CN100467587C - Pseudomonas zjwp-14 and its uses in asymmetrical hydrolyzed preparation of L-cysteine - Google Patents
Pseudomonas zjwp-14 and its uses in asymmetrical hydrolyzed preparation of L-cysteine Download PDFInfo
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- CN100467587C CN100467587C CNB2006100539328A CN200610053932A CN100467587C CN 100467587 C CN100467587 C CN 100467587C CN B2006100539328 A CNB2006100539328 A CN B2006100539328A CN 200610053932 A CN200610053932 A CN 200610053932A CN 100467587 C CN100467587 C CN 100467587C
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- pseudomonas
- zjwp
- halfcystine
- wet thallus
- enzyme liquid
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- 241000589516 Pseudomonas Species 0.000 title claims abstract description 34
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 title abstract description 23
- 239000004201 L-cysteine Substances 0.000 title abstract description 8
- 235000013878 L-cysteine Nutrition 0.000 title abstract description 8
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- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an application of pseudomonas to prepare L-cysteine, wherein the pseudomonas zjwp-14(Pseudomonas sp. zjwp-14) is reserved in the Chinese typical culture medium reserving center with reserving number at CCTCC M. 206104, which comprises the following steps: culturing zjwp-14 to obtain wet thalline; disposing the rough enzyme liquid as enzyme source; adopting DL-2-amino acid-thiazoline-4-carboxyl acid as substrate; proceeding microbe transmitting reaction in the transmitting liquid at 20-52 deg. c for 1-9h; obtaining the reacting liquid with L-cysteine; separating; purifying to obtain the L-cysteine.
Description
(1) technical field
The present invention relates to the microorganism novel bacterial: pseudomonas (Pseudomonas sp.) zjwp-14 and prepare application in the L-halfcystine at asymmetric hydrolysis.
(2) background technology
The sulfydryl that the L-halfcystine is had has the important physical function, is widely used in industries such as medicine, food, makeup and feed.In addition, the L-halfcystine also can be used as important intermediate feed and is used to the derivative for preparing gsh, tryptophane and prepare multiple L-halfcystine.The production method of L-halfcystine mainly contains natural product extraction method, chemical synthesis and enzyme transforming process.Domestic present production mainly adopts human or animal's hair raw material elder generation behind acid hydrolysis or alkali hydrolysis method extraction L-Gelucystine, makes the L-halfcystine through electrolytic reduction again, and this method often is subjected to the restriction of raw material sources, and easily sneaks into other amino acid in the product.Along with the enhancing of people's environmental consciousness, the particularly appearance of ground mad cow disease such as Europe in recent years forces European Community's declaration L-halfcystine that July 1, forbidding was made by the animal hair raw material from 1998.Chemical synthesis not only step is more, and the product that obtains is generally the racemic modification of DL type, still needs just to obtain having the L-halfcystine of physiologically active after splitting.Because it is good that enzyme transforming process has stereoselectivity, can obtain the product of single configuration; The reaction conditions gentleness; Advantage such as environmentally friendly and come into one's own day by day (Yang Jinkui etc. microbial process is produced the progress of L-halfcystine. external medical microbiotic fascicle, 2001,22 (4): 179).
Enzyme transforming process is produced the L-halfcystine can select to adopt three kinds of substrates: (1) O-acetylserine; (2) 3-chloro-L-L-Ala; (3) DL-2-amino-thiazolyl-quinoline-4-carboxylic acid (DL-ATC).People such as Sano screened some pseudomonas (Pseudomonas sp.) from soil in 1977, can be the L-halfcystine with the DL-ATC asymmetric hydrolysis.Except that pseudomonas, many bacteriums also can transform the synthetic L-halfcystine of DL-ATC.Its pathways metabolism of the different strains of Pseudomonas there are differences, when adopting Pseudomonas putida AJ3865 and Pseudomonas sp.ON-4a bacterial strain to transform, intermediate product is nitrogen-carboxamide-L-halfcystine (N-carbamoyl-L-cysteine is called for short L-NCC); And the conversion intermediate product of Pseudomonas sp.CU6 and Pseudomonas thiazolinophilum bacterial strain is sulphur-carboxamide-L-halfcystine (S-carbamoyl-L-cysteine is called for short L-SCC).People such as Liu Zhong take soil sample from producing the DL-ATC environment, therefrom separate obtaining a strain pseudomonas (Pseudomonas sp.) TS1138 bacterial strain, can transform DL-ATC and generate the L-halfcystine.(Liu Zhong etc. synthetic L-halfcystine of microbial enzyme method and L-Gelucystine. the microbiology circular, 2003,30 (6): 16-21.) noted earlier can be pseudomonas Pseudomonas sp..ON-4a, Pseudomonas putida genus pseudomonasputida (pseudomonas putida) strain of L-halfcystine with the DL-ATC asymmetric hydrolysis.
