CN105177076A - Method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid) - Google Patents
Method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid) Download PDFInfo
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Abstract
The invention discloses a method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid). Fusion protein (SEQ ID NO.1) containing DL-ATC racemase and L-ATC hydrolase and nitrogen-carbamoyl-L-cysteine hydrolase (SEQ ID NO.4) are subjected to immobilization by means of polyvinyl alcohol-sodium alginate composite carrier through a crosslinking-embedding-crosslinking process, a co-immobilized enzyme system is obtained, and DL-ATC can be converted to generate L-cysteine. Sequences of genes of the fusion protein and nitrogen-carbamoyl-L-cysteine hydrolase are encoded to be shown in SEQ ID NO.2 and SEQ ID NO. 5 respectively, the genes are constructed to pET-28a, and high-purity target protein is obtained through induced expression and purification. The immobilized enzyme system conversion efficiency and the stability are high, and the method has important industrial application value in synthesis of L-cysteine.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of immobilized enzyme and transform DL-2-amino-△
2-thiazoline-4-carboxylic acid (DL-ATC) synthesizes the method for Cys.
Background technology
Cys is the amino acid uniquely in 20 multiple amino acids of constitutive protein matter with reductibility group sulfydryl (-SH), has important physiological function.Widespread use in medicine, foodstuff additive and makeup at present.China is Cys big producing country, accounts for 80% of global ultimate production, and product has not only captured domestic market, and exports in a large number all over the world.At present, domestic production Cys mainly relies on the Keratin sulfate in the hair of human or animal to extract after CYSTINE through acid hydrolysis, then obtains Cys through electrolytic reduction.The method yield is low, and energy consumption is high, and hydrolytic process produces a large amount of irritant gas, and liquid waste disposal difficulty, environmental pollution is serious.In recent years, along with the development of Cys production technology, progressively instead of hair-hydrolyzation with microbe transformation method in the world and prepare Cys.With DL-ATC enzymatic conversion method most advantage in microbe transformation method.DL-ATC enzymatic conversion method utilizes saccharase system (comprising DL-ATC racemase, L-ATC lytic enzyme, sulphur-carboxamide-Cys lytic enzyme, nitrogen one carboxamide-Cys lytic enzyme) contained in microorganism cells to be Cys by DL-ATC bio-transformation.This method is that the people such as Japanese Sano in 1977 propose, and the pseudomonas (Pseudomonassp.) that they screen from soil can by DL-2-amino-△
2-thiazoline-4-carboxylic acid (DL-ATC) bio-transformation is Cys.The advantages such as this method has that high specificity, production technique are simple, side reaction and by product is few, product is homogeneous, easy extraction, are suitable for synthesizing with the more difficult amino acid of fermentative Production very much.
Although microbial enzyme method transforms this technology of DL-ATC synthesis Cys and has comparatively ripe application in the world, but it is not long (because L-ATC lytic enzyme is not high in the thermostability of physiological temp for work-ing life owing to there is thalline, volatility, its transformation period only has 100 hours), enzyme in enzymatic reaction approach is inducible enzyme, long to the substrate time of response, in wild strain, enzyme amount is not enough, vigor is relatively low etc. makes the continuous seepage ability of whole set process greatly be limited, production cost remains high, this method general is only for the generation of medical grade high purity Cys.Particularly there is the catabolic pathway of halfcystine in wild mushroom, the Cys accumulated in conversion process is decomposed, and discharges a large amount of H
2s gas, has had a strong impact on productive rate.And at present due to the shortage of strain gene group information and some are unknown, very large to the genetic engineering modified work difficulty of wild pseudomonas, be in progress not always.
As can be seen here, found the novel method that a set of enzymatic conversion method DL-ATC that is efficient, high yield synthesizes Cys and just seem very important.
Summary of the invention
The wild-type pseudomonas method that utilizes that the object of the invention is to solve prior art existence transforms DL-2-amino-△
2-thiazoline-4-carboxylic acid (DL-ATC) synthesizes the technical problem run in Cys technique, provides a kind of more efficient immobilized enzyme to transform the method for DL-ATC synthesis Cys.
