CN108517306A - A kind of method that bioanalysis prepares L-cysteine - Google Patents

Abstract

The present invention relates to a kind of pseudomonas putidas（Pseudomonsas putida）Method HW 3 bacterial strains and be used to prepare L cysteines belongs to application of biological engineering field.The technical scheme is that：One plant of pseudomonas putida strain（Pseudomonsas putida）HW 3, culture presevation number is CGMCC No.15553.The depositary institution of the bacterial strain is China Committee for Culture Collection of Microorganisms's common micro-organisms center.By screening provide one plant can bioconversion DL ATC prepare the bacterial strain HW 3 of L cysteines, be identified as pseudomonas putida.During the bacterial strain bioconversion prepares L cysteines, L cysteines yield is high, and reaction condition is mild, and controllably, environmental protection and energy saving have good prospects for commercial application.

Description

A kind of method that bioanalysis prepares L-cysteine

Technical field

The invention belongs to application of biological engineering field, it is related to a kind of utilizing pseudomonas putida（Pseudomonas putida）HW-3 microbe transformation methods（Enzyme process）The method for preparing L-cysteine.

Background technology

L-cysteine is a kind of important sulfur-containing amino acid, be widely used in sulfur-containing compound for example methionine, thiamine, The synthesis of biotin and coacetylase.In addition, L-cysteine ensures that protein is correctly folded, assembled and ties up by forming disulfide bond Hold the stability of albumen；Some researches show that the L-cysteines in periplasmic can improve tolerance of the Escherichia coli to hydrogen peroxide Property.In view of important physiological action of the L-cysteine in cellular process, it is also usually used in medicine intermediate, food additive Add the industries such as agent and cosmetics.

The production method of cysteine substantially has hair hydrolysis liquid extraction method, chemical synthesis and bioanalysis（Microorganism sends out Ferment method and enzyme process）.Hydrolyze method and chemical synthesis yield are relatively low, and operating procedure is more, and production process generates a large amount of not tractable Waste causes environmental pollution, faces larger environmental protection pressure；Bioanalysis prepares cysteine, has high specificity, produces work The advantages that skill is simple, side reaction and few by-product.

Currently, being widely used the method and wild mushroom fermentation the preparation method for preparing L-cysteine with engineering bacteria fermentation It compares, there is higher substrate conversion efficiency, but substrate conversion efficiency only has about 30%, still in reduced levels, this cannot meet L- The industrialization production requirements of cysteine.

By continually developing the potential catalysis of microbial enzyme, make the chemical synthesis process and microbial enzyme of technical maturity Method is combined, and a kind of chemistry that brand-new chemical technology intersects, be combined with each other, interpenetrating with biotechnology has had been established Biotechnology.DL-2- amino-△ 2- thiazoline -4- the carboxylic acids produced with chemical synthesis（DL-2-Amino-△2- thiazoline-4-Carboxylic Acid , DL-ATC）L-cysteine, which is produced, through microbial enzyme method conversion just causes people Concern.This method transformation efficiency is high, and product purification is easy, compared with fermentation method, there is larger advantage.Japan has reported It is the production technology that raw material produces through microbial enzyme method L-cysteine with DL-ATC.

Invention describes a kind of methods preparing cysteine using pseudomonas putida microbe transformation method, through in batches Fed-batch fermentation, and penetrant treating thalline is used, L-cysteine specific yield improves 7 times, and final product accumulates in conversion fluid Tired concentration can reach 24.6 g/L.And permeability cell is prepared with zymotic fluid, simplifies operating process, is more easy to realize industrialization.

Invention content

Goal of the invention

The purpose of the present invention is overcoming the shortcomings of that chemical method and fermentation method prepare L-cysteine technique, plant height selectivity is provided Catalysis DL-ATC biosynthesis L-cysteines pseudomonas putidaPseudomonas putida HW-3, and utilizing should Bacterial strain prepares L-cysteine.

The technical scheme is that：

One plant of pseudomonas putida HW-3, culture presevation number is CGMCC No.15553.During the depositary institution of the bacterial strain is State's Microbiological Culture Collection administration committee common micro-organisms center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Number Institute of Microorganism, Academia Sinica, culture presevation date are on 04 03rd, 2018.The Classification And Nomenclature of bacterial strain of the present invention It is：Pseudomonas putida, Latin literary fame are known as：Pseudomonas putida.

