CN101555502B - Method for preparing L-tryptophan by enzymatic conversion - Google Patents
Method for preparing L-tryptophan by enzymatic conversion Download PDFInfo
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- CN101555502B CN101555502B CN2009100293478A CN200910029347A CN101555502B CN 101555502 B CN101555502 B CN 101555502B CN 2009100293478 A CN2009100293478 A CN 2009100293478A CN 200910029347 A CN200910029347 A CN 200910029347A CN 101555502 B CN101555502 B CN 101555502B
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 106
- 229960004799 tryptophan Drugs 0.000 title claims abstract description 101
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 46
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 18
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims abstract description 79
- 229960001153 serine Drugs 0.000 claims abstract description 39
- 241000894006 Bacteria Species 0.000 claims abstract description 16
- 229940024606 amino acid Drugs 0.000 claims abstract description 16
- 150000001413 amino acids Chemical class 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 9
- 238000010353 genetic engineering Methods 0.000 claims abstract description 6
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 3
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims description 31
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- 239000000243 solution Substances 0.000 claims description 24
- 150000002475 indoles Chemical class 0.000 claims description 16
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 11
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 5
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
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- 239000005715 Fructose Substances 0.000 claims description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 4
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
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- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 3
- 238000005903 acid hydrolysis reaction Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- -1 polyoxyethylene octyl phenyl ether Polymers 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 239000008351 acetate buffer Substances 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 2
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- 235000013379 molasses Nutrition 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 2
- 229920002114 octoxynol-9 Polymers 0.000 claims description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims 1
- 229930195722 L-methionine Natural products 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 claims 1
- 239000000284 extract Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 claims 1
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims 1
- 239000002699 waste material Substances 0.000 claims 1
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- 102000003960 Ligases Human genes 0.000 abstract 1
- 108090000364 Ligases Proteins 0.000 abstract 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 34
- 239000006035 Tryptophane Substances 0.000 description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 238000000855 fermentation Methods 0.000 description 11
- 230000004151 fermentation Effects 0.000 description 11
- 230000009466 transformation Effects 0.000 description 11
- 238000000746 purification Methods 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 102100040653 Tryptophan 2,3-dioxygenase Human genes 0.000 description 6
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- INPQIVHQSQUEAJ-UHFFFAOYSA-N 5-fluorotryptophan Chemical compound C1=C(F)C=C2C(CC(N)C(O)=O)=CNC2=C1 INPQIVHQSQUEAJ-UHFFFAOYSA-N 0.000 description 3
- 102000002667 Glycine hydroxymethyltransferase Human genes 0.000 description 3
- 108010043428 Glycine hydroxymethyltransferase Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 101150050470 ase gene Proteins 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 2
- 125000001041 indolyl group Chemical group 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
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- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical class CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-Serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 description 1
- 229930195711 D-Serine Natural products 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
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- FQXBMKZVLSACGS-UHFFFAOYSA-N n,n-dimethylmethanamine;hexadecane;hydrobromide Chemical compound Br.CN(C)C.CCCCCCCCCCCCCCCC FQXBMKZVLSACGS-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the technical field of medicine and biology and particularly relates to a method for preparing L-tryptophan by enzymatic conversion. The method, of which L-tryptophan is prepared from mixed amino acids containing L-serine, comprises the following steps: mixing the high-activity genetic engineering bacteria of L-tryptophan synthetase or L-tryptophanase with the conversion solution of the mixed amino acid containing L-serine; carrying out the enzymatic reaction at the temperature of 30 to 45 DEG C; and then, separating the converted products by the isoelectric point crystallization method or the method combining isoelectric point crystallization and ion exchange resin to obtain high-purity L-tryptophan. The method solves the problems of the separation and overall development of L-serine in the mixed amino acids by obtaining L-serine products with wider applicable range and higher added value, therefore, the invention has the advantages of wide material availability, low price, convenient operation, short conversion time, low production cost and the like.
Description
One, technical field
The invention belongs to the medical biotechnology field, be specifically related to the enzymatic conversion preparation method of L-tryptophane.