Object of the present invention is achieved through the following technical solutions:
Immobilized enzyme transforms the method that DL-ATC synthesizes Cys, comprises the steps:
(1) preparation of co-immobilization enzyme system
The fusion rotein AtcAB be made up of DL-ATC racemase, be rigidly connected peptide and L-ATC lytic enzyme and nitrogen-carboxamide-Cys hydrolase A tcC is joined in the aqueous solution containing polyvinyl alcohol and sodium alginate, after abundant mixing, add glutaraldehyde solution and carry out crosslinking reaction.Reaction solution is dropwise instilled in calcium chloride solution, leaches microsphere.Microsphere is immersed in calcium chloride solution again and carries out hardening treatment.Be cross-linked with glutaraldehyde solution again after leaching microsphere, then leach microsphere, for subsequent use after washing.
The aminoacid sequence of described fusion rotein AtcAB is as shown in SEQIDNO.1, and the nucleotide sequence of the gene of this fusion rotein of encoding is as shown in SEQIDNO.2; The aminoacid sequence of described nitrogen-carboxamide-Cys hydrolase A tcC is as shown in SEQIDNO.4; Encode the nucleotide sequence of gene of this nitrogen-carboxamide-Cys lytic enzyme as shown in SEQIDNO.5.
(2) co-immobilization enzyme system transforms DL-ATC and generates Cys
The microsphere containing co-immobilization enzyme system step (1) obtained joins containing reactant DL-ATC, KH
2pO
4, sorbyl alcohol system in carry out being obtained by reacting Cys.
Fusion rotein described in step (1) is prepared preferably by the method comprised the steps:
1) DNA fragmentation of sequence as shown in SEQIDNO.3 is connected to structure on pET-28a (+) carrier by NdeI, EcoRI and obtains expression vector pET-atcAB.
2) carry out abduction delivering after expression vector pET-atcAB being proceeded to e. coli bl21 (DE3), carry out cytoclasis after having expressed, use Ni
2+the method purifying of affinity chromatography obtains target protein.
Nitrogen-carboxamide-Cys lytic enzyme described in step (1) is prepared preferably by the method comprised the steps:
1) DNA fragmentation of sequence as shown in SEQIDNO.6 is connected to structure on pET-28a (+) carrier by NdeI, EcoRI and obtains expression vector pET-atcC.
2) carry out abduction delivering after expression vector pET-atcC being proceeded to e. coli bl21 (DE3), carry out cytoclasis after having expressed, use Ni
2+the method purifying of affinity chromatography obtains target protein.
Described expression vector pET-28a (+) is Novagen Products, can arbitrarily buy from market; Described e. coli bl21 (DE3) bacterial strain is Promega Products, also can arbitrarily buy from market.
The mass ratio of the fusion rotein described in step (1) and nitrogen-carboxamide-Cys lytic enzyme is preferably 1:0.2-1:1.
The aqueous solution containing polyvinyl alcohol and sodium alginate described in step (1) is preferably pH7.5, the aqueous solution containing 80g/L polyvinyl alcohol and 15g/L sodium alginate.
The concentration of the calcium chloride solution described in step (1) is preferably 20g/L.
In step (1), first time is cross-linked glutaraldehyde solution used, and the massfraction of glutaraldehyde is preferably 10%, and the volume of the glutaraldehyde solution added is preferably the 3%-8% of crosslinking reaction system cumulative volume, and the time of crosslinking reaction is preferably 3h.
The crosslinked glutaraldehyde solution used of second time in step (1), the massfraction of glutaraldehyde is preferably 0.02%, and the time of crosslinking reaction is preferably 3h.
In step (1), the washing spherular solution that leaches is preferably pH7.5, containing 15g/LKH
2pO
4the aqueous solution.