(3) summary of the invention
But the new bacterial strain of microorganism that the purpose of this invention is to provide the synthetic L-halfcystine of a kind of bio-transformation DL-ATC: pseudomonas zjwp-14, and the method for utilizing the synthetic L-halfcystine of this bacterial strain biocatalysis DL-ATC asymmetric hydrolysis.
For reaching goal of the invention the technical solution used in the present invention be:
Pseudomonas (Pseudomonas sp.) zjwp-14 is preserved in Chinese typical culture collection center, deposit number CCTCC NO.M 206104, preservation date on September 29th, 2006.
Bacterial classification source: pseudomonas zjwp-14 (below be abbreviated as zjwp-14) is that separation and Culture obtains near the soil the auspicious symbol in Hangzhou town wild strain HJ-14 is through uviolizing, microwave irradiation, Co
60-gamma-ray irradiation mutagenic treatment, and DL-ATC substrate resistance screening obtains.Uviolizing power is 15W, irradiation distance 30cm, and irradiation time is 30～150s; Microwave irradiation is low fire screen, and ice bath was handled 20～120 seconds; Co
60-gamma-ray irradiation dosage range is at 0.25KGy～2.0KGy.The separation method of wild strain HJ-14 is: will gather soil sample and adopt the nutrient solution that contains DL-ATC to carry out the microbial enrichment cultivation, it is on the solid medium of only nitrogen source that nutrient solution is coated on DL-ATC after dilution, and culture temperature is 25～36 ℃, cultivates 2～4d.With the HJ-14 bacterial strain is starting strain, obtains the zjwp-14 bacterial strain through mutagenesis pedigree seed selection as follows.
Starting strain HJ14 → ultraviolet mutagenesis → microwave irradiation → Co
60-γ irradiation → substrate DL-ATC resistance screening → natural separation → bacterial strain zjwp-14
The growth characteristics of zjwp-14: zjwp-14 is a non-zymocyte, and growth rapidly grows tiny, point-like, circle, moistening, milky bacterium colony behind 30 ℃ of first culture 24h, and bacterium colony is long to diameter 2-3mm behind the 48h, surface wettability, and be unified into a slice.
The form of zjwp-14: be Gram-negative, short and small bacillus.Diameter is 0.5-1.0um, and long is 2um, and microphotograph is seen Fig. 1 behind the gramstaining.
The biochemical characteristic of pseudomonas zjwp-14: bacterial oxidation enzyme positive, the catalase test positive.Adopt API biochemical identification lath (French biomerium company) and classical manual method to be identified.The result shows that zjwp-14 is similar to Pseudomonas mendocina, but coincidence rate is less than 90%.(API20NE, RapID andVITEK-Ams60) divide appraising datum to sum up and see Table 1 with the full automatic microorganism Biochemical Analyzer.
Table 1:zjwp-14 biochemical characteristic (in the table-expression is negative; + expression is positive)
| Biochemical title | English | Qualification result | Biochemical title | English | Qualification result |
| Catalase | Catalase | + | N.F,USP MANNITOL | MAN | - |
| Oxydase | Oxidase | + | The D-sorbyl alcohol | SOR | - |
| The N-acetylglucosamine | NAG | - | Salicin | SAL | - |
| D-ribose | RIB | - | 5-ketone group grape hydrochlorate | 5KG | - |
| Malonate | MNT | - | Valerate | VALT | + |
| Acetate | ACE | + | Citrate trianion | CIT | + |
| The DL-lactic acid salt | LAT | + | Histidine | HIS | + |
| The L-L-Ala | ALA | + | Glycogen | GLAG | - |
| 3-hydroxyl-butyrates | 3BU | + | 3-hydroxy-benzoic acid salt | mOBE | - |
| Propionic salt | PROP | + | 2-ketone group grape hydrochlorate | 2KG | - |
| Caprate | CAP | + | Inositol | INO | - |
| Suberate | SUB | - | The L-Fucose | FUC | - |
| D-glucose | GLU | + | Methylene-succinic acid | ITA | - |
| Rhamnosyl | Rhamnose | - | The D-melibiose | MEL | - |
| Sucrose | SAC | - | L-arabinose | ARA | - |
| Maltose | MAL | - | The L-Serine | SER | + |
| 4-hydroxy-benzoic acid salt | pOBE | - | The L proline(Pro) | PRO | + |
The 16S rDNA characteristic of zjwp-14: the sequence with the 16S rDNA of bacterium can more accurately be identified genus and the kind of bacterium.With the sequence of the PCR product of the 16S rDNA of zjwp-14 and the 16S rDNA sequence alignment among the gene pool NCBI, some bacterium in discovery and the Pseudomonas, as Pseudomonas sp.BS 99.7% homology is arranged, the partial nucleotide sequence of the 16S rDNA of zjwp-14 is as follows:
ATTGACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTAGGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGGCTCATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGG。
Pseudomonas zjwp-14 of the present invention can be used for DL-2-amino-thiazolyl-quinoline-4-carboxylic acid (DL-2-amino-△
2-thiazolin-4-carbonic acid, DL-ATC) asymmetric hydrolysis prepares the L-halfcystine.
