CN104212738B - Bacillus amyloliquefaciens WZZ002 and application - Google Patents
Bacillus amyloliquefaciens WZZ002 and application Download PDFInfo
- Publication number
- CN104212738B CN104212738B CN201410380289.4A CN201410380289A CN104212738B CN 104212738 B CN104212738 B CN 104212738B CN 201410380289 A CN201410380289 A CN 201410380289A CN 104212738 B CN104212738 B CN 104212738B
- Authority
- CN
- China
- Prior art keywords
- ester
- tertbutyloxycarbonyl
- boc
- wzz002
- bacillus amyloliquefaciens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 28
- -1 amino-acid ester Chemical class 0.000 claims abstract description 45
- 239000000758 substrate Substances 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 19
- 230000004151 fermentation Effects 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- 239000002609 media Substances 0.000 claims description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000001963 growth media Substances 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 10
- 239000000376 reactant Substances 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229940041514 Candida albicans extract Drugs 0.000 claims description 6
- GUBGYTABKSRVRQ-YOLKTULGSA-N Maltose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)O[C@H]1CO)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 GUBGYTABKSRVRQ-YOLKTULGSA-N 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 239000003054 catalyst Substances 0.000 claims description 6
- 239000006071 cream Substances 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 239000008346 aqueous phase Substances 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L Iron(II) sulfate Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000012071 phase Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound C1=CC=[Tl]C=C1 HNBDQABBWNOTRU-UHFFFAOYSA-N 0.000 claims description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims 2
- 229910052564 epsomite Inorganic materials 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 11
- 102000004190 Enzymes Human genes 0.000 abstract description 11
- 235000001014 amino acid Nutrition 0.000 abstract description 7
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 238000006555 catalytic reaction Methods 0.000 abstract description 4
- 201000002574 conversion disease Diseases 0.000 abstract description 4
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 abstract 3
- QVHJQCGUWFKTSE-YFKPBYRVSA-N (2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-YFKPBYRVSA-N 0.000 description 10
- 229940088598 Enzyme Drugs 0.000 description 10
- 230000001580 bacterial Effects 0.000 description 10
- 239000007789 gas Substances 0.000 description 9
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 5
- 150000002148 esters Chemical class 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000002194 synthesizing Effects 0.000 description 5
- QVHJQCGUWFKTSE-RXMQYKEDSA-N (2R)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)[C@@H](C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-RXMQYKEDSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- DWKPPFQULDPWHX-UHFFFAOYSA-N methyl 2-aminopropanoate Chemical compound COC(=O)C(C)N DWKPPFQULDPWHX-UHFFFAOYSA-N 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229920001670 16S ribosomal RNA Polymers 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 230000024881 catalytic activity Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- ZFNFXIVKGTXSMW-UHFFFAOYSA-N ethyl 2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CCOC(=O)C(C)NC(=O)OC(C)(C)C ZFNFXIVKGTXSMW-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000006011 modification reaction Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 210000004215 spores Anatomy 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000003805 vibration mixing Methods 0.000 description 2
- IVBOUFAWPCPFTQ-SFYZADRCSA-N (3S)-3-azaniumyl-4-oxo-4-[[(2R)-1-oxo-1-[(2,2,4,4-tetramethylthietan-3-yl)amino]propan-2-yl]amino]butanoate Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C)C(=O)NC1C(C)(C)SC1(C)C IVBOUFAWPCPFTQ-SFYZADRCSA-N 0.000 description 1
- QVHJQCGUWFKTSE-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)C(C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-UHFFFAOYSA-N 0.000 description 1
- FDWFFCURSPACFQ-UHFFFAOYSA-N 2-[(2-phenylacetyl)amino]propanoic acid Chemical compound OC(=O)C(C)NC(=O)CC1=CC=CC=C1 FDWFFCURSPACFQ-UHFFFAOYSA-N 0.000 description 1
- VMAQYKGITHDWKL-UHFFFAOYSA-N 5-methylimidazolidine-2,4-dione Chemical compound CC1NC(=O)NC1=O VMAQYKGITHDWKL-UHFFFAOYSA-N 0.000 description 1
- 239000004377 Alitame Substances 0.000 description 1
- 229950010030 DL-Alanine Drugs 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N DL-alanine Chemical compound CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid zwitterion Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- HPHUVLMMVZITSG-ZCFIWIBFSA-N Levetiracetam Chemical compound CC[C@H](C(N)=O)N1CCCC1=O HPHUVLMMVZITSG-ZCFIWIBFSA-N 0.000 description 1