Preferred, step (1) is: get fusion rotein and nitrogen-carboxamide-Cys lytic enzyme in mass ratio for the ratio of the amount of 1:0.2-1:1 joins pH7.5, containing in the aqueous solution of 80g/L polyvinyl alcohol and 15g/L sodium alginate, be 4-10mg/mL to total protein final concentration, after abundant mixing, the massfraction adding certain volume be 10% glutaraldehyde solution make the volume fraction of this solution in crosslinking reaction system reach 3%-8%, fully after mixing at 4 DEG C crosslinking reaction 3h.Reaction solution is dropwise added dropwise in the calcium chloride solution of 20g/L, leaches microsphere.Microsphere is placed in the calcium chloride solution of 20g/L, then soaks sclerosis 1h.At 4 DEG C, be cross-linked 3h again with the glutaraldehyde solution that massfraction is 0.02% after leaching microsphere, then leach microsphere.With pH be 7.5 containing 15g/LKH
2pO
4solution washing 3 times, be placed in 4 DEG C for subsequent use.
The reaction system of step (2) is preferably: the microsphere concentration containing co-immobilization enzyme system is 50-200g/L, and reactant DL-ATC concentration is 5-20g/L, KH
2pO
4concentration is 0.5-2g/L, and sorbitol concentration is 10-200g/L, pH is 6-8.
The condition optimization of the reaction described in step (2) is: temperature of reaction is 28-37 DEG C, and mixing speed is 100-200 rev/min, and the reaction times is 2-6h.
Under the reaction conditions described in step (2), the transformation efficiency that DL-ATC is converted into Cys reaches 75-90%.
After conversion reaction completes, the microsphere containing co-immobilization enzyme system can be reclaimed by filtering, the microsphere KH of recovery
2pO
4concentration is 2g/L, sorbitol concentration to be 200g/L, pH be 7.5 lavation buffer solution washing 2-3 time after, again can set up above-mentioned conversion reaction system with it, carry out conversion reaction.Iterative cycles like this, achieves co-immobilization enzyme system and repeatedly transforms DL-ATC synthesis Cys.Under condition of the present invention, when co-immobilization enzyme system uses eight cycles, still keep the enzymatic conversion vigor of more than 70%, for the suitability for industrialized production realizing Cys is laid a good foundation.
Synthesizing a co-immobilization enzyme system for Cys for transforming DL-ATC, being obtained by above-mentioned steps (1).
The present invention by from the DL-ATC racemase (atcA) of pseudomonas, L-ATC lytic enzyme (atcB) gene by codon optimized, add one section of peptide sequence that is rigidly connected in the middle of two gene orders after optimization.Form the amalgamation and expression frame of atcA+atcB, carry out the expression and purification of fusion rotein after being cloned into expression vector pET-28a (+) by chemosynthesis full genome; Secondly, nitrogen one carboxamide-Cys lytic enzyme (atcC) gene is crossed codon optimized after also carry out the expression and purification of fusion rotein after being cloned into expression vector pET-28a (+) by chemosynthesis full genome; 3rd, utilized by above-mentioned two kinds of recombinant proteins polyvinyl alcohol-sodium alginate complex carrier to carry out the co-immobilization of enzyme by crosslinked-embedded-cross-linked technique, the obtained immobilized multienzyme system that can transform DL-ATC and synthesize Cys.Finally, determine the condition of immobilized multienzyme system catalytic conversion reaction, thus form the novel method that complete immobilized enzyme transforms DL-ATC synthesis Cys.
Compare with the existing traditional method utilizing pseudomonas conversion DL-ATC to prepare Cys, advantage and the effect of the inventive method are as follows:
(1) the key gene atcA in path for transformation and atcB gene are carried out codon optimized after, this two gene carries out joining end to end and carries out amalgamation and expression, expression efficiency high (target protein accounts for about 25% of total protein of cell) by the utilization peptide that is rigidly connected; Fusion rotein exists with soluble form, has two kinds of enzyme activities, is equivalent to once express to obtain two kinds of target enzymes; Utilize and histidine-taggedly carry out Ni
2+affinity chromatography, purification efficiency is high, and affinitive layer purification can reach the purity of 90%;
(2) the key gene atcC of the 3rd in path for transformation is carried out codon optimized after carry out full genome synthesis, successfully in intestinal bacteria, achieve high expression level (target protein accounts for about 35% of total protein of cell), target protein exists in cell with soluble activating form, utilizes histidine-taggedly to carry out Ni
2+affinity chromatography, purification efficiency is high, and affinitive layer purification can reach the purity of more than 90%;
(3) sodium alginate-polyvinyl alcohol complex carrier is utilized, adopt the co-immobilization technology of glutaraldehyde secondary crosslinking, co-immobilization has been carried out to above-mentioned recombinant protein, efficiently solve that native enzyme activity is low, the problem of poor stability, overcome the defect that in common immobilization technology, immobilized enzyme is easily revealed simultaneously, improve operational stability, co-immobilization enzyme uses the transformation efficiency still keeping more than 70% during eight cycles.