Described being applied as: carrying out conventional wet thallus or the treated crude enzyme liquid that obtains of wet thallus of cultivating acquisition with pseudomonas zjwp-14 is the enzyme source, with DL-2-amino-thiazolyl-quinoline-4-carboxylic acid is substrate, in conversion fluid, under 20～52 ℃, carry out microbial transformation reaction 1～9h, react completely to such an extent that contain the reaction solution of L-halfcystine, make the L-halfcystine through separation and Extraction.
Described substrate DL-2-amino-thiazolyl-quinoline-4-carboxylic acid initial mass concentration is 0.5%～2.5%.
Described enzyme source is a wet thallus, and described wet thallus is from fermenting enzyme liquid, OD after 10 times of the described fermenting enzyme liquid dilutions
600Be 0.4～0.6, described wet thallus addition is 0.5～5:1 by fermenting enzyme liquid with conversion fluid volume ratio.
Concrete, described application is to carry out seed culture and fermentation culture with pseudomonas zjwp-14, from fermenting enzyme liquid the centrifugal wet thallus that obtains as the enzyme source, OD after 10 times of the described fermenting enzyme liquid dilutions
600Be 0.4～0.6, with the phosphate buffer soln that contains 0.1～1.5% DL-ATC is conversion fluid, described wet thallus addition is 0.5～5:1 by fermenting enzyme liquid with conversion fluid volume ratio, the control invert point is 20～52 ℃, carry out microbial transformation reaction 1～9h, react completely to such an extent that contain the reaction solution of L-halfcystine, make the L-halfcystine through separation and Extraction.
Described application obtains wet thallus as follows:
(1) slant culture: the pseudomonas zjwp-14 that picking is preserved is transferred to slant medium, cultivates 24～48h for 30 ℃ and gets the activatory bacterial classification; Described slant medium is formed: DL-2-amino-thiazolyl-quinoline-4-carboxylic acid 1.0～5.0g/L, glycerine 10～30g/L, yeast extract 2.0～10g/L, peptone 2.0～10g/L, NaCl 1.0～5.0g/L, agar 15～25g/L, pH7.0～8.0; 121 ℃ of sterilization 20min, sterilization postcooling bevel;
(2) seed culture: one ring pseudomonas zjwp-14 is transferred to seed culture medium with the transfering loop picking, and 30 ℃, shaking speed 200r/min cultivates 10～24h and gets seed culture fluid; Described seed culture medium is formed: DL-2-amino-thiazolyl-quinoline-4-carboxylic acid 1.0～5.0g/L, glycerine 5.0～30.0g/L, yeast extract 5.0～30g/L, peptone 5.0～30g/L, extractum carnis 5.0～30g/L, NaCl 1.0～5.0g/L, pH7.0～8.0; 121 ℃ of sterilization 20min, the sterilization postcooling is made seed culture medium;
(3) producing enzyme cultivates: get the seed culture fluid inoculation, inoculum size is 0.5～5%, 25～35 ℃, and shaking speed 100～250r/min carries out shake-flask culture 10～24h in producing the enzyme substratum, obtain fermenting enzyme liquid; Described product enzyme substratum is formed (with the seed substratum); OD after 10 times of the fermenting enzyme liquid dilutions that obtains by above-mentioned condition
600Be 0.4～0.6.
(4) wet thallus is collected in fermenting enzyme liquid is centrifugal, washing.