- 229940040461 Lipase Drugs 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 229940049954 Penicillin Drugs 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- IPVQLZZIHOAWMC-QXKUPLGCSA-N Perindopril Chemical compound C1CCC[C@H]2C[C@@H](C(O)=O)N(C(=O)[C@H](C)N[C@@H](CCC)C(=O)OCC)[C@H]21 IPVQLZZIHOAWMC-QXKUPLGCSA-N 0.000 description 1
- 229960002582 Perindopril Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 235000019409 alitame Nutrition 0.000 description 1
- 108010009985 alitame Proteins 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 108010003977 aminoacylase I Proteins 0.000 description 1
- 238000005284 basis set Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 229960000626 benzylpenicillin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000000875 corresponding Effects 0.000 description 1
- 230000004059 degradation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 150000004715 keto acids Chemical class 0.000 description 1
- 229960004002 levetiracetam Drugs 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 102000004882 lipase Human genes 0.000 description 1
- 108090001060 lipase Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- GJDICGOCZGRDFM-ZCFIWIBFSA-N methyl (2R)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound COC(=O)[C@@H](C)NC(=O)OC(C)(C)C GJDICGOCZGRDFM-ZCFIWIBFSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000003287 optical Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 230000000707 stereoselective Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Abstract
The invention discloses strain new strains bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZZ002 and prepare the application in Boc D amino-acid ester and Boc L aminoacid at catalysis raceme Boc DL amino-acid ester；The regioselectivity of enzyme of the present invention is strong, and reaction conversion ratio is high, and downstream separation is simple, and energy consumption is low, and environmental pollution is little, is suitable for industrialized production.
Description
(1) technical field
The present invention relates to a kind of enzyme process and decompose the method that raceme Boc-DL-amino-acid ester prepares chiral intermediate, especially
Relate to carrying out stereo selectivity under the ester hydrolase effect that bacillus amyloliquefaciens WZZ002 (CCTCC M 2013366) produces
Hydrolysis synthesizing optical pure Boc-D-amino-acid ester and the amino acid whose method of Boc-L-.
(2) background technology
N-tertbutyloxycarbonyl-D-amino-acid ester (being called for short Boc-D-amino-acid ester) and (letter of N-tertbutyloxycarbonyl-l-amino acid
Claim Boc-L-aminoacid) it is a kind of important biochemical reagents, protect monomer for Peptide systhesis and aminoacid.Boc-D-aminoacid
Ester can obtain corresponding D-aminoacid and l-amino acid by chemical hydrolysis with Boc-L-amino-acid ester.D-aminoacid is as non-
Natural amino acid, at medicine and pesticide synthesis, food, the aspect such as feedstuff tool has been widely used.Wherein D-alanine one weight
The medicine intermediate wanted, is widely used in the fields such as medicine and food additive, is mainly used in synthesizing new sweeting agent alitame (L-
Aspartoyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-aminopropanamide) and some chirality pharmaceutical intermediate compound is former
Material.Wherein L-norvaline is the important intermediate of resisting hypertension class medicine perindopril.C4H9NO2 is antuepileptic
The important intermediate of thing levetiracetam.
At present, the synthesis Boc-D-alanine ester of document report is to obtain after substrate B oc replaces esterification by D-alanine.
Wherein the synthetic method of D-alanine is mainly chemical resolution method.Chemical resolution process routes also exists that yield is low, cost is high, step
The most various, product purity is relatively low, the problem such as seriously polluted.And enzyme process is prepared D-alanine method and is mainly had: (1) is with chemosynthesis
5-methyl hydantoin be substrate, utilize glycolylurea lyases to prepare；(2) acetyl or the phenylacetyl-DL-Alanine of chemosynthesis is the end
Thing, utilizes amino-acylase or the acylated fractionation of penicillin；(3) with DL-Alanine as substrate, microbial degradation method；(4) with third
Keto acid is substrate, utilizes aminotransferase to prepare.At present bioanalysis is prepared D-alanine to there is enzyme selectivity the highest, produces into
This height, the problems such as precursor substrate synthesis is difficult.
(3) summary of the invention
The present invention seeks to by screening yielding lipase microorganism, it is provided that the producing microbial enzyme bacterium of a strain Cheap highly effective
Strain, with raceme Boc-DL-amino-acid ester as reaction substrate, in catalysis stereo selective hydrolysis Boc-DL-amino-acid ester, L-is different
Structure body thus prepare Boc-D-amino-acid ester and the amino acid whose method of Boc-L-.