The present invention be applied in the suitability for industrialized production of Cys, can greatly improve production efficiency and production cost, it has important industrial application value.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The preparation of embodiment 1DL-ATC racemase/L-ATC lytic enzyme fusion rotein
1, material
Bacterial classification: e. coli bl21 (DE3), purchased from Promega company.
Plasmid: plasmid pET28a (+) is purchased from Miao Ling bio tech ltd, Wuhan.
LB liquid nutrient medium: peptone 10g/L, yeast powder 5g/L, NaCl10g/L.
Kalamycin resistance is dull and stereotyped: the LB solid medium containing 30mg/L kantlex, 1.5% agar powder.
Kalamycin resistance LB substratum: the LB liquid nutrient medium containing 30mg/L kantlex.
2, method
(1) structure of atcAB expression vector
The DL-ATC racemase gene (atcA) (GenBanK accession number is: BAD15357) deriving from pseudomonas Pseudomonassp.BS pnca gene group and L-ATC hydrolase gene (atcB) (its sequence information is present in one section of GenBanK accession number be one section of size of AB176845 be in the DNA sequence dna of 10Kb) are carried out codon optimized, after optimization two add one section of coding in the middle of gene orders is rigidly connected the sequence of peptide, form the amalgamation and expression frame of atcA/atcB, the nucleotide sequence of this amalgamation and expression frame is as shown in SEQIDNO.2, the protein then called after AtcAB of this section of amalgamation and expression frame coding, its aminoacid sequence is as shown in SEQIDNO.1.This sequence designations is opt-atcAB by the complete genome sequence of chemosynthesis as shown in SEQIDNO.3 after 5 ' of this expression cassette is held and 3 ' end introduces restriction enzyme site NdeI and EcoRI respectively.The synthesis of opt-atcAB complete genome sequence transfers to Jin Sirui bio tech ltd to complete, and during delivery, the gene fragment opt-atcAB of synthetic is connected on carrier pUC57.Object fragment is reclaimed after carrier pUC57 NdeI and EcoRI containing opt-atcAB fragment is carried out double digestion, for subsequent use.Adopt NdeI and EcoRI to carry out double digestion to expression vector pET28a (+) simultaneously, and the opt-atcAB gene obtained after double digestion is connected in pET28a (+) carrier, transformation of E. coli TOP10, construction of expression vector pET-atcAB.Cut through enzyme and confirm that after expression vector establishment is errorless to extract plasmid for subsequent use with sequencing.
(2) preparation of BL21 (DE3) competent cell
1) the coli strain BL21 (DE3) on picking LB flat board, overnight incubation.
2) the bacterium liquid of overnight incubation is proceeded in the triangular flask of the 300mL that 50mLLB liquid nutrient medium is housed according to the inoculum size of 1% (V/V) cultivate, OD
600to about 0.4 time stop cultivate, put 20min on ice, 4 DEG C, the centrifugal 10min of 4000g.Abandon supernatant, add the CaCl of ice-cold 100mM
2solution suspension, leaves standstill 30min on ice.Centrifugal concentrating, obtains BL21 (DE3) competent cell, is put in-70 DEG C of preservations.
(3) conversion of pET-atcAB expression vector
1) proceeded to by the expression vector pET-atcAB of 50ng in BL21 (DE3) competent cell prepared by 100 μ L steps (2), mixing, puts 30min on ice, 42 DEG C of heat shock 90s, leaves standstill 2min on ice.
2) the LB liquid nutrient medium of 900 μ L is added, 37 DEG C, 100 turns/min cultivates 1h.
3) be coated with kalamycin resistance dull and stereotyped, overnight incubation, picking list bacterium colony extracts plasmid after cultivating and carries out digestion verification, and obtain engineering strain BL21 (DE3)+atcAB, this bacterial strain can abduction delivering recombination fusion protein AtcAB.