Two, background technology
Tryptophane (tryptophan) has another name called pantonine-indolepopionic acid, is the seed amino acid that Hopkins and Cole found and were separated in 1902.It has D-and two kinds of isomer of L-type, and naturally occurring have only the L-tryptophane.The L-tryptophane plays an important role to the growing of humans and animals, metabolism, therefore is widely used in fields such as medicine, food and feed.
Preparing method according to present reported in literature L-tryptophane mainly contains following three kinds:
1, fermentation method
Fermentation production of L-tryptophan can be divided into substantially: direct fermentation and precursor add fermentation method.
L-tryptophane commonly used produces bacterium in the direct fermentation has: Corynebacterium glutamicum, brevibacterium flavum, Bacillus subtilus, Beijing rod bacillus etc.(JP 1974,20:391) wait 5-fluorotryptophan (5-FT) resistant mutant by the Bacillus subtilus seed selection, can accumulate L-tryptophane 9.6g/L like Fukui.(JP 1982,5:694) further transform variant, make it have 5-FT and the dual resistance of 8-azaguanine (8-AG), and even flow adds anthranilic acid, can accumulate L-tryptophane 15.6g/L for Nakayama etc.People such as Oikawa (JP 2000342294,2000) invention is a kind of to be the method that starting raw material is produced the L-tryptophane with lactic acid, promptly earlier lactic acid is oxidized to pyruvic acid, again with indoles and ammonium ion react the L-tryptophane.People such as Hatakeyama (US 5776740,1998) adopt single stage method to produce the L-tryptophane, i.e. synthetic L-tryptophane in the microorganism cells aqueous solution that contains glycocoll, formaldehyde, indoles and tool serine hydroxymethylase.At present, be used for the seed selection that novel tryptophane produces bacterium with external Genetic Manipulative Technology in the body.
2, enzyme transforming process
The Enzymatic transformation method has advantages such as the end product accumulation volume is high, reaction time is short, the separation purification is easy, and it is the cheap L-of a production tryptophane effective means the most.The investigator makes up and has screened high yield L-tryptophanase and L-tryptophane synth(et)ase genetic engineering bacterium both at home and abroad, lays a good foundation for producing the L-tryptophane with enzyme engineering technology.In amino acid major country of production Japan, press in major company of a few family such as Mitsui east, Mitsubishi's oiling and three happy etc. all adopts the Enzymatic transformation method to produce the L-tryptophane.According to bibliographical information (Nikkei biotechnology; 1987, (1): 8) intestinal bacteria tryptophane synth(et)ase gene and serine hydroxymethylase (SHTase) gene have been cloned by Japan three happy companies, use glycocoll to be the synthetic L-Serine of raw material; Add indoles again and produce tryptophane, output is 90g/L.
Ishiwata etc. adopt the synthetic DL-serine of chemical method, use the pseudomonasputida racemase that the D-Serine is the L type transition, add the substrate indoles, carry out Enzymatic transformation with the intestinal bacteria tryptophane synth(et)ase, and the product tryptophane is 110g/L.Yukawa etc. (Process Biotech., 1987, (12): 165) made up the e. coli k-12 that has the tryptophane synth(et)ase gene, Enzymatic transformation L-Serine and indoles get L-tryptophane 200g/L.(Trends in Biotechnology, 1985,8 (3): 64-68) cloned tryptophan gene, be that the substrate Enzymatic transformation is produced L-tryptophane 200g/L with L-Serine and indoles to the Hamilton of Genex company etc.
(contemporary Chinese is used pharmaceutical journal to the gentle grade of domestic Wei; 1999; 16 (6): 37-39) utilizing the round pcr JM105 tryptophan gene that increased, make up tryptophan gene engineering bacteria WW-4, is substrate Enzymatic transformation product L-tryptophane 49g/L with L-Serine and indoles; L-Serine transformation efficiency is 84.2%, and the indoles transformation efficiency is 93.6%.(the biotechnology communication, 2006,17 (1): 12-14) make up tryptophane synth(et)ase genetic engineering bacterium BA3, its tryptophane synth(et)ase activity is 3.7 times of contrast bacterium to Zhang Xumei etc.