The technical solution used in the present invention is:
The present invention provides a strain new strains--bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
WZZ002, is preserved in China typical culture collection center, preservation address: China, Wuhan, Wuhan University, and postcode 430072 is protected
Hide numbering CCTCC M 2013366, preservation date on August 8th, 2013.
The invention still further relates to a kind of described bacillus amyloliquefaciens WZZ002 and split N-tertbutyloxycarbonyl-DL-aminoacid
Application in ester, concrete described application is: cultivate, so that bacillus amyloliquefaciens WZZ002 is fermented, the wet thallus lyophilizing obtained
After dry mycelium be catalyst, with raceme N-tertbutyloxycarbonyl-DL-amino-acid ester (i.e. Boc-DL-amino acid methyl ester) as the end
Thing, in the phosphate buffer that pH value is 6～9 (preferable ph is 7.0), carries out converting instead under 20～50 DEG C (preferably 30 DEG C)
Should, after reaction terminates, by reactant liquor post processing, it is thus achieved that N-tertbutyloxycarbonyl-D-amino-acid ester (Boc-D-amino-acid ester) and N-
Tertbutyloxycarbonyl-l-amino acid (Boc-L-aminoacid).
Further, the quality consumption of described dry mycelium is 1～50g/L buffer, preferably 10～20g/L, the described initial end
Substrate concentration is 1～200g/L buffer, preferably 50～100g/L.
Further, the most described substrate is N-tertbutyloxycarbonyl-DL-Alanine ester (Boc-DL-alanine ester), the tertiary fourth of N-
Oxygen carbonyl-DL-2-aminobutyric acid ester (Boc-DL-2-aminobutyric acid ester), N-tertbutyloxycarbonyl-DL-norvaline ester (Boc-
DL-norvaline ester), N-tertbutyloxycarbonyl-DL-methionine ester (Boc-DL-methionine ester), N-tertbutyloxycarbonyl-DL-dried meat ammonia
Acid esters (Boc-DL-proline ester), N-tertbutyloxycarbonyl-DL-serine ester (Boc-DL-serine ester), N-tertbutyloxycarbonyl-
DL-LEUCINE ester (Boc-DL-leucine ester), N-tertbutyloxycarbonyl-DL-isoleucine ester (Boc-DL-isoleucine ester), N-
Tertbutyloxycarbonyl-DL-threonine ester (Boc-DL-threonine ester), N-tertbutyloxycarbonyl-DL-Trp ester (Boc-DL-color ammonia
Acid esters) or N-tertbutyloxycarbonyl-DL-phenylalanine ester (Boc-DL-phenylalanine ester).
Further, catalyst of the present invention is prepared as follows:
(1) slant culture
Bacillus amyloliquefaciens WZZ002 is seeded to slant medium, cultivates 24h at 30 DEG C, it is thus achieved that inclined-plane bacterium colony；Tiltedly
Face culture medium final concentration composition: NaNO33.0g/L, KH2PO41.0g/L, MgSO4·7H2O0.5g/L, KCl0.5g/L, FeSO4·
7H2O0.01g/L, Boc-DL-alanine ester 10mmol/L, solvent is deionized water, pH7.0；
(2) seed culture
The inclined-plane colony inoculation that step (1) is obtained in seed culture medium, 30 DEG C, 200r/min cultivate 24h, it is thus achieved that plant
Sub-liquid；Seed culture medium final concentration forms: peptone 5g/L, Carnis Bovis seu Bubali cream 5g/L, yeast extract 1g/L, maltose 5g/L,
NaCl0.5g/L, agar 23g/L, solvent is deionized water, pH7.0；
(3) fermentation culture
Aseptically, seed liquor is inoculated in fermentation medium with the inoculum concentration of volumetric concentration 5～20%
(250mL triangular flask, liquid amount is 50mL), 30 DEG C, 200r/min cultivate 24h, it is thus achieved that fermentation liquid, fermentation liquid is centrifuged, abandons
Clear liquid, obtains bacillus amyloliquefaciens WZZ002 wet thallus, wet thallus is suspended in the phosphate-buffered of pH7.0,0.2mol/L
In liquid, stir, lyophilization under the conditions of-40 DEG C, obtain dry mycelium；Fermentation medium consists of: peptone 5g/L, beef
Cream 5g/L, yeast extract 1g/L, MgSO41mmol/L, solvent is deionized water, pH7.0.