(4) expression and purification of recombinant protein
The single bacterium colony of picking engineering strain BL (DE3)+atcAB is also inoculated in 100mL kalamycin resistance LB substratum, in 37 DEG C of overnight incubation.After taking out bacterium liquid, be inoculated in 100mL kalamycin resistance LB substratum by 1:100 (V/V), be cultured to OD in 37 DEG C
600when=0.6, adding 1mol/LIPTG to final concentration is 1mmol/L, and shake bacterium in 37 DEG C and cultivate, induced fusion albumin A tcAB expresses.After induction 4h, under 8000r/min, centrifugal 10min collects thalline.By this thalline 20mL phosphate buffered saline buffer (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH
2pO
4, 1.44g/LNa
2hPO
4, pH=7.4) wash 3 times and use 10mL sample-loading buffer (20mMNa
3pO
4, 0.5MNaCl; 10mM imidazoles, pH7.4) resuspended after carry out ultrasonication, operational condition is: 50HZ, 200W, ultrasonic 3S, and interval 5S, works 100 times.Ultrasonic complete after, the centrifugal 15min of 12000g carries out electrophoresis detection after collecting precipitation and supernatant respectively.Find that recombination fusion protein AtcAB is present in thalline in solubility expression mode, the analysis of laser thin layer scanning shows, and target protein accounts for about 25% of bacterial protein.
Carried out filtering rear HisTrapaffinitycolumns (GEhealthcare Products) by the filter membrane of the ultrasonication supernatant liquor of above-mentioned acquisition with 0.45 μm, method to specifications carries out the purifying of recombination fusion protein.Concrete grammar is as follows:
1) be filled distilled water with 5mL syringe, turn on the stopper of post, with the joint provided, post is connected with syringe, wash post with 1mL/min flow velocity.
2) by 10mL sample-loading buffer balance, 1mL/min flow velocity.
3) by fusion rotein (the ultrasonication supernatant liquor after filtration) loading, 1mL/min flow velocity.
4) use 10mL sample-loading buffer, wash post with 1mL/min flow velocity.
5) with 20mL lavation buffer solution (20mMNa
3pO
4, 0.5MNaCl, 40mM imidazoles, pH7.4), wash post with 1mL/min flow velocity.
6) with 10mL elution buffer (20mMNa
3pO
4, 0.5MNaCl, 300mM imidazoles, pH7.4), with 1mL/min flow velocity wash-out, be in charge of collection, often pipe 1mL, 12%SDS-PAGE detect, and merge the sample containing target protein in elution fraction, freeze-drying saves backup.Recombinant protein is after this affinity chromatography purifying, and SDS-PAGE shows its purity more than 90%.
Embodiment 2 is recombinated the preparation of nitrogen one carboxamide-Cys lytic enzyme
1, material
Bacterial classification: e. coli bl21 (DE3), purchased from Promega company.
Plasmid: plasmid pET28a (+) is purchased from Miao Ling bio tech ltd, Wuhan.
LB liquid nutrient medium is: peptone 10g/L, yeast powder 5g/L, NaCl10g/L.
Kalamycin resistance is dull and stereotyped: the LB solid medium containing 30mg/L kantlex, 1.5% agar powder.
Kalamycin resistance LB substratum: the LB liquid nutrient medium containing 30mg/L kantlex.
2, method
(1) structure of atcC expression vector;
The nitrogen one carboxamide-Cys hydrolase gene (atcC) DNA sequence dna of to be one section of size of AB176845 be 10Kb (its sequence information is present in one section of GenBanK accession number in) deriving from pseudomonas Pseudomonassp.BS pnca gene group is carried out codon optimized, gene order after optimization is as shown in SEQIDNO.5, the protein then called after AtcC of this fragment gene sequence encoding, its aminoacid sequence is as shown in SEQIDNO.4.5 ' end of gene after optimization and 3 ' end introduce the complete genome sequence of chemosynthesis as shown in SEQIDNO.6 after restriction enzyme site NdeI and EcoRI respectively, are opt-atcC by this sequence designations.The synthesis of opt-atcC complete genome sequence transfers to Jin Sirui bio tech ltd to complete, and during delivery, the gene fragment opt-atcC of synthetic is connected on carrier pUC57.Object fragment is reclaimed after carrier pUC57 NdeI and EcoRI containing opt-atcC fragment is carried out double digestion, for subsequent use.Adopt NdeI and EcoRI to carry out double digestion to expression vector pET28a (+) simultaneously, the opt-atcC gene obtained after double digestion is connected in pET28a (+) carrier, and transformation of E. coli TOP10, construction of expression vector pET-atcC.Cut through enzyme and confirm that after expression vector establishment is errorless to extract plasmid for subsequent use with sequencing.