3, chemical synthesis
The chemosynthesis approach of tryptophane is a lot, mainly contain with the indoles be raw material and in building-up process, generate two big types of indole rings (the amino acid magazine, 1983,4:29-33).But above-mentioned two kinds of method combination colour propylhomoserins or synthetic route are long, maybe need adopt the route reaction step of expensive special material or structure indole ring many, and it is many to relate to raw material, and processing condition are harsh, and operation easier is big, and is not suitable for large-scale industrial production.
Three, summary of the invention
The problem that the present invention need solve provides a kind of method for preparing the L-tryptophane efficiently, cheaply.
At present; In Industrial processes such as China's amino acid production field and food fermentation; Sub product is for can not directly be used for the kilnitamin that is rich in the L-Serine of food and medicine aspect in a large number, and L-Serine cost is high, efficient is low and this kilnitamin direct separation prepares.The present invention adopts the kilnitamin be rich in the L-Serine to prepare the raw material of L-tryptophane as enzyme process, and reaction conditions is gentle, and tryptophane synth(et)ase or tryptophanase specificity are strong; Transformation efficiency is high; Can reach conversion L-Serine is the purpose that is easy to isolating L-tryptophane, has realized the comprehensive utilization of industry byproduct, and cost is low; Technical process is simple, is fit to suitability for industrialized production.
The present invention can reach through following technical scheme:
The enzymatic conversion preparation method of L-tryptophane the steps include:
(1) the genetically engineered bacteria strain of tryptophane synth(et)ase or tryptophanase (BA3 or WW-4) is cultivated in the substratum that contains isopropylthiogalactoside (IPTG, Sigma company) or lactose, induces the tryptophane synth(et)ase or the tryptophanase that produce high vigor;
(2) adopt free cell method, after will containing the enzyme cell and being rich in the kilnitamin conversion fluid mixing of L-Serine, add damping fluid and tensio-active agent again, under 30～45 ℃ of conditions, carry out enzymic transformations.
(3) resultant of reaction is separated, obtain highly purified L-tryptophane.
Culture medium carbon source in the above-mentioned steps (1) adopts glucose, SANMALT-S, sucrose, fructose and lactose; Its concentration is 1～20g/L; Nitrogenous source adopts organic nitrogen source, and like Carnis Bovis seu Bubali cream, yeast extract paste, steeping water, peptone, soya-bean cake hydrolyzed solution and/or protein hydrolystate, its concentration is 1～30g/L; Add IPTG or lactose-induced, its concentration is 0.05～10g/L.
The conversion fluid buffer system of above-mentioned steps (2) is phosphoric acid buffer, borate buffer and carbonic acid buffer, makes conversion fluid pH7.5～9.5, the righttest conversion fluid pH8～9; In conversion fluid, add tensio-active agent, like tween-80, hexadecane trimethyl ammonium bromide (CTAB), polyoxyethylene octyl phenyl ether (OP), its concentration is 0.005～5g/L.
The method of L-tryptophane is to adopt isoelectric point crystallizing method or isoelectric point crystallizing to separate the method that combines with ion exchange resin in the middle separating reaction system of above-mentioned steps (3).
The present invention compared with prior art has following advantage:
(1) the present invention's genetic engineering bacterium (BA3 or WW-4) of adopting tryptophane synth(et)ase or tryptophanase in preferred substratum, cultivate can high yield generation tryptophane synth(et)ase or tryptophanase; Make reaction that very high transformation efficiency and speed of reaction arranged, L-Serine molar yield reaches more than 82%.
(2) the present invention adopts the kilnitamin that is rich in the L-Serine as the preparation raw material; Reaction conditions is gentle; Tryptophane synth(et)ase or tryptophanase specificity are strong, and transformation efficiency is high, has made full use of industrial by-products; Solve the difficult problem of L-Serine high efficiency separation, had good economic benefit and environmental benefit.
(3) other amino acid physico-chemical property in L-tryptophane and the kilnitamin is widely different, just can separate preparation with simple iso-electric point method, and cost is low, and technical process is simple, is fit to suitability for industrialized production.
Advantages such as it is abundant, cheap (4) to have raw material sources, and transformation time is short, and is easy and simple to handle, and production cost is low.