Since the halfcystine instability, and Gelucystine is more stable, and the halfcystine that the present invention obtains bio-transformation extracts after being oxidized to Gelucystine.Concrete grammar is: after transforming end, with reaction solution centrifugal (10,000rpm, 10min, 4 ℃), supernatant liquor adds the FeSO of trace
4, behind ventilation vibration 20～30h on the reciprocating type shaking table, be adjusted to about pH2 with dense HCl solution, be concentrated into finite concentration through rotary evaporation, cooling back centrifugal (10,000rpm, 20min, 4 ℃), supernatant liquor is as the upper prop sample.Sample is added in the ion exchange column that 732 resins are housed with the velocity flow of constant flow pump with 0.5mL/min, use deionized water drip washing, use the ammoniacal liquor wash-out of 0.5～1.0mol/L again, flow velocity is 0.5mL/min, at any time the L-cystine in the monitoring stream fluid in the elution process, merge each higher pipe of content after wash-out finishes, vacuum-drying obtains solid sample.Sample is through polarimetry, and confirming really is L type product.The thin plate tomographic map of comparative preparation sample and standard substance, mass spectrum are defined as same substance.
Preferably, described application is as follows: described being applied as: with the wet thallus 0.1M that fermenting enzyme liquid is directly made, the phosphoric acid buffer of pH8.0 cleans, OD after described fermenting enzyme liquid dilutes 10 times
600Be 0.4～0.6, wet thallus is changed in the described damping fluid again, described wet thallus addition by fermenting enzyme liquid and conversion fluid volume than being 2:1, adding substrate DL-2-amino-thiazolyl-quinoline-4-carboxylic acid concentration simultaneously, to make substrate initial mass concentration be 1%, in 42 ℃, water-bath 6h, after reaction finishes the reaction solution separation and Extraction is made L-Gelucystine form and deposit, described separation and Extraction step is: reaction solution is centrifugal, gets supernatant liquor and adds FeSO
4Be enough to make the L-halfcystine to be oxidized to the L-Gelucystine, this moment, preferably bubbling air impelled acceleration FeSO
4The L-halfcystine is oxidized to the L-Gelucystine, behind vibration 20～30h, be adjusted to pH2 with dense HCl solution, evaporation concentration, the cooling back is centrifugal, and supernatant liquor stream is added in the ion exchange column that 732 resins are housed, use deionized water drip washing, use the ammoniacal liquor wash-out of 0.5～1.0mol/L again, merge the higher effluent liquid of L-cystine after wash-out finishes, vacuum-drying obtains described L-Gelucystine.
Beneficial effect of the present invention is mainly reflected in: but provide the new asymmetric hydrolysis DL-ATC of a strain to produce L-halfcystine microbial strains, pseudomonas provided by the invention (Pseudomonas sp.) zjwp-14 is different from the pseudomonasputida of mentioning in the background technology, be applied to prepare that L-halfcystine stereoselectivity is good, reaction conditions is gentle, environmentally friendly, the productive rate height.
(4) description of drawings
Fig. 1 is a microphotograph behind the gramstaining of pseudomonas zjwp-14;
Fig. 2 is the scanning spectrogram of different strains converted product;
Fig. 3 is embodiment 2 product infrared spectrums;
Fig. 4 is the infrared standard spectrogram of L-Gelucystine;
Fig. 5 is the cell concentration of different incubation times;
Fig. 6 is for producing the enzyme time curve.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Separation and Culture obtains near the auspicious symbol in Hangzhou town the soil wild pseudomonas strain HJ-14, HJ-3, HJ-6, HJ-21, HJ-17, HJ-10, carry out primary dcreening operation by following sequential grammar respectively:
Enrichment medium I (g/L): glycerine 10.0, DL-ATC 3.0, FeSO
47H
2O 0.01, KH
2PO
41.0, MgSO
47H
2O 0.5, pH7.0.121 ℃ of sterilization 20min, the inoculation of sterilization postcooling is inoculated into a small amount of soil sample in the substratum, and 200rpm rotary type shaking table is cultivated 2d for 30 ℃, as the seed of further enrichment culture.
Enrichment medium II (g/L): glycerine 10.0, DL-ATC 6.0, FeSO
47H
2O 0.01, KH
2PO
41.0, MgSO
47H
2O 0.5, pH7.0.121 ℃ of sterilization 20min, the inoculation of sterilization postcooling.To insert medium ii through the pregnant solution of enrichment for the first time, 200rpm rotary type shaking table is cultivated 2d for 30 ℃, as the seed of primary dcreening operation flat board.