(2) preparation of competent cell
1) the coli strain BL21 (DE3) in picking LB flat board, overnight incubation.
2) the bacterium liquid of overnight incubation is proceeded in the triangular flask of the 300mL that 50mLLB liquid nutrient medium is housed according to the inoculum size of 1% (V/V) cultivate, OD
600to about 0.4 time stop cultivate, put 20min on ice, 4 DEG C, the centrifugal 10min of 4000g.Abandon supernatant, add the CaCl of ice-cold 100mM
2solution suspension, leaves standstill 30min on ice.Centrifugal concentrating, obtains BL21 (DE3) competent cell, is put in-70 DEG C of preservations.
(3) conversion of pET-atcC expression vector
1) proceeded to by the expression vector pET-atcC of 50ng in BL21 (DE3) competent cell prepared by 100 μ L steps (2), mixing, puts 30min on ice, 42 DEG C of heat shock 90s, leaves standstill 2min on ice.
2) the LB liquid nutrient medium of 900 μ L is added, 37 DEG C, 100 turns/min cultivates 1h.
3) be coated with kalamycin resistance dull and stereotyped, overnight incubation, picking list bacterium colony extracts plasmid after cultivating and carries out digestion verification, and obtain engineering strain BL21 (DE3)+atcC, this bacterial strain can abduction delivering recombinant protein A tcC.
(4) expression and purification of recombinant protein
The single bacterium colony of picking engineering strain BL (DE3)+atcC is also inoculated in 100mL kalamycin resistance LB substratum, in 37 DEG C of overnight incubation.After taking out bacterium liquid, be inoculated in 100mL kalamycin resistance LB substratum by 1:100 (V/V), be cultured to OD in 37 DEG C
600when=0.6, adding 1mol/LIPTG to final concentration is 1mmol/L, shakes bacterium and cultivates, induction expression of recombinant proteins in 37 DEG C.After induction 4h, under 8000r/min, centrifugal 10min collects thalline.By this thalline 20mL phosphate buffered saline buffer (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH
2pO
4, 1.44g/LNa
2hPO
4, pH=7.4) wash 3 times and use 10mL sample-loading buffer (20mMNa
3pO
4, 0.5MNaCl; 10mM imidazoles, pH7.4) resuspended after carry out ultrasonication, operational condition is: 50HZ, 200W, ultrasonic 3S, and interval 5S, works 100 times.Ultrasonic complete after, the centrifugal 15min of 12000g carries out electrophoresis detection after collecting precipitation and supernatant respectively.Find that recombinant protein A tcC is present in thalline in solubility expression mode, the analysis of laser thin layer scanning shows, and target protein accounts for about 35% of bacterial protein.
Carried out filtering rear HisTrapaffinitycolumns (GEhealthcare Products) by the filter membrane of the ultrasonication supernatant liquor of above-mentioned acquisition with 0.45 μm, method to specifications carries out the purifying of recombinant protein.Concrete grammar is as follows:
1) be filled distilled water with 5mL syringe, turn on the stopper of post, with the joint provided, post is connected with syringe, wash post with 1mL/min flow velocity.
2) by 10mL sample-loading buffer balance, 1mL/min flow velocity.
3) by recombinant protein (the ultrasonication supernatant liquor after filtration) loading, 1mL/min flow velocity.
4) use 20mL sample-loading buffer, wash post with 1mL/min flow velocity.
5) with 20mL lavation buffer solution (20mMNa
3pO
4, 0.5MNaCl, 40mM imidazoles, pH7.4), wash post with 1mL/min flow velocity.