The method of reactant liquor post processing of the present invention is: after conversion reaction is complete, conversion reaction liquid is filtered to remove bacterium
Body, filtrate extracts (preferably 2 times) with organic solvent, obtains organic facies and aqueous phase respectively, and organic facies decompression is distilled off solvent, obtains
Obtain the N-tertbutyloxycarbonyl-D-alanine methyl ester product of high-optical-purity；Described organic solvent is normal hexane, hexamethylene or acetic acid
Ethyl ester；Aqueous phase regulation pH value (preferably adds hydrochloric acid regulation) to after 2.0, extracts with extractant, takes the organic facies after extraction and obtain
Boc-L-aminoacid, described extractant is normal hexane, hexamethylene or ethyl acetate.
Enzyme is lived and is defined as: the enzyme amount at 30 DEG C, needed for the BOC-DL-alanine ester of catalyzing hydrolysis 1 μm ol per minute.Enzyme
Condition determination method of living is: take 0.2g dry mycelium, adds the phosphoric acid containing final concentration 5g/L Boc-DL-amino-acid ester in thalline
Buffer solution (pH value is 7.0) 10ml, under the conditions of 30 DEG C, speed of agitator 200rpm, after reaction 20min, it is filtered to remove enzyme, takes filter
Liquid gas chromatography determination Boc-D-alanine ester, Boc-L-alanine ester, Boc-D-alanine and Boc-L-alanine
Content.
The present invention, with the Boc-DL-amino-acid ester shown in formula I as reaction substrate, (i.e. solves starch spore bar at catalyst
Bacterium WZZ002 fermentation culture obtain dry mycelium, this dry mycelium produce microorganism ester hydrolase) effect under, carry out selective hydrolysis
The prepared described Boc-D-amino-acid ester (II) of reaction and Boc-L-aminoacid (III):
In formula I, (II) and (III), R group is the alkyl of C1～C4, R1Group is the side-chain radical of a-amino acid, excellent
Elect methyl as.
The present invention provides a strain new strains--bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
WZZ002 and catalysis raceme Boc-DL-amino-acid ester prepare the application in Boc-D-amino-acid ester and Boc-L-aminoacid,
The regioselectivity that described bacillus amyloliquefaciens WZZ002 produces enzyme is strong, and reaction conversion ratio is high, and downstream separation is simple, energy consumption
Low, environmental pollution is little, is suitable for industrialized production.
(4) accompanying drawing explanation
Fig. 1 racemic modification Boc-DL-methyl lactamine standard substance gas chromatogram；
The gas chromatogram of Fig. 2 Boc-L-methyl lactamine standard substance；
The gas chromatogram of Fig. 3 microorganism catalysis product Boc-D-methyl lactamine.
Fig. 4 is bacterial strain WZZ002 phylogenetic evolution tree.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
Embodiment 1: the screening of bacterial strain and qualification
1, the screening of bacterial strain WZZ002
Weigh wet soil sample 1g (obtaining soil near Shang Tang river, Hangzhou, Zhejiang province city) and be suspended in 10mL sterile physiological salt
In water, vibration mixing；Connect 3mL suspension in 50mL liquid enrichment medium, 30 DEG C, cultivate 4～5d under 200r/min after,
Bacterium solution muddy for sterile working switching 1mL, to fresh liquid enrichment medium, is enriched with 3～4 times continuously.In enrichment medium,
With Boc-DL-alanine ester as sole carbon source, formula is as follows: NaNO33.0g/L, KH2PO41.0g/L, MgSO4·7H2O0.5g/
L, KCl0.5g/L, FeSO4·7H2O0.01g/L, Boc-DL-alanine ester 10mmol/L, solvent is deionized water, pH7.0.Will
After enrichment, bacterium solution is through normal saline gradient dilution, and coating separates plating medium, separating for several times, obtains single bacterium colony.Separate flat board
Culture medium consists of: enrichment medium adds agar 23g/L (final concentration), pH7.0.
Single colony inoculation of picking isolated, in slant medium, cultivates 24h at 30 DEG C, from slant medium picking one
Inoculating loop colony inoculation to seed culture medium (liquid amount is 50ml), 30 DEG C, 200r/min cultivate 24h, aseptically,
Take 3mL seed liquor to be inoculated in the fermentation medium of 50mL, 30 DEG C, 200r/min cultivate 24h, it is thus achieved that fermentation liquid.Slant culture
Basis set one-tenth is with to separate plating medium consistent；Seed culture based formulas is: peptone 5g/L, Carnis Bovis seu Bubali cream 5g/L, yeast extract 1g/L,
Maltose 5g/L, NaCl0.5g/L, solvent is deionized water, pH7.0；Fermentation medium consists of: peptone 5g/L, Carnis Bovis seu Bubali cream
5g/L, yeast extract 1g/L, MgSO41mmol/L, solvent is deionized water, pH7.0.