Bacterial strain provided by the invention is that screening obtains as follows：

Yantai, 1.0 g of Weihai soil sample is taken to be placed in shaking table 160 rpm, 30 DEG C of enrichment cultures 3 times, 24 h every time.Then bacterium will be diluted Liquid is coated on solid plate, after growing single bacterium colony, is inoculated into slant medium as primary dcreening operation strain.Primary dcreening operation strain is transferred to Fluid nutrient medium is cultivated, and centrifugation obtains thalline, after being washed for several times with PBS buffer solution, carries out the biology system of L-cysteine It is standby, the higher bacterial strain of vigor is chosen after testing as strain.

The present invention provides the identification of bacterial strain：

Colonial morphology：30 DEG C of 48 h of culture, obtain pale on plating medium, round, flat colony, surface wettability light Sliding, edge blurry is identified as gram-positive bacteria.

It, will using the 16S rDNA sequences of the primer amplification bacterial strain HW-3 of design using the cell total DNA of extraction as template PCR product carries out the agarose gel electrophoresis of 1 %, and electrophoresis result, which is shown, has amplified a segment for being about 1.4 kb, such as schemes 1, meet expected results.Through the genetic fragment for being 1385 bp is sequenced.

Discovery is compared in data in the sequencing result and Genbank of gene order, is provided in the present invention Microbial strains HW-3 withPseudomonas putida（KT970643.1）Homology highest（Homology, 100%/ 1365bps, based on 16S rDNA）, according to microbial molecules science of heredity identity principle, based on the same of 16S rDNA sequences Source property is higher than 95 %, and identification bacterium substantially belongs to control bacterium.In conjunction with bio-chemical characteristics, identify that the microbial strains belong to stench vacation Monad.

Bacterial strain in the present invention is used to prepare L-cysteine, using DL-ATC as raw material, with the HW- of fermented culture acquisition 3 thalline are as catalyst, and in pH 6.0-8.5,20-40 DEG C of progress bioconversion is final to obtain product L-cysteine.

It is as follows：

The first step：The preparation of fermentation seed liquid

HW-3 is inoculated into seed culture medium, shaking speed is 100-200 rpm, and 15-60 h are cultivated under the conditions of 20-45 DEG C, Obtain fermentation seed liquid.Culture medium constituent wherein used herein is：Maltose 1.0-30.0 g/L, DL-ATC 0.1-3.0 G/L, ammonium chloride 0.2-2.0 g/L, magnesium chloride 0.05-0.4 g/L, frerrous chloride 0.05-0.5 g/L, yeast extract 0.2- 10.0 g/L, ox bone peptone 2-20.0 g/L, disodium hydrogen phosphate 0.2-2.0 g/L, sodium chloride 0.1-1.2 g/L, solvent are Water.

Second step：Thalline ferments and bioconversion

Fermentation seed liquid is inoculated into the inoculum concentration of 1-8% in fermentation medium, condition of culture is：Shaking speed is 100-200 15-60 h are cultivated under the conditions of rpm, 20-45 DEG C.

Fermentation medium constituent is：Maltose 1.0-30.0 g/L, DL-ATC 0.1-3.0 g/L, molasses 0.2- 2.0 g/L, magnesium chloride 0.05-0.4 g/L, frerrous chloride 0.05-0.5 g/L, ammonium sulfate 1.0-10.0 g/L, disodium hydrogen phosphate 0.1-2.0 g/L, sodium chloride 0.1-1.2 g/L, manganese chloride 0.02-0.2 g/L, solvent is water.

After fermentation, the bleeding agent chloroform of 0.05-3.5 % is added into zymotic fluid, 0.2- is handled at 10-50 DEG C 4.0 h prepare permeability cell.

Permeability cell preparation process can be carried out in shaking flask, can also be carried out in fermentation tank, to increase thalline and bleeding agent Contact, enhance treatment effect, processing procedure will be shaken or be stirred.When shaking flask prepares permeability cell, places it in and shake Concussion processing, shaking speed 60-200 rpm in bed；When fermentation tank prepares permeability cell, stirring is opened, speed of agitator is 100-300 rpm。

In triangular flask or fermentation tank, using permeability cell as biocatalyst, it is added final concentration of 0.05-3.0 g/L's Ferment at constant temperature converts 5-40 h at 20-60 DEG C after DL-ATC.