Plate culture medium (g/L): DL-ATC 3.0, glycerine 10.0, and NaCl 5.0, agar 2.0, pH7.0.121 ℃ of sterilization 20min, the inoculation of sterilization postcooling.To be coated on the flat board after the thalline dilution through the secondary enrichment, cultivate 2d for 30 ℃.Picking list bacterium colony switching inclined-plane.
Slant medium (g/L): DL-ATC 2.0, glycerine 10.0, and yeast extract 5.0, peptone 5.0, NaCl 5.0, agar 20.0, pH7.0.121 ℃ of sterilization 20min, the sterilization postcooling is inoculated back 30 ℃ and is cultivated 1d.
Seed culture and fermentation: substratum (g/L) DL-ATC 3.0, glycerine 20.0, yeast extract 5.0, peptone 5.0, extractum carnis 5.0, NaCl 5.0, pH7.0.Liquid amount is the bottled 50ml substratum of 250ml triangle, 121 ℃ of sterilization 20min, and sterilization postcooling inoculation inclined-plane seed, the 200rpm shaking table is cultivated 12h for 30 ℃, as seed or fermenting enzyme liquid.
Thalline turbidity scope was OD after fermenting enzyme liquid diluted 10 times
6000.4～0.6, and centrifugal 10min (4,000rpm), collect thalline, (0.1M pH8.0) cleans twice, and thalline is changed in the identical damping fluid with phosphoric acid buffer, making cell concentration is the twice of former fermenting enzyme liquid, add concentration simultaneously and be 1% DL-ATC substrate, in 42 ℃, behind the water-bath 2h, add 1M HCl, centrifugal remove behind the thalline supernatant liquor.Supernatant liquor adopts the acid ninhydrine method to carry out the preliminary qualitative and quantitative analysis of product.The result as shown in Figure 2.
Conclusion: the converted product of bacterial strain HJ-14, HJ-3, HJ-6, HJ-21, HJ-17, HJ-10 is consistent with the scanning curve of standard specimen halfcystine, the converted product that can judge these bacterial strains is a halfcystine, wherein the converted product of HJ-14 illustrates that in the absorption maximum at 560nm place its cysteine content is the highest.
Embodiment 2:
Select bacterial strain HJ-14 according to the result among the embodiment 1, carry out fermentation culture after, add substrate DL-ATC (final concentration is 1%), in 42 ℃, behind the water-bath 6h, with reaction solution centrifugal (10,000rpm, 10min, 4 ℃), supernatant liquor adds the FeSO of trace
4, behind ventilation vibration 20～30h on the reciprocating type shaking table, being adjusted to about pH2 with dense HCl solution, rotary evaporation concentrates, cooling, centrifugal (10,000rpm, 20min, 4 ℃), supernatant liquor is as the upper prop sample.Sample is added in 732 resin columns with the velocity flow of constant flow pump with 0.5mL/min, after deionized water drip washing, ammoniacal liquor with 0.5mol/L carries out wash-out, flow velocity is 0.5mL/min, at any time the L-cystine in the monitoring stream fluid in the elution process, merge each higher pipe of content after wash-out finishes, vacuum-drying obtains solid sample.
To extract product and L-Gelucystine standard substance are dissolved in 0.01M HCl, and carry out tlc analysis, developping agent is a propyl carbinol: acetate: water=2:1:1, extract the R of product
fBe 0.400, the R of L-Gelucystine standard substance
fBe 0.402, the product of acquisition has identical R with L-Gelucystine standard substance
fValue.L-halfcystine standard substance R
fBe 0.593, product R in the enzymatic reaction liquid
fBe 0.596, prove that converted product really is a halfcystine.
Fig. 3, shown in Figure 4 is seen in the infrared spectrogram contrast that transforms sample and standard substance.
The rotational analysis of sample and standard substance:
-0.634 ,-0.628 ,-0.627 reading substitution formula in the polarimeter is calculated the specific rotatory power of this product, the product that obtains is measured 3 times, its reading is respectively:, get 3 times mean value :-0.629.The substitution formula calculates:
-0.628 ,-0.635 ,-0.633 the value that L-Gelucystine standard substance 3 times are measured is:, get mean value 3 times :-0.632, and the substitution formula calculates:
By calculation result as can be known, product behind the purifying and L-Gelucystine standard substance have very approaching specific rotatory power, prove that the Gelucystine that obtains is a L type isomer, can determine that the halfcystine that is generated by the DL-ATC enzymatic reaction also is a L type isomer.
Embodiment 3:
The wild strain HJ-14 that embodiment 1 screening obtains is through uviolizing, microwave irradiation, Co
60-gamma-ray irradiation mutagenic treatment, and DL-ATC substrate resistance screening (detailed step as previously mentioned) obtain pseudomonas (Pseudomonas sp.) zjwp-14.