Four, embodiment
Embodiment one
Keratin sulfate source L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophane synth(et)ase engineering strain is cultivated in following 1000ml substratum (g/ml): steeping water 0.5%, NaCl 0.5%, KH
2PO
40.16%, MgSO
4.7H
2O 0.04%, FeSO
4.7H
2O0.01%, Carnis Bovis seu Bubali cream 2%, SANMALT-S 0.5%, glucose 0.5%, IPTG 0.005%, pH7.2.37 ℃ are shaken a bottle shaking culture 12h, and the centrifugal 15min of 4000rpm gets wet cell 25g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L sodium carbonate buffer (pH9.5) preparation; Conversion fluid is for containing feather acid hydrolysis liquid (amino acid total content 15%), 22.8g indoles and 0.05%OP solution (g/ml) 6mL of 3.8%L-Serine (g/ml), 37 ℃ of enzymatic reaction 36h.L-tryptophane concentration is 6.3% in the reaction solution, is 87% to L-Serine molar yield.
3. with the centrifugal 10min of reaction solution 4000rpm, collect wet cell and solid L-tryptophane mixture 68g, with 300mL pure water dissolved solids mixture; Drip 6mol/L NaOH and transfer pH12～13, stir and be warmed up to 60～80 ℃ fully, add activated carbon decolorizing and remove thalline the dissolving of solid L-tryptophane; Filtrating is transferred pH to 5.9 with 6mol/L HCl, and acidizing fluid is cooled to room temperature, the crystallization vacuum filtration of separating out; With the washing of a small amount of pure water, dry 21.2g L-tryptophane.
4. 550ml centrifuged supernatant and 350ml L-tryptophane crystalline mother solution are merged, mixed solution is transferred pH to 3.5 with 6mol/LHCl, adds pure water and is diluted to upward JK008 cationic resin column absorption L-tryptophane of 1600ml; Adsorb saturated after, earlier with 400ml washed resin post, use 800ml 4% ammoniacal liquor wash-out then; Collection contains L-tryptophane elutriant, is warmed up to 70～80 ℃ of decolourings, and concentrating under reduced pressure is to 200ml; Add long-pending 95% alcohol of triploid, stir, put the refrigerator hold over night; The crystallization of separating out is filtered the back with a small amount of pure water washing, the dry L-tryptophane 10.3g of getting, and crystalline mother solution recycles.
Embodiment two
Keratin sulfate source L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophan gene engineering bacteria is cultivated in following 1000ml substratum (g/ml): steeping water 0.5%, Carnis Bovis seu Bubali cream 2%, protein hydrolystate 0.5%, SANMALT-S 0.5%, lactose 0.5%, IPTG 0.007%, K
2HPO
40.5%, MgSO
4.7H
2O 0.03%, and NaCl 0.5%, pH7.2.37 ℃ are shaken a bottle shaking culture 14h, and the centrifugal 15min of 4000rpm gets wet cell 23.8g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L sodium carbonate buffer (pH9.5) preparation; Conversion fluid is for containing hair acid hydrolysis liquid (amino acid total content 16%), 25.2g indoles and 0.5% tween-80 (g/ml) the solution 6mL of 4.2%L-Serine (g/ml), 39 ℃ of enzymatic reaction 32h.L-tryptophane concentration is 7.1% in the reaction solution, is 90% to L-Serine molar yield.
3.L-the tryptophane separation purification method with embodiment one, gets L-tryptophane 33.7g.
Embodiment three
Plant origin L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophane synth(et)ase engineering strain is cultivated in following 1000ml substratum (g/ml): peptone 1%, Carnis Bovis seu Bubali cream 0.3%, SANMALT-S 0.5%, sucrose 0.3%, NaCl 0.5%, KH
2PO
40.14%, K
2HPO
40.5%, MgSO
4.7H
2O 0.05%, and IPTG 0.005%, pH7.0.37 ℃ are shaken a bottle shaking culture 13h, and the centrifugal 15min of 4000rpm gets wet cell 25.3g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L borate buffer (pH9.0) preparation; Conversion fluid is for containing molasses extracting solution (amino acid total content 14%), 12g indoles and 0.05%CTAB (g/ml) the solution 6mL of 2%L-Serine (g/ml), 39 ℃ of enzymatic reaction 32h.L-tryptophane concentration is 3.3% in the reaction solution, is 87% to L-Serine molar yield.