After microbe conversion, zymotic fluid is taken to carry out 8000-12000 rpm, centrifuge 1-5 min, collects centrifuged supernatant. Microbial activity, screening operation bacterial strain are measured with acid ninhydrin method.

Fermented and cultured and biotransformation can carry out in shaking flask, can also be carried out in fermentation tank.Conversion process should be noted Meaning control pH is 6.0-8.5.

Enzyme activity defines：Under these conditions, conversion of substrate generates the enzyme amount needed for 1 μ g product L-cysteines in 1 h As an enzyme activity unit (U).Enzyme activity is indicated (U/mL) with the enzyme activity unit number contained by every milliliter of zymotic fluid.

Beneficial effects of the present invention are：Providing one plant by screening being capable of bioconversion DL-ATC preparation L-cysteine Bacterial strain HW-3, be identified as pseudomonas putida.During the bacterial strain bioconversion prepares L-cysteine, L-cysteine Yield is high, and reaction condition is mild, and controllably, environmental protection and energy saving have good prospects for commercial application.Substrate conversion efficiency can reach 83%.

【Description of the drawings】

The bacterial strain HW-3 16S rDNA sequence agarose gel electrophoresis figures of Fig. 1 PCR amplifications

Fig. 2 chloroforms concentration influences permeability cell viability

Influence of Fig. 3 chloroform processing times to permeability cell viability

Influences of Fig. 4 differences pH to permeability cell viability

Influence of Fig. 5 concentration of substrate to permeability cell viability

【Specific implementation mode】

With reference to specific implementation example, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：Microbe to screen

Enrichment culture is carried out to soil sample sample with seed culture medium, seed culture medium group becomes：Maltose 20.0 g/L, DL-ATC 1.0 g/L, 2.0 g/L of ammonium chloride, 0.4 g/L of magnesium chloride, 0.5 g/L of frerrous chloride, 10.0 g/L of yeast extract, ox bone albumen 20.0 g/L of peptone, 2.0 g/L of disodium hydrogen phosphate, 1.2 g/L of sodium chloride, solvent is water.

In the random soil sampling in Weihai Yantai periphery, and indicate sample time and place.

It takes 1.0 g soil samples to be placed in 100 mL triangular flasks, 20 mL physiological saline is added, adds 10 beades, is placed in shaking table 40 min of upper shake culture.0.5 mL soil supensions are taken to be added in enriched medium, shaking speed is 160 rpm, 30 DEG C of items 36 h are cultivated under part.After enrichment culture repeats 3 times, takes 0.1 mL culture solutions to be coated on solid plate and carry out separation training It supports.Solid plate culture medium constituent is：5.0 g/L of yeast powder, 10.0 g/L of peptone, 10.0 g/L of sodium chloride, agar 20.0 g/L, solvent are water, pH 7.0.It is cultivated under the conditions of 28 DEG C to single bacterium colony is grown, as primary dcreening operation strain library.Primary dcreening operation bacterium Kind of single bacterium colony is crossed on slant medium, is grown and is inoculated into seed culture concentration after bacterium colony, and shaking speed is 160 rpm, and 30 36 h are cultivated under the conditions of DEG C, obtain seed liquor.Seed liquor is connected to the inoculum concentration of 2 % in 80 mL fermentation mediums, item is cultivated Part is：Shaking speed is 200 rpm, and 48 h are cultivated under the conditions of 30 DEG C.Fermentation medium constituent is：20.0 g/ of maltose 1.0 g/L of L, DL-ATC, 1.0 g/L of molasses, 0.2 g/L of magnesium chloride, 0.1 g/L of frerrous chloride, 6.0 g/L of ammonium sulfate, phosphoric acid 1.2 g/L of disodium hydrogen, 0.8 g/L of sodium chloride, 0.1 g/L of manganese chloride, solvent is water.

After conversion, zymotic fluid centrifuges 2 min at 10000 rpm, collects centrifuged supernatant.With acid ninhydrin Method measures microbial activity（All elder generation's bowls, biochemistry Instrumental Analysis and technology, Chemical Industry Press）, screening operation bacterial strain. The original enzyme activities of pseudomonas putida HW-3 that acquisition is screened in the present invention are 85.3 U/mL, vigor highest, as work bacterium Kind.