Taking 10mL fermentation liquid in 10mL centrifuge tube, 6000rpm is centrifuged 15min, abandons supernatant, add 2mL0.2mol/L,
The phosphate buffer of pH7.0, vibration mixing, add final concentration 5.26g/L substrate B OC-DL-methyl lactamine.30 DEG C,
Under 200r/min, convert 2h.Taking out centrifuge tube, add 2mL ethyl acetate, fully vibrate, 6000rpm is centrifuged 15min.Take upper strata
Organic facies 1mL, is dried except water with a small amount of anhydrous sodium sulfate, with gas chromatographic detection asymmetric hydrolysis product Boc-D-alanine first
The gas phase adsorption peak of ester and Boc-L-alanine, and calculate enantiomeric excess value (e.e.), screening enantiomeric excess value reaches
The bacterial strain of 98%, i.e. obtains bacterial strain WZZ002.Boc-DL-methyl lactamine and Boc-L-methyl lactamine standard substance and conversion produce
Shown in gas chromatogram as Fig. 1-Fig. 3 of thing Boc-D-methyl lactamine.
Enantiomeric excess value computing formula:
Wherein, e.e.s represents the enantiomeric excess value of Boc-D-methyl lactamine, e.e.pRepresent Boc-L-alanine
Enantiomeric excess value, AD1, AL1, AL2And AD2Be respectively Boc-D-alanine ester, Boc-L-alanine ester, Boc-L-alanine,
The peak area of the gas chromatographic detection of Boc-D-alanine.
Substrate conversion efficiency computing formula:
In formula (3), c is substrate conversion efficiency.
Analytical conditions for gas chromatography: use Agilent6890 type gas chromatograph；Chiral gas chromatography post BGB-174
(0.25mm×30m)；Carrier gas N2(70kPa), split ratio 50:1, injector temperature 250 DEG C, detector temperature 250 DEG C；Post case liter
Temperature program: initial temperature 100 DEG C, keeps 3min, 5 DEG C/min to be warming up to 200 DEG C, keeps 2min；Sample size 1.5 μ L, air stream
Amount: 300mL/min；Make-up gas flow: N225mL/min；Hydrion flame detector.Boc-D-methyl lactamine and Boc-L-
Methyl lactamine goes out peak at 13.9min and 14.2min respectively, and hydrolyzate Boc-D-alanine and Boc-L-alanine exist respectively
22.2min and 22.5min goes out peak.
2, the qualification of bacterial strain WZZ002
(1) physiological and biochemical property: bacterial strain WZZ002 is gram positive bacteria, obligate aerobic, grows pH change more sensitive,
Optimum pH is 7.0.Utilize inorganic nitrogen-sourced ability poor.Growth and product enzyme are by Cu2+、Mn2+、Zn2+Deng suppression, by Mg2 +、K+Deng promotion.
(2) 16S rDNA sequence
Through student on commission work biological engineering (Shanghai) limited company, order-checking is identified, the 16S rDNA sequence of bacterial strain WZZ002
Shown in SEQ ID NO.1:
GCGCTTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTG
GGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTC
AGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCA
CCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG
GAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGG
ATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAG
CCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGG
CTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTG
AGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGG
CGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC
GCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCT
GGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT
CGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCA
GAGTGACAGGTGGTGCATGGTGTCGTCAGCTCGTGTCATGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT
GATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT
CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGG
TTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGT
AATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTA
ACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGAAA
According to physio-biochemical characteristics and molecular biology identification, and bacterial strain WZZ002 phylogenetic evolution tree (Fig. 4), will
Bacterial strain WZZ002 is accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens), named solution starch spore bar
Bacterium (Bacillus amyloliquefaciens) WZZ002, is preserved in China typical culture collection center (address China, force
The Chinese, Wuhan University, 430072), deposit number CCTCC M 2013366, preservation date on August 8th, 2013.
Embodiment 2: the preparation of catalyst
(1) slant culture
Bacillus amyloliquefaciens WZZ002 is seeded to slant medium, cultivates 24h at 30 DEG C, it is thus achieved that inclined-plane bacterium colony.Tiltedly
Face culture medium forms with embodiment 1.
(2) seed culture
The inclined-plane colony inoculation that step (1) is obtained in the seed culture medium of 50mL, 30 DEG C, 200r/min cultivate 24h,
Obtain seed liquor.Seed culture medium forms with embodiment 1.