Slant medium (g/L): DL-ATC 2.0, glycerine 10.0, and yeast extract 5.0, peptone 5.0, NaCl 5.0, agar 20.0, pH7.0.121 ℃ of sterilization 20min, the sterilization postcooling inserts Pseudomonas sp.zjwp-14, cultivates 1d for 30 ℃.
Seed culture medium (g/L): DL-ATC 3.0, glycerine 20.0, and yeast extract 5.0, peptone 5.0, extractum carnis 5.0, NaCl 5.0, pH7.0.Liquid amount is the bottled liquid 50ml of 250ml triangle, sterilizes 20 minutes for 121 ℃, and the sterilization postcooling inserts a ring inclined-plane seed, and the 200rpm shaking table is cultivated 12h for 30 ℃, as seed.
Produce enzyme substratum (g/L): DL-ATC 3.0, glycerine 20.0, and yeast extract 5.0, peptone 5.0, extractum carnis 5.0, NaCl 5.0, pH7.0.Liquid amount is the bottled liquid 50ml of 250ml triangle, sterilizes 20 minutes for 121 ℃, and the sterilization postcooling inserts the seed of 1ml, the 200rpm shaking table, and 30 ℃ of cultivations every 2h, are pressed embodiment 1 method mensuration thalline turbidity and enzyme and are lived.Result such as Fig. 5, shown in Figure 6.
Embodiment 4:
Use Pseudomonas sp.zjwp-14, press example 3 method enzymatic productions, behind the fermentation culture 12h, get 10ml fermenting enzyme liquid, the wet thallus that obtains after centrifugal is added in the 20ml test tube adorns 0.1M, pH8.0 phosphoric acid buffer 5ml, contain substrate DL-ATC amount and be respectively 0.1%, 0.3%, 0.6%, 1.0%, 1.3%, 1.6% carries out conversion reaction under 42 ℃ of water-baths, finish behind the reaction 2h, add 1ml HCl (1M) inactivator source, the centrifugal thalline of removing of reaction solution gets supernatant liquor.Supernatant liquor is measured with the acid ninhydrine method.Result such as table 2:
Table 2: the influence of different concentration of substrate to transforming
| ATC concentration (%) | L-cysteine(mg/ml) |
| 0.1 | 0.103 |
| 0.3 | 0.411 |
| 0.6 | 0.845 |
| 1.0 | 2.047 |
| 1.3 | 2.038 |
| 1.6 | 2.033 |
Embodiment 5:
Use Pseudomonas sp.zjwp-14, after pressing example 3 method fermentation culture 12h, getting wet thallus that the fermenting enzyme liquid of 2.5～25ml obtains after centrifugal is added in the 20ml test tube and adorns 0.1M, pH8.0 phosphoric acid buffer 5ml, initial substrate DL-ATC amount is carried out conversion reaction for the 0.05g/ pipe under 42 ℃ of water-baths.The 2h afterreaction finishes, and adds 1mlHCl (1M) inactivator source, and the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor is measured with the acid ninhydrine method, result such as table 3.
Table 3: the influence of different biomasses to transforming
| Biomass (fermenting enzyme liquid is long-pending) | L-cysteine(mg/ml) | Enzyme (U/ml) alive |
| 2.5 | 0.352 | 352.0 |
| 5.0 | 1.451 | 725.5 |
| 10 | 2.047 | 1023.5 |
| 15 | 2.045 | 1022.5 |
| 20 | 2.031 | 1015.1 |
| 25 | 2.028 | 1014.0 |
Embodiment 6:
Use Pseudomonas sp.zjwp-14, press example 3 method enzymatic productions, carry out conversion reaction by example 5 methods, contain substrate DL-ATC amount in the 5ml reaction system and be 0.05g, metal ion content is respectively 0.2mM and 2mM, take a sample behind the reaction 2h, carry out the acid ninhydrine content analysis, the result is as shown in table 4.