Embodiment 2：It is prepared by microbial cells

It using the inclined-planes the HW-3 single bacterium colony filtered out as strain, is inoculated into seed culture medium, 160 rpm of shaking speed, at 30 DEG C Under the conditions of culture obtain the seed liquor of purebred bacterial strain.3-12 h are logarithmic phase in incubation after measured, choose the kind of logarithmic phase Sub- liquid is transferred.

It takes in the 80 mL fermentation mediums that 2 % seed liquors are inoculated into, shaking speed is 160 rpm, is trained under the conditions of 30 DEG C 48 h are supported, the zymotic fluid containing a large amount of thalline is obtained.

Embodiment 3：The selection of bleeding agent

It is 2.5 % to select three kinds of different penetrant treating pseudomonas putida HW-3, osmotic agent concentration, and processing time is 2.5 h, 30 DEG C for the treatment of temperature, the results are shown in Table 1.Enzyme activity relative value is control with original strain, and original strain vigor is set as 100 %。

The comparison that the different bleeding agents of table 1 influence permeability cell viability

The result shows that chloroform is best to the function and effect of permeability cell, it may act on the lipid knot on cell wall and cell membrane Structure has especially dissolved the phospholipid layer on cell membrane, has increased cell permeability, increases contact of the raw material with enzyme, to carry High enzyme activity.

Embodiment 4：Various dose chloroform prepares influence of the permeability cell to enzyme activity

In 100 mL systems, zymotic fluid is handled with 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% chloroform respectively, 2.5 h are handled at 30 DEG C and prepare permeability cell, and according to the vigor of permeability cell come the quality of evaluation condition, specific data are shown in figure 2.As shown in Figure 2,2.5 % chloroform handle 2.5 h of somatic cells, 536 % of permeability cell relative viability, relative activity highest, Therefore the dosage of selected area update strategy agent is 2.5 %.Enzyme activity relative value is control with original strain, and original strain vigor is set as 100 %.

Embodiment 5：Chloroform handles different time and prepares influence of the permeability cell to enzyme activity

In 100 mL systems, zymotic fluid is handled with 2.5 % chloroforms, handle 0.5 respectively at 30 DEG C, 1.0,1.5,2.0,2.5, 3.0,3.5 h prepare permeability cell, and according to the vigor of permeability cell come the quality of evaluation condition, specific data are shown in Fig. 3.By Fig. 3 It is handled it is found that handling 2.5 h, 614 % of relative activity of permeability cell, relative activity highest, therefore selection with the chloroform of 2.5 % Time is 2.5 h.Enzyme activity relative value is control with original strain, and original strain vigor is set as 100 %.

Embodiment 6：Permeability cell transformation prepares L-cysteine appropriate pH

In 100 mL systems, the DL-ATC of 2.5 % is added, at 30 DEG C, respectively in different pH（6.0、6.5、7.0、7.5、8.0、 8.5）Bioconversion is carried out in buffer solution.As shown in Figure 4, vigor is higher under the conditions of pH is 7.0-8.0, saturating when pH is 7.5 Property 648 % of cell relative viability, therefore select pH at 7.5 using permeability cell transformation prepare L-cysteine.Enzyme activity is opposite Value is control with original strain, and original strain vigor is set as 100 %.

Embodiment 7：Permeability cell transformation prepares L-cysteine suitable substrate concentration

In 100 mL systems, 30 DEG C, under pH 7.5, be separately added into the DL- of 0.75,1.0,1.25,1.5,1.75,2.0 g/L ATC, buffer solution in carry out bioconversion.As shown in Figure 5, substrate DL-ATC concentration is in 1.5 g/L, the phase of permeability cell To vigor highest, reach 721 %, therefore selects concentration of substrate for 1.5 g/L.Enzyme activity relative value is control with original strain, former Beginning bacterial strain vigor is set as 100 %.

Embodiment 8：The upper tank of pseudomonas putida ferments and prepared by the biology of L-cysteine

By 2% inoculum concentration, 160 mL seed liquors is taken to be seeded in 8.0 L fermentation mediums, setting speed of agitator is 200 rpm, Ventilatory capacity is 200 L/h, and control fermentation pH stablizes 7.5, and 2.5 % are added into fermentation tank after 48 h are cultivated under the conditions of 30 DEG C Bleeding agent, handle 2.5 h, prepare permeability cell.The DL-ATC that final concentration of 1.5 g/L is added constitutes transformation system. Stream plus suitable DL-ATC during the fermentation, it is about 1.5 g/L to keep DL-ATC concentration.It is converted by the feed supplement of 40 h, production Object concentration can reach 24.6 g/L, and substrate conversion efficiency reaches 83%.