(3) fermentation culture
Aseptically, take 3mL seed liquor and be inoculated in the fermentation medium of 50mL that (250mL triangular flask, liquid amount is
50mL), 30 DEG C, 200r/min cultivate 24h, it is thus achieved that fermentation liquid.Fermentation liquid 6000rpm is centrifuged 15min after terminating by cultivation, abandons
Clear liquid, obtains bacillus amyloliquefaciens WZZ002 wet thallus 8.5g, adds the phosphate buffer of 10ml, pH7.0,0.2mol/L
Stir, lyophilization under the conditions of-40 DEG C, obtain dry mycelium 0.8g.Through enzyme activity determination, this dry mycelium specific enzyme activity power is
5.0U/g。
Embodiment 3:
Bacillus amyloliquefaciens WZZ002 dry mycelium 0.1g embodiment 2 method prepared adds containing 20g/L Boc-
In the 10mL pH7.0 phosphate buffer of DL-Alanine methyl ester, react after 1h under the conditions of 30 DEG C, reaction convert after reactant liquor from
The heart, takes supernatant, adds isopyknic ethyl acetate and extracts after 2 times, merges organic facies, and decompression is distilled off solvent, obtains
Boc-D-methyl lactamine product；After aqueous phase is by interpolation hydrochloric acid to pH2.0, extracts by extractant ethyl acetate, take organic facies
Obtain Boc-L-alanine.Use the enantiomeric excess value of method detection Boc-D-methyl lactamine described in embodiment 1
E.e.99.9%, Boc-L-alanine e.e.99.4%, substrate conversion efficiency 50.9%.
Embodiment 4:
Bacillus amyloliquefaciens WZZ002 dry mycelium 0.2g embodiment 2 method prepared adds containing 100g/L Boc-
Reactant liquor in 10mL, pH7.0 phosphate buffer of DL-Alanine methyl ester, after reacting 3h under the conditions of 30 DEG C, after reaction is converted
Centrifugal, other operations are with embodiment 3, enantiomeric excess value e.e.99.6%, the Boc-L-alanine of Boc-D-methyl lactamine
E.e.99.8%, substrate conversion efficiency 50.2%.
Embodiment 5:
Bacillus amyloliquefaciens WZZ002 dry mycelium 0.5g embodiment 2 method prepared adds containing 200g/L Boc-
In 10mL, pH7.0 phosphate buffer of DL-Alanine methyl ester, react after 3h under the conditions of 30 DEG C, reaction convert after reactant liquor from
The heart, other operations are with embodiment 3, enantiomeric excess value e.e.99.9%, the Boc-L-alanine of Boc-D-methyl lactamine
E.e.99.5%, substrate conversion efficiency 49.8%.
Embodiment 6:
The bacillus amyloliquefaciens WZZ002 dry mycelium 0.5g respectively prepared by embodiment 2 method be sequentially added into containing
100g/L Boc-DL-methyl lactamine, Boc-DL-alanine ethyl ester, Boc-DL-alanine propyl ester and Boc-DL-alanine fourth
In 10mL, pH7.0 phosphate buffer of ester, after reacting 3h under the conditions of 30 DEG C, the reactant liquor after reaction converts is centrifuged, other operations
With embodiment 3, respectively obtaining Boc-L-alanine ester compound, result is as shown in table 1.
The different substrate conversion efficiency of table 1
| Substrate title | Substrate conversion efficiency | Boc-D-alanine ester e.e. | Boc-L-alanine e.e. |
| Boc-DL-methyl lactamine | 50.1% | 99.9% | 99.5% |
| Boc-DL-alanine ethyl ester | 50.1% | 99.9% | 99.4% |
| Boc-DL-alanine propyl ester | 50.5% | 99.6% | 99.2% |
| Boc-DL-alanine butyl ester | 50.2% | 99.5% | 99.0% |
Embodiment 7:
Bacillus amyloliquefaciens WZZ002 dry mycelium 0.5g embodiment 2 method prepared adds containing 100g/L, difference
In the 10mL pH7.0 phosphate buffer of substrate, after reacting 5h under the conditions of 30 DEG C, the reactant liquor after reaction converts is centrifuged, other behaviour
Making with embodiment 3, reaction result is as shown in table 2.
The conversion ratio of the different substrate of table 2
The above, be only presently preferred embodiments of the present invention, and not the technology contents to the present invention makees any form
On restriction.Any simple modification that above example is made by every technical spirit according to the present invention, equivalent variations with repair
Decorations, each fall within protection scope of the present invention.