Table 4: the influence that the different metal ions enzyme is lived
| Time (h) | L-cysteine(mg/ml) | Enzyme (U/ml) alive |
| MnSO 4 0.2 2 | 3.072 3.616 | 1536 1808 |
| ZnSO 4 0.2 2 | 2.011 1.452 | 1006 726.0 |
| CoCl 6 0.2 2 | 1.998 2.025 | 999.0 1013 |
| MySO 4 0.2 2 | 1.833 2.065 | 916.5 1033 |
| CuSO 4 0.2 2 | 1.865 1.564 | 932.5 782.0 |
| FeSO 4 0.2 2 | 1.975 2.045 | 987.5 1023 |
| CaCl 2 0.2 2 | 1.963 1.812 | 981.5 906.0 |
| Contrast | 2.047 | 1024 |
Embodiment 7:
Use Pseudomonas sp.zjwp-14, press example 3 method enzymatic productions, contain substrate DL-ATC amount in the 5ml reaction system and be 0.05g, carry out conversion reaction by example 6 methods, transform in 20-52 ℃ water-bath and react 2h, after reaction finishes, carry out acid ninhydrine and analyze content.Result such as table 5.
Table 5 invert point curve
| Invert point (℃) | L-cysteine(mg/ml) |
| 20 | 1.452 |
| 25 | 1.759 |
| 30 | 2.658 |
| 37 | 3.105 |
| 42 | 3.616 |
| 47 | 0.256 |
| 52 | 0 |
Embodiment 8:
Use Pseudomonas sp.zjwp-14, the enzymatic production method is carried out the conversion reaction method with example 6 with example 3, contains substrate DL-ATC amount in the 50ml reaction system and is 0.5g.In the sampling of reaction different time, carry out acid ninhydrine and analyze content and calculate conversion yield.The result is as shown in table 6.
Table 6: transformation time curve
| Time (h) | L-cysteine(mg/ml | Mole conversion yield (%) |
| 0 | 0 | 0.0 |
| 1 | 1.45 | 19.6 |
| 2 | 3.616 | 49.0 |
| 3 | 3.868 | 52.4 |
| 4 | 4.025 | 54.5 |
| 5 | 4.447 | 60.2 |
| 6 | 4.582 | 62.1 |
| 7 | 4.585 | 62.1 |
| 8 | 4.552 | 61.6 |
| 9 | 4.559 | 61.7 |
Sequence table .ST25
SEQUENCE LISTING
＜110〉Zhejiang Polytechnical University
＜120〉pseudomonas zjwp-14 and prepare application in the L-halfcystine at asymmetric hydrolysis
<130>
<160>1
<170>PatentIn version 3.2
<210>1
<211>1456
<212>DNA
<213>Pseudomonas sp.zjwp-14
<400>1
Claims (9)
1. pseudomonas (Pseudomonas sp.) zjwp-14 is preserved in Chinese typical culture collection center, and preserving number is: CCTCC M.206104.
2. pseudomonas zjwp-14 as claimed in claim 1 prepares the application in the L-halfcystine at DL-2-amino-thiazolyl-quinoline-4-carboxylic acid asymmetric hydrolysis.
3. application as claimed in claim 2, it is characterized in that described being applied as: carrying out conventional wet thallus or the treated crude enzyme liquid that obtains of wet thallus of cultivating acquisition with pseudomonas zjwp-14 is the enzyme source, with DL-2-amino-thiazolyl-quinoline-4-carboxylic acid is substrate, in conversion fluid, under 20～52 ℃, carry out microbial transformation reaction 1～9h, react completely to such an extent that contain the reaction solution of L-halfcystine, make the L-halfcystine through separation and Extraction.
4. application as claimed in claim 3 is characterized in that described substrate DL-2-amino-thiazolyl-quinoline-4-carboxylic acid initial mass concentration is 0.5%～2.5%.
5. application as claimed in claim 3 is characterized in that described enzyme source is a wet thallus, and described wet thallus is from fermenting enzyme liquid, OD after 10 times of the described fermenting enzyme liquid dilutions
600Be 0.4～0.6, described wet thallus addition is 0.5～5:1 by fermenting enzyme liquid with conversion fluid volume ratio.
6. application as claimed in claim 5 is characterized in that described application is to carry out seed culture and fermentation culture with pseudomonas zjwp-14, from fermenting enzyme liquid the centrifugal wet thallus that obtains as the enzyme source, OD after 10 times of the described fermenting enzyme liquid dilutions
600Be 0.4～0.6, with the phosphate buffer soln that contains 0.1～1.5% DL-ATC is conversion fluid, described wet thallus addition is 0.5～5:1 by fermenting enzyme liquid with conversion fluid volume ratio, the control invert point is 20～52 ℃, carry out microbial transformation reaction 1～9h, react completely to such an extent that contain the reaction solution of L-halfcystine, make the L-halfcystine through separation and Extraction.