Gene order table

CAAGTCGAGC GGATGACGGG AGCTTGCTCC TTGATTCAGC GGCGGACGGG TGAGTAATGC 0060

CTAGGAATCT GCCTGGTAGT GGGGGACAAC GTTTCGAAAG GAACGCTAAT ACCGCATACG 0120

TCCTACGGGA GAAAGCAGGG GACCTTCGGG CCTTGCGCTA TCAGATGAGC CTAGGTCGGA 0180

TTAGCTAGTT GGTGGGGTAA TGGCTCACCA AGGCGACGAT CCGTAACTGG TCTGAGAGGA 0240

TGATCAGTCA CACTGGAACT GAGACACGGT CCAGACTCCT ACGGGAGGCA GCAGTGGGGA 0300

ATATTGGACA ATGGGCGAAA GCCTGATCCA GCCATGCCGC GTGTGTGAAG AAGGTCTTCG 0360

GATTGTAAAG CACTTTAAGT TGGGAGGAAG GGCAGTAAGT TAATACCTTG CTGTTTTGAC 0420

GTTACCGACA GAATAAGCAC CGGCTAACTC TGTGCCAGCA GCCGCGGTAA TACAGAGGGT 0480

GCAAGCGTTA ATCGGAATTA CTGGGCGTAA AGCGCGCGTA GGTGGTTTGT TAAGTTGGAT 0540

GTGAAAGCCC CGGGCTCAAC CTGGGAACTG CATCCAAAAC TGGCAAGCTA GAGTACGGTA 0600

GAGGGTGGTG GAATTTCCTG TGTAGCGGTG AAATGCGTAG ATATAGGAAG GAACACCAGT 0660

GGCGAAGGCG ACCACCTGGA CTGATACTGA CACTGAGGTG CGAAAGCGTG GGGAGCAAAC 0720

AGGATTAGAT ACCCTGGTAG TCCACGCCGT AAACGATGTC AACTAGCCGT TGGAATCCTT 0780

GAGATTTTAG TGGCGCAGCT AACGCATTAA GTTGACCGCC TGGGGAGTAC GGCCGCAAGG 0840

TTAAAACTCA AATGAATTGA CGGGGGCCCG CACAAGCGGT GGAGCATGTG GTTTAATTCG 0900

AAGCAACGCG AAGAACCTTA CCAGGCCTTG ACATGCAGAG AACTTTCCAG AGATGGATTG 0960

GTGCCTTCGG GAACTCTGAC ACAGGTGCTG CATGGCTGTC GTCAGCTCGT GTCGTGAGAT 1020

GTTGGGTTAA GTCCCGTAAC GAGCGCAACC CTTGTCCTTA GTTACCAGCA CGTAATGGTG 1080

GGCACTCTAA GGAGACTGCC GGTGACAAAC CGGAGGAAGG TGGGGATGAC GTCAAGTCAT 1140

CATGGCCCTT ACGGCCTGGG CTACACACGT GCTACAATGG TCGGTACAGA GGGTTGCCAA 1200

GCCGCGAGGT GGAGCTAATC TCACAAAACC GATCGTAGTC CGGATCGCAG TCTGCAACTC 1260

GACTGCGTGA AGTCGGAATC GCTAGTAATC GCGAATCAGA ATGTCGCGGT GAATACGTTC 1320

CCGGGCCTTG TACACACCGC CCGTCACACC ATGGGAGTGG GTTGCACCAG AAGTAGCTAG 1380

TCTAA 1385。

Sequence table

<110>Disha Pharmaceutical Industry Group Corp., Ltd. of Weihai Disu Pharmaceutical Co., Ltd.

<120>A kind of method that bioanalysis prepares L-cysteine

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1385

<212> DNA

<213>Pseudomonas putida bacterium (Pseudomonas putida)

<400> 1

caagtcgagc ggatgacggg agcttgctcc ttgattcagc ggcggacggg tgagtaatgc 60

ctaggaatct gcctggtagt gggggacaac gtttcgaaag gaacgctaat accgcatacg 120

tcctacggga gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga 180

ttagctagtt ggtggggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga 240

tgatcagtca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 300

atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg 360

gattgtaaag cactttaagt tgggaggaag ggcagtaagt taataccttg ctgttttgac 420

gttaccgaca gaataagcac cggctaactc tgtgccagca gccgcggtaa tacagagggt 480

gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggtttgt taagttggat 540

gtgaaagccc cgggctcaac ctgggaactg catccaaaac tggcaagcta gagtacggta 600

gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt 660

ggcgaaggcg accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac 720

aggattagat accctggtag tccacgccgt aaacgatgtc aactagccgt tggaatcctt 780

gagattttag tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg 840

ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900

aagcaacgcg aagaacctta ccaggccttg acatgcagag aactttccag agatggattg 960

gtgccttcgg gaactctgac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020

gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgtaatggtg 1080

ggcactctaa ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat 1140

catggccctt acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa 1200

gccgcgaggt ggagctaatc tcacaaaacc gatcgtagtc cggatcgcag tctgcaactc 1260

gactgcgtga agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc 1320

ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagctag 1380

tctaa 1385

Claims (6)

1. one plant of pseudomonas putida strain HW-3, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, culture presevation number is CGMCC No. 15553.

2. applications of the pseudomonas putida strain HW-3 described in claim 1 in preparing L-cysteine.

3. the method that pseudomonas putida strain HW-3 according to claim 1 prepares L-cysteine, which is characterized in that with DL-2- amino-△ 2- thiazoline -4- carboxylic acids are raw material, and the HW-3 thalline obtained using fermented culture are as catalyst, in pH 6.0-8.5,20-40 DEG C of progress bioconversions, it is final to obtain product L-cysteine.

4. the method that pseudomonas putida strain HW-3 according to claim 1 prepares L-cysteine, which is characterized in that packet Include following steps：

The first step：The preparation of fermentation seed liquid：

HW-3 is inoculated into seed culture medium, shaking speed is 100-200 rpm, and 15-60 h are cultivated under the conditions of 20-45 DEG C, Obtain fermentation seed liquid；

Second step：Thalline ferments and bioconversion：

Fermentation seed liquid is inoculated into the inoculum concentration of 1-8% in fermentation medium, condition of culture is：Shaking speed is 100-200 15-60 h are cultivated under the conditions of rpm, 20-45 DEG C；

After fermentation, the bleeding agent chloroform of 0.05-3.5 % is added into zymotic fluid, 0.2-4.0 h are handled at 10-50 DEG C, Prepare permeability cell；

Permeability cell preparation process can be carried out in shaking flask, can also be carried out in fermentation tank, to increase connecing for thalline and bleeding agent It touches, enhances treatment effect, processing procedure will be shaken or be stirred；

When shaking flask prepares permeability cell, places it in concussion in shaking table and handle, shaking speed 60-200 rpm；In fermentation tank system When standby permeability cell, stirring is opened, speed of agitator is 100-300 rpm；

In triangular flask or fermentation tank, using permeability cell as biocatalyst, the DL- of final concentration of 0.05-3.0 g/L is added Ferment at constant temperature converts 5-40 h at 20-60 DEG C after ATC.

5. the method that pseudomonas putida strain HW-3 according to claim 4 prepares L-cysteine, which is characterized in that institute Stating seed culture medium constituent is：Maltose 1.0-30.0 g/L, DL-ATC 0.1-3.0 g/L, ammonium chloride 0.2-2.0 g/ L, magnesium chloride 0.05-0.4 g/L, frerrous chloride 0.05-0.5 g/L, yeast extract 0.2-10.0 g/L, ox bone peptone 2- 20.0 g/L, disodium hydrogen phosphate 0.2-2.0 g/L, sodium chloride 0.1-1.2 g/L, solvent is water.

6. the method that pseudomonas putida strain HW-3 according to claim 4 prepares L-cysteine, which is characterized in that institute Stating fermentation medium constituent is：Maltose 1.0-30.0 g/L, DL-ATC 0.1-3.0 g/L, molasses 0.2-2.0 g/L, Magnesium chloride 0.05-0.4 g/L, frerrous chloride 0.05-0.5 g/L, ammonium sulfate 1.0-10.0 g/L, disodium hydrogen phosphate 0.1-2.0 G/L, sodium chloride 0.1-1.2 g/L, manganese chloride 0.02-0.2 g/L, solvent is water.