Claims (7)
1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZZ002, is preserved in Chinese Typical Representative culture and protects
Center, Tibetan, preservation address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC M 2013366, preservation day
August 8 2013 phase.
2. bacillus amyloliquefaciens WZZ002 described in a claim 1 is in splitting N-tertbutyloxycarbonyl-DL-amino-acid ester
Application.
Apply the most as claimed in claim 2, it is characterised in that described application is: with bacillus amyloliquefaciens WZZ002 through sending out
It is catalyst that ferment cultivates the dry mycelium after the wet thallus lyophilizing obtained, with raceme N-tertbutyloxycarbonyl-DL-amino-acid ester as the end
Thing, in the phosphate buffer that pH value is 6～9, carries out conversion reaction, after reaction terminates, after reactant liquor at 20～50 DEG C
Process, it is thus achieved that N-tertbutyloxycarbonyl-D-amino-acid ester and N-tertbutyloxycarbonyl-l-amino acid.
Apply the most as claimed in claim 3, it is characterised in that the quality consumption of described dry mycelium is 1～50g/L buffer, institute
The initial concentration stating substrate is 1～200g/L buffer.
Apply the most as claimed in claim 3, it is characterised in that described substrate is N-tertbutyloxycarbonyl-DL-Alanine ester, uncle N-
Butoxy carbonyl-DL-2-aminobutyric acid ester, N-tertbutyloxycarbonyl-DL-norvaline ester, N-tertbutyloxycarbonyl-DL-methionine ester,
N-tertbutyloxycarbonyl-DL-proline ester, N-tertbutyloxycarbonyl-DL-serine ester, N-tertbutyloxycarbonyl-DL-LEUCINE ester, N-
Tertbutyloxycarbonyl-DL-isoleucine ester, N-tertbutyloxycarbonyl-DL-threonine ester, N-tertbutyloxycarbonyl-DL-Trp ester or
N-tertbutyloxycarbonyl-DL-phenylalanine ester.
Apply the most as claimed in claim 3, it is characterised in that described catalyst is prepared as follows: (1) slant culture
Bacillus amyloliquefaciens WZZ002 is seeded to slant medium, cultivates 24h at 30 DEG C, it is thus achieved that inclined-plane bacterium colony；Inclined-plane is trained
Support base final concentration composition: NaNO33.0g/L, KH2PO41.0g/L, MgSO4·7H2O 0.5g/L, KCl 0.5g/L, FeSO4·
7H2O 0.01g/L, Boc-DL-alanine ester 10mmol/L, agar 23g/L, solvent is deionized water, pH 7.0；
(2) seed culture
The inclined-plane colony inoculation that step (1) is obtained in seed culture medium, 30 DEG C, 200r/min cultivate 24h, it is thus achieved that seed
Liquid；Seed culture medium final concentration forms: peptone 5g/L, Carnis Bovis seu Bubali cream 5g/L, yeast extract 1g/L, maltose 5g/L, NaCl
0.5g/L, solvent is deionized water, pH 7.0；
(3) fermentation culture
Aseptically, seed liquor is inoculated in fermentation medium with the inoculum concentration of volumetric concentration 5～20%, 30 DEG C,
200r/min cultivates 24h, it is thus achieved that fermentation liquid, is centrifuged by fermentation liquid, abandons supernatant, obtains the wet bacterium of bacillus amyloliquefaciens WZZ002
Body, is suspended in wet thallus in the phosphate buffer of pH 7.0,0.2mol/L, stirs, and lyophilization obtains dry bacterium
Body；Fermentation medium consists of: peptone 5g/L, Carnis Bovis seu Bubali cream 5g/L, yeast extract 1g/L, MgSO41mmol/L, solvent for go from
Sub-water, pH 7.0.