3.L-the tryptophane separation purification method with embodiment one, gets L-tryptophane 14.8g.
Embodiment four
Plant origin L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophan gene engineering bacteria is cultivated in following 1000ml substratum (g/ml): peptone 1%, yeast extract paste 0.3%, Carnis Bovis seu Bubali cream 0.4%, lactose 1.5%, glucose 0.5%, NaCl 0.5%, KH
2PO
40.15%, K
2HPO
40.45%, MgSO
4.7H
2O 0.03%, pH7.2.37 ℃ are shaken a bottle shaking culture 12h, and the centrifugal 15min of 4000rpm gets wet cell 23.9g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L sodium-acetate buffer (pH8.8) preparation; Conversion fluid is for containing corn steep liquor (amino acid total content 15%), 18.6g indoles and 0.5% tween-80 (g/ml) the solution 6mL of 3.1%L-Serine (g/ml), 37 ℃ of enzymatic reaction 36h.L-tryptophane concentration is 5.1% in the reaction solution, is 88% to L-Serine molar yield.
3.L-the tryptophane separation purification method with embodiment one, gets L-tryptophane 23.6g.
Embodiment five
Fermentation source L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophane synth(et)ase genetic engineering bacterium is cultivated in following 1000ml substratum (g/ml): soya-bean cake hydrolyzed solution 3%, peptone 0.5%, yeast extract paste 0.3%, lactose 1%, sucrose 0.5%, SANMALT-S 0.5%, NaCl 0.5%, KH
2PO
40.06%, MgSO
4.7H
2O 0.05%, FeSO
4.7H
2O 0.01%, pH7.2.37 ℃ are shaken a bottle shaking culture 14h, and the centrifugal 15min of 4000rpm gets wet cell 25.8g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L acetate buffer solution (pH9.0) preparation; Conversion fluid is for containing citric acid fermentation extracting solution (amino acid total content 14%), 15g indoles and 0.5% tween-80 (g/ml) the solution 6mL of 2.5%L-Serine (g/ml), 35 ℃ of enzymatic reaction 38h.L-tryptophane concentration is 4.1% in the reaction solution, is 85% to L-Serine molar yield.
3.L-the tryptophane separation purification method with embodiment one, gets L-tryptophane 18.2g.
Embodiment six
Fermentation source L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophan gene engineering bacteria is cultivated in following 1000ml substratum (g/mi): peptone 1%, Carnis Bovis seu Bubali cream 0.3%, steeping water 0.3%, sucrose 0.5%, fructose 0.5%, IPTG 0.01%, and NaCl 0.5%, MgSO
4.7H
2O 0.04%, K
2HPO
40.55%, pH7.0.37 ℃ are shaken a bottle shaking culture 12h, and the centrifugal 15min of 4000rpm gets wet cell 23.7g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L sodium borate buffer liquid (pH9.0) preparation; Conversion fluid is for containing L-glutamic acid fermentation extracting solution (amino acid total content 16%), 18g indoles and 0.05%CTAB (g/ml) the solution 6mL of 3%L-Serine (g/ml), 42 ℃ of enzymatic reaction 30h.L-tryptophane concentration is 4.9% in the reaction solution, is 85% to L-Serine molar yield.
3.L-the tryptophane separation purification method with embodiment one, gets L-tryptophane 22.7g.
Embodiment seven
Fermentation source L-serine by enzymatic transforms preparation L-tryptophane method, and its preparation process is following:
1. the tryptophan gene engineering bacteria is cultivated in following 1000ml substratum (g/ml): peptone 1%, steeping water 0.4%, Carnis Bovis seu Bubali cream 0.3%, fructose 0.5%, SANMALT-S 0.5%, IPTG 0.01%, NaCl0.7%, K
2HPO
40.65%, pH7.0.37 ℃ are shaken a bottle shaking culture 12h, and the centrifugal 15min of 4000rpm gets wet cell 22.5g.
2. wet cell is joined in the 600ml conversion fluid of 0.1mol/L sodium borate buffer liquid (pH9.0) preparation; Conversion fluid is for containing L-fermenting lysine extracting solution (amino acid total content 15%), 18g indoles and 0.05%CTAB (g/ml) the solution 6mL of 2.9%L-Serine (g/ml), 40 ℃ of enzymatic reaction 32h.L-tryptophane concentration is 4.8% in the reaction solution, is 86% to L-Serine molar yield.
3.L-the tryptophane separation purification method with embodiment one, gets L-tryptophane 21.5g.
Claims (5)
1. the enzymatic conversion preparation method of a L-tryptophane is characterized in that being made up of following steps:
(1) has the genetic engineering bacterium of L-tryptophane synth(et)ase or L-tryptophanase, in the substratum that contains IPTG or lactose, cultivate, produce L-tryptophane synth(et)ase or L-tryptophanase;
(2) use free cell method; To contain the enzyme cell mixes with the kilnitamin conversion fluid that contains the L-Serine; Wherein in the conversion fluid L-Serine to account for total aminoacid weight ratio content be 20%～50%, add damping fluid, an amount of Vitazechs, tensio-active agent and indoles again, under 35～42 ℃ of conditions, carry out enzymatic reaction; Resultant of reaction is separated, obtain the L-tryptophane; The kilnitamin conversion fluid of the described L-of containing Serine derives from feather and/or the hair acid hydrolysis liquid through separation and Extraction, and L-L-glutamic acid, L-Methionin and/or Hydrocerol A extract the back waste liquid, or corn steep liquor and/or molasses.
2. L-tryptophane enzyme process conversion preparation method according to claim 1 is characterized in that the used culture medium carbon source of step (1) adopts glucose, SANMALT-S, sucrose and fructose, and its mass concentration is 1～20g/L; Nitrogenous source adopts Carnis Bovis seu Bubali cream, yeast extract paste, steeping water, soya-bean cake hydrolyzed solution or protein hydrolystate, and its mass concentration is 1～30g/L, adds IPTG or lactose-induced, and the IPTG mass concentration is 0.05～0.1g/L, and the lactose mass concentration is 5～10g/L.
3. L-tryptophane enzyme process conversion preparation method according to claim 1 is characterized in that the described damping fluid of step (2) is phosphoric acid buffer, acetate buffer solution, borate buffer or carbonic acid buffer, makes conversion fluid pH 7.5～9.5.
4. L-tryptophane enzyme process conversion preparation method according to claim 1 is characterized in that the described tensio-active agent of step (2) is tween-80, cetyl trimethylammonium bromide or polyoxyethylene octyl phenyl ether.
5. L-tryptophane enzyme process conversion preparation method according to claim 1 is characterized in that it is that isoelectric point crystallizing method or isoelectric point crystallizing separate the method that combines with ion exchange resin that step (2) is separated the method for L-tryptophane.
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| CN102517352B (en) * | 2011-12-28 | 2014-09-24 | 南京大学 | Method for preparing L-cysteine through enzymatic conversion |
| CN103224972B (en) * | 2013-05-09 | 2015-02-04 | 湖北新生源生物工程股份有限公司 | L-tyrosine preparation method through enzymatic conversion |
| CN103333928B (en) * | 2013-07-05 | 2015-12-23 | 宁夏伊品生物科技股份有限公司 | The method of L-Trp is produced with the fermentation using bacteria that fbp expression of enzymes weakens and/or enzymic activity reduces |
| CN103333929B (en) * | 2013-07-05 | 2016-02-17 | 宁夏伊品生物科技股份有限公司 | The method of L-Trp is produced with the fermentation using bacteria changing fbp enzyme controlling element |
| CN104017838B (en) * | 2014-06-18 | 2016-08-31 | 宿州学院 | A kind of enzymatic conversion preparation method of 2-methyl-L-tryptophan |
| CN104928327B (en) * | 2015-06-03 | 2018-06-05 | 武汉轻工大学 | A kind of preparation method of L-Trp |
| CN105693592B (en) * | 2016-03-03 | 2019-03-19 | 天津科技大学 | A kind of carry disease germs from fermentation liquid crystallizes the process of high efficiency extraction L-Trp |
| CN111362860B (en) * | 2020-04-03 | 2022-04-15 | 通辽梅花生物科技有限公司 | Method for extracting tryptophan from fermentation liquor |
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