7. application as claimed in claim 3 is characterized in that described application obtains wet thallus as follows:
(1) slant culture: the single bacterium colony switching of picking pseudomonas zjwp-14 slant medium, cultivate 24～48h for 30 ℃ and get the activatory bacterial classification; Described slant medium is formed: DL-2-amino-thiazolyl-quinoline-4-carboxylic acid 1.0～5.0g/L, glycerine 10～30g/L, yeast extract 2.0～10g/L, peptone 2.0～10g/L, NaCl 1.0～5.0g/L, agar 15～25g/L, pH7.0～8.0; 121 ℃ of sterilization 20min, sterilization postcooling bevel;
(2) seed culture: one ring pseudomonas zjwp-14 is transferred to seed culture medium with the transfering loop picking, and 30 ℃, shaking speed 200r/min cultivates 10～24h and gets seed culture fluid; Described seed culture medium is formed: DL-2-amino-thiazolyl-quinoline-4-carboxylic acid 1.0～5.0g/L, glycerine 5.0～30.0g/L, yeast extract 5.0～30g/L, peptone 5.0～30g/L, extractum carnis 5.0～30g/L, NaCl 1.0～5.0g/L, pH7.0～8.0; 121 ℃ of sterilization 20min, the sterilization postcooling is made seed culture medium;
(3) producing enzyme cultivates: get the seed culture fluid inoculation, inoculum size is 0.5%～5%, 25～35 ℃, and shaking speed 100～250r/min carries out shake-flask culture 10～24h in producing the enzyme substratum, obtain fermenting enzyme liquid; Described product enzyme substratum is formed with the seed substratum;
(4) wet thallus is collected in fermenting enzyme liquid is centrifugal, washing.
8. pseudomonas zjwp-14 as claimed in claim 6 prepares the application in the L-halfcystine at DL-2-amino-thiazolyl-quinoline-4-carboxylic acid asymmetric hydrolysis, it is characterized in that described being applied as: the wet thallus 0.1M that fermenting enzyme liquid is directly made, the phosphoric acid buffer of pH8.0 cleans, OD after described fermenting enzyme liquid dilutes 10 times
600Be 0.4～0.6, wet thallus is changed in the described damping fluid again, described wet thallus addition by fermenting enzyme liquid and conversion fluid volume than being 2:1, adding substrate DL-2-amino-thiazolyl-quinoline-4-carboxylic acid concentration simultaneously, to make substrate initial mass concentration be 1%, in 42 ℃, water-bath 6h, react completely to such an extent that contain the reaction solution of L-halfcystine, make the L-halfcystine through separation and Extraction.
9. pseudomonas zjwp-14 as claimed in claim 1 prepares the application in the L-Gelucystine at DL-2-amino-thiazolyl-quinoline-4-carboxylic acid asymmetric hydrolysis, it is characterized in that preparing on the basis of L-halfcystine reaction solution through pseudomonas zjwp-14 asymmetric hydrolysis DL-2-amino-thiazolyl-quinoline-4-carboxylic acid, further with L-halfcystine reaction solution separation and Extraction, described separation and Extraction step is: reaction solution is centrifugal, gets supernatant liquor and adds FeSO
4Be enough to make the L-halfcystine to be oxidized to the L-Gelucystine, behind vibration 20～30h, regulating the pH value with dense HCl solution is 2, evaporation concentration, the cooling back is centrifugal, and supernatant liquor stream is added in the ion exchange column that 732 resins are housed, use deionized water drip washing, use the ammoniacal liquor wash-out of 0.5～1.0mol/L again, merge the higher effluent liquid of L-cystine after wash-out finishes, vacuum-drying obtains described L-Gelucystine.
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| CN108517306A (en) * | 2018-05-09 | 2018-09-11 | 威海迪素制药有限公司 | A kind of method that bioanalysis prepares L-cysteine |
| CN114276947B (en) * | 2021-10-12 | 2024-07-23 | 湖北远大生物技术有限公司 | Method for preparing L-cysteine by enzymatic conversion and application thereof |
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| Title |
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| 微生物转化DL-ATC生产L-半胱氨酸菌种筛选及产酶条件研究. 王普等.药物生物技术，第12卷第5期. 2005 |
| 微生物转化DL-ATC生产L-半胱氨酸菌种筛选及产酶条件研究. 王普等.药物生物技术，第12卷第5期. 2005 * |
| 微生物酶法合成L-半胱氨酸和L-胱氨酸. 刘忠等.微生物学通报，第30卷第6期. 2003 |
| 微生物酶法合成L-半胱氨酸和L-胱氨酸. 刘忠等.微生物学通报，第30卷第6期. 2003 * |
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