Apply the most as claimed in claim 3, it is characterised in that the method for described reactant liquor post processing is: conversion reaction is complete
After, conversion reaction liquid is filtered to remove thalline, filtrate extracts with organic solvent, obtains organic facies and aqueous phase respectively, organic subtracts each other
Pressure is distilled off solvent, it is thus achieved that N-tertbutyloxycarbonyl-D-amino-acid ester；Described organic solvent is normal hexane, hexamethylene or acetic acid
Ethyl ester；Aqueous phase adjusts pH value to after 2.0, extracts with extractant, takes the organic facies after extraction and obtain N-tertbutyloxycarbonyl-L-amino
Acid, described extractant is normal hexane, hexamethylene or ethyl acetate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410380289.4A CN104212738B (en) | 2014-08-04 | Bacillus amyloliquefaciens WZZ002 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410380289.4A CN104212738B (en) | 2014-08-04 | Bacillus amyloliquefaciens WZZ002 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104212738A CN104212738A (en) | 2014-12-17 |
| CN104212738B true CN104212738B (en) | 2017-01-04 |
Family
ID=
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881952A (en) * | 2014-04-03 | 2014-06-25 | 大连理工大学 | Bacillus amyloliquefaciens and an application thereof as well as method for preparing kelp feed |
| RO129512A2 (en) * | 2012-12-07 | 2014-06-30 | Institutul De Cercetare-Dezvoltare Pentru Protecţia Plantelor | Strain of bacillus amyloliquefaciens which can be used as agricultural inoculant in the substrata with high phytosanitary risk and as improver for lands contaminated with hydrocarbons |
| CN103951736A (en) * | 2014-05-09 | 2014-07-30 | 山东省农业科学院农产品研究所 | Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RO129512A2 (en) * | 2012-12-07 | 2014-06-30 | Institutul De Cercetare-Dezvoltare Pentru Protecţia Plantelor | Strain of bacillus amyloliquefaciens which can be used as agricultural inoculant in the substrata with high phytosanitary risk and as improver for lands contaminated with hydrocarbons |
| CN103881952A (en) * | 2014-04-03 | 2014-06-25 | 大连理工大学 | Bacillus amyloliquefaciens and an application thereof as well as method for preparing kelp feed |
| CN103951736A (en) * | 2014-05-09 | 2014-07-30 | 山东省农业科学院农产品研究所 | Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product |
Non-Patent Citations (2)
| Title |
|---|
| Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens;X.H. Chen et al.;《Journal of Biotechnology》;20091231;第140卷;第27-37页 * |
| 解淀粉芽孢杆菌（Bacillus amyloliquefaciens）的研究进展;车晓曦等;《北京农业》;20100131;第7-10页 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104342390B (en) | A kind of Sinorhizobium meliloti strain and combinations thereof and application | |
| CN110527646A (en) | Tropical bacillus WZZ018 and its application | |
| CN104774792B (en) | The Methyldmonas of one plant of enduring high-concentration methanol and its application | |
| CN106282041A (en) | One plant height produces the bacillus subtilis mutant strain of surface element and utilizes this mutant to carry out the method that semi-solid ferment produces surface element | |
| CN104745509B (en) | Sugar vacation anthropi WZZ003 and its application are not understood | |
| CN101535467A (en) | A levorotatory lactonohydrolase producing strain and its use for producing chiral oxyacid | |
| CN102181370B (en) | South China sea mangrove endophytic fungus BL321 | |
| CN109762768A (en) | Bacillus B8W22 and its application | |
| CN104531577B (en) | Arthrobacter nicotinovorans WYG001 and application thereof in preparation of N-BOC-L-homoserine lactone | |
| CN101113423B (en) | Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion | |
| CN109504634A (en) | Bacillus WZZ006 and its application | |
| CN105316257A (en) | lysinibacillus xylanilyticus and method for preparing alpha-ketoacid with same | |
| CN102994429B (en) | Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain | |
| CN104630116A (en) | Streptomyces sp. and application thereof | |
| CN110438015A (en) | The method that the dried immature fruit of citron orange endogenetic fungus and its fermentation for producing hesperidinase produce hesperidinase | |
| CN102485879A (en) | Fermentation medium used for producing WF 11899A | |
| CN104212738B (en) | Bacillus amyloliquefaciens WZZ002 and application | |
| Fu et al. | Continuous fermentation of a prodigiosin-producing Serratia marcescens strain isolated from soil | |
| CN103589665B (en) | Celebrating sheng, a reed pipe wind instrument rhodococcus and the application in preparation (S)-4-chloro-3-hydroxyl ethyl butyrate thereof | |
| CN113337432B (en) | Methylophilus for producing pyrroloquinoline quinone and application thereof | |
| CN106086090B (en) | A kind of method that two-step microbial conversion method prepares R-MA | |
| CN108220188A (en) | A kind of molten bacillus and its application | |
| CN104250621A (en) | Sea streptomyces cinereoruber and method of applying same to prepare aurone compound | |
| CN102199546B (en) | Agromyces sp. and application thereof in preparation of (S)-epichlorohydrin through hydrolysis | |
| CN102559540B (en) | Flavobacterium aquati and application thereof in preparation of L-tryptophan by microbial transformation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |