 1 | -1 | 1 | 1 | -1 | 1 | -1 | 3154.11 |
| 10 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | 3178.16 |
| 11 | 1 | 1 | -1 | 1 | 1 | -1 | 1 | -1 | -1 | -1 | 1 | 3239.72 |
| 12 | 1 | 1 | 1 | -1 | 1 | 1 | -1 | 1 | -1 | -1 | -1 | 3161.27 |
To three important factors, i.e. xylan, yeast powder, potassium primary phosphate, has carried out the center combination design based on Box-Behnken, has designed totally 17 groups of experimental points of 3 levels of 3 factors, completes response surface analysis optimization, as shown in table 2.
The experiment of table 2 strain X yn-1 fermentation condition Box-Behnken unitized design
In analytic process, set up Quadratic response surface regression model, to analyze optimal response factor level, obtained matching quadratic regression equation as follows:
Secondary respective face regression model is significant (R
2=0.9884), model-fitting degree is fine, illustrates that this model returns significantly, so the theoretical prediction that this model can be optimized for strain X yn-1 enzyme ferment condition.By data analysis, model proposes the Optimal compositions of fermentation medium condition of this process: xylan 10.7g, yeast powder 5.6g, peptone 2g, NH
4nO
34g, (NH
4)
2sO
46g, KH
2pO
40.9g, MgSO
47H
2o1g, NaCl5g, distilled water 1000mL, the enzymatic production highest level of model prediction reaches 5653.11IU/mL, compared to the 3245.5IU/mL of initial fermentation level, utilizes this optimization method can significantly improve the enzymatic production ability of strain X yn-1.
Fire resistant xylanase is produced in embodiment 4 fermentations
Slant medium: adopt beef-protein medium, specifically formula: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 20g, is dissolved in 1000mL distilled water pH7.4-7.6.
LB substratum: peptone 10g, yeast extract 5g, NaCl10g, agar 20g, is dissolved in 1000mL distilled water, pH7.0～7.2,121 DEG C of sterilizings, 20min.
Fermention medium: xylan 10.7g, yeast powder 5.6g, peptone 2g, NH
4nO
34g, (NH
4)
2sO
46g, KH
2pO
40.9g, MgSO
47H
2o1g, NaCl5g, distilled water 1000mL, pH nature;
First strain X yn-1 is seeded to slant medium, is placed at 37 DEG C, cultivate 48～72h.Choose single bacterium colony to 250mL Erlenmeyer flask from inclined-plane, LB substratum is housed in Erlenmeyer flask, liquid amount is 25mL/250mL Erlenmeyer flask, is placed in the shaking table of 200～250r/min under 37 DEG C of conditions, cultivates 12h, obtains first order seed.By the counting to first order seed, the inoculum size of controlled fermentation is that (bacteria suspension concentration is 5 × 10 to 1mL
6individual/mL).
Producing enzymic fermentation culture condition is: fermention medium consumption 25mL/250mL Erlenmeyer flask, and 45 DEG C of culture temperature, shaking speed 200r/min, bacteria suspension concentration is 5 × 10
6individual/mL, every bottle of inoculum size is 1mL, initial pH7.0, fermentation time is 72h, fermentation ends is measured the enzyme of fire resistant xylanase in fermented liquid and is lived.Zytase (Xylanase) measuring method reference literature (Bailey M J, Peter B, and Kaisa P.Inter laboratory testing of methods for assay of xylanase activity[J] .Journal of Biotechnology, 1992,23 (3): 257-270.), xylanase activity unit of force (IU) is defined as 1min hydrolyzed xylan substrate and generates the required enzyme amount of 1 μ mol wood sugar and be defined as an enzyme activity unit.Found that fermentation level is up to 5678.5IU/mL, by fermented liquid centrifugal treating 5min under the rotating speed of 5500r/min, collect supernatant liquor and be fermenting enzyme liquid.
The application that embodiment 5 strain X yn-1 produce zytase degrading maize straws hemicellulose
1. the extraction of Technique of Hemicellulose from Cornstalk
The maize straw reclaiming carries out pulverization process, sieves and obtains the stalk that mean particle size is 2mm.By straw sample washing and drying after treatment.Take 10g and dry straw sample, add wherein 1% NaOH solution 100mL, and stir 60min under 65 DEG C of conditions.After reaction finishes, add ethanol/water (50/50 volume ratio) solvent, and by filtered and recycled filtrate.Regulate filtrate pH to 6.0, and be placed in 4 DEG C of standing 30min, by solution centrifugal 5min under 5000r/min condition, remove supernatant liquor subsequently, reclaim precipitation washing and drying and obtain experiment Technique of Hemicellulose from Cornstalk sample.
2. strain X yn-1 produces zytase degrading maize straws hemicellulose
Based on the research of the above-mentioned fermenting process for strain X yn-1, determine that this bacterial strain produces the optimal culture condition of zytase, and the application of producing enzyme with this.According to the process described in embodiment 4, strain X yn-1 to be produced to enzyme and cultivate and obtain corresponding fermenting enzyme liquid, the enzyme work of measuring fire resistant xylanase in fermented liquid reaches 5678.5IU/mL.Take Technique of Hemicellulose from Cornstalk sample prepared by 0.5g, and add zytase liquid with the enzymolysis ratio of 100～150IU/g hemicellulose, under 55 DEG C of conditions, carry out enzymolysis 72h.After reaction finishes, filter and collect enzymolysis solution, utilize HPLC method to measure each reducing sugar content in enzymolysis solution, and calculate the enzymolysis efficiency of this process with this.Experimental result shows, utilizes the enzymolysis solution of strain X yn-1 fermentation preparation to carry out the degraded of Technique of Hemicellulose from Cornstalk, its xylo-bioses yield nearly 60%.
Sequence table
<110> Nanjing University of Technology
Spherical bacillus and the application thereof of a <120> high-yield thermostable zytase
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1455
<212> DNA
<213> artificial sequence
<400> 1
aggttcgggg tgctatactg caagtcgagc gaaagagaag gagcttgctc ctttgacgtt 60
agcggcggac gggtgagtaa cacgtgggca acctacctta tagtttggga taactccggg 120
aaaccggggc taataccgaa taatctgttc cacctcatgg tggaacactg aaagacggtt 180
tcggctgtcg ctataagatg ggcccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc acaatgggcg aaagcctgat 360
ggagcaacgc cgcgtgagtg aagaaggttt tcggatcgta aaactctgtt gtaagggaag 420
aacaagtaca gtagtaactg gctgtacctt gacggtacct tattagaaag ccacggctaa 480
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540
taaagcgcgc gcaggcggtc ctttaagtct gatgtgaaag cccacggctc aaccgtggag 600
ggtcattgga aactggggga cttgagtgca gaagaggaaa gtggaattcc aagtgtagcg 660
gtgaaatgcg tagagatttg gaggaacacc agtggcgaag gcgactttct ggtctgtaac 720
tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840
ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc cgttgaccac tgtagagata tagtttcccc ttcgggggca acggtgacag 1020
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080
gcaacccttg atcttagttg ccatcattta gttgggcact ctaaggtgac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtgctaca atggacgata caaacggttg ccaactcgcg agagggagct aatccgataa 1260
agtcgttctc agttcggatt gtaggctgca actcgcctac atgaagccgg aatcgctagt 1320
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac ctttggagcc agccgccgaa 1440
gggtggataa aattt 1455
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
agagtttgat cmtggctcag 20
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<400> 3
ggytaccttg ttacgact 18
Claims (8)
1. a plant height produces the spherical bacillus of fire resistant xylanase, its Classification And Nomenclature is spherical Methionin genus bacillus (Lysinibacillus sphaericus) Xyn-1, be preserved in Chinese Typical Representative culture collection center C CTCC on May 5th, 2014, deposit number is CCTCC M 2014183.
2. the spherical bacillus of high-yield thermostable zytase claimed in claim 1 produces the application of zytase in fermentation.
3. the application of the spherical bacillus of high-yield thermostable zytase claimed in claim 1 in degrading straw hemicellulose.
4. the spherical bacillus of high-yield thermostable zytase according to claim 2 produces the application of zytase in fermentation, it is characterized in that comprising the steps:
1) slant culture: spherical Methionin genus bacillus Xyn-1 is seeded on slant medium, at 37 DEG C, cultivates 48 ~ 72 h;
2) seed culture: the single bacterium colony on picking step 1) inclined-plane, in LB substratum, is placed in the shaking table of 200 ~ 250 r/min under 37 DEG C of conditions, cultivates 12 h, obtains seed liquor;
3) enzymatic production: by step 2) in seed liquor be seeded in fermention medium, inoculum size is 1 ~ 10% (v/v), the 72 h preparations of 45 DEG C of bottom fermentations are containing the fermented liquid of fire resistant xylanase.
5. the spherical bacillus of high-yield thermostable zytase according to claim 4 produces the application of zytase in fermentation, it is characterized in that: described in step 1), slant medium is: extractum carnis 3.0 g, peptone 10.0 g, NaCl 5.0 g, agar 20 g, be dissolved in 1000 mL distilled water pH 7.4-7.6.
6. the spherical bacillus of high-yield thermostable zytase according to claim 4 produces the application of zytase in fermentation, it is characterized in that: step 2) described LB substratum is: peptone 10 g, yeast extract 5 g, NaCl 10 g, agar 20 g, be dissolved in 1000 mL distilled water pH 7.0 ~ 7.2.
7. the spherical bacillus of high-yield thermostable zytase according to claim 4 produces the application of zytase in fermentation, it is characterized in that: described in step 3), fermention medium is: xylan 10.7 g, yeast powder 5.6 g, peptone 2 g, NH
4nO
34 g, (NH
4)
2sO
46 g, KH
2pO
40.9 g, MgSO
47H
2o 1 g, NaCl 5 g, distilled water 1000 mL, pH nature.
8. the application of the spherical bacillus of high-yield thermostable zytase according to claim 3 in degrading straw hemicellulose, it is characterized in that: spherical Methionin genus bacillus Xyn-1 fermentation is obtained to the fermented liquid containing fire resistant xylanase, enzymolysis ratio with 100 ~ 150 IU/g hemicelluloses adds the fermented liquid containing fire resistant xylanase, enzymolysis 72 h under 55 DEG C of conditions.
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| CN105567605A (en) * | 2016-02-19 | 2016-05-11 | 黑龙江省科学院大庆分院 | Lysinibacillus sp. and application thereof, degumming auxiliary containing lysinibacillus sp. and preparation method of degumming auxiliary |
| CN107460151A (en) * | 2017-09-29 | 2017-12-12 | 南京师范大学 | A kind of spherical lysine bacillus and its application for preventing and treating Meloidogyne incognita |
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| CN105039215A (en) * | 2015-07-14 | 2015-11-11 | 湖南龙腾生物科技有限公司 | Bacillus capable of producing xylanase and its application and screening method |
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| CN105567605A (en) * | 2016-02-19 | 2016-05-11 | 黑龙江省科学院大庆分院 | Lysinibacillus sp. and application thereof, degumming auxiliary containing lysinibacillus sp. and preparation method of degumming auxiliary |
| CN107460151A (en) * | 2017-09-29 | 2017-12-12 | 南京师范大学 | A kind of spherical lysine bacillus and its application for preventing and treating Meloidogyne incognita |
| CN107460151B (en) * | 2017-09-29 | 2020-07-14 | 南京师范大学 | Bacillus sphaericus and application thereof in preventing and treating meloidogyne incognita |
| CN108410753A (en) * | 2018-02-06 | 2018-08-17 | 安徽师范大学 | A kind of method of composite bacteria agent of degrading straw and preparation method thereof, degrading straw |
| CN108410753B (en) * | 2018-02-06 | 2021-04-13 | 安徽师范大学 | Composite microbial inoculum for degrading straw, preparation method thereof and method for degrading straw |
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