6) with 10mL elution buffer (20mMNa
3pO
4, 0.5MNaCl, 100mM imidazoles, pH7.4), with 1mL/min flow velocity wash-out, be in charge of collection, often pipe 1mL, 12%SDS-PAGE detect, and merge the sample containing target protein in elution fraction, freeze-drying saves backup.Recombinant protein is after this affinity chromatography purifying, and SDS-PAGE shows its purity more than 90%.
The structure of embodiment 3 co-immobilization multi-enzyme system
What the recombination fusion protein AtcAB50mg obtained of the Example 1 and recombinant protein A tcC25mg obtained of embodiment 2 joined 12.5mL simultaneously adjusts pH in the aqueous solution of 7.5 containing 80g/L polyvinyl alcohol and 15g/L sodium alginate with 0.1NNaOH, be 6mg/mL to total protein final concentration, after abundant mixing, adding 750 μ L massfractions is the glutaraldehyde solution of 10%, fully after mixing at 4 DEG C crosslinking reaction 3h.Reaction solution No. 6 needle applicators are dropwise added dropwise in the calcium chloride solution of 20g/L, leach microsphere.Microsphere is placed in the calcium chloride solution of 20g/L, then soaks sclerosis 1h.At 4 DEG C, be cross-linked 3h again with the glutaraldehyde solution that 400mL massfraction is 0.02% after leaching microsphere, then leach microsphere.With pH be 7.5 containing 15g/LKH
2pO
4washings wash 3 times, be placed in 4 DEG C for subsequent use.
The catalytic conversion reaction of embodiment 4 co-immobilization enzyme system
Set up the conversion reaction system that volume is 1L, conversion reaction system and reaction conditions as follows: containing the microsphere 150g containing co-immobilization enzyme system obtained in embodiment 3 in system, reactant DL-ATC15g, KH
2pO
41.5g, sorbyl alcohol 100g, conversion reaction system pH are 7.5; Temperature of reaction is 30 DEG C, and mixing speed is 150 revs/min, and the reaction times is 1h.
After having reacted, extract reaction solution the content detecting Cys, concrete detection method sees reference document: Gaitonde, M.K., Aspectrophotometricmethodforthedirectdeterminationofcyst eineinthepresenceofothernaturallyoccurringaminoacids.Bio chemistryJournal, 1967,104,627-633., according to the molecule mol ratio of product Cys and substrate DL-ATC, calculates transformation efficiency.At this point in the reaction, the transformation efficiency that DL-ATC is converted into Cys reaches 90%.
After conversion reaction completes, microsphere can be reclaimed by filtering, the microsphere KH of recovery
2pO
4concentration is 2g/L, sorbitol concentration to be 200g/L, pH be 7.5 lavation buffer solution wash 3 times, again set up above-mentioned conversion reaction system with it, carry out conversion reaction.Iterative cycles like this, realizes co-immobilization enzyme system and repeatedly transforms DL-ATC synthesis Cys.
With this understanding, when immobilized enzyme system uses eight cycles, the transformation efficiency of reaction system is still more than 70%, and the transformation efficiency in each cycle is as following table:
| Life cycle | Transformation efficiency (%) |
| 1 | 90.4 |
| 2 | 88.6 |
| 3 | 86.5 |
| 4 | 84.3 |
| 5 | 82.1 |
| 6 | 77.2 |
| 7 | 74.2 |
| 8 | 70.3 |
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. immobilized enzyme transforms the method that DL-ATC synthesizes Cys, it is characterized in that comprising the steps:
(1) preparation of co-immobilization enzyme system
The fusion rotein be made up of DL-ATC racemase, be rigidly connected peptide and L-ATC lytic enzyme and nitrogen-carboxamide-Cys lytic enzyme are joined in the aqueous solution containing polyvinyl alcohol and sodium alginate, after abundant mixing, add glutaraldehyde solution and carry out crosslinking reaction; Reaction solution is instilled in calcium chloride solution, leach microsphere; Microsphere is immersed in calcium chloride solution again and carries out hardening treatment; Be cross-linked with glutaraldehyde solution again after leaching microsphere, then leach microsphere, for subsequent use after washing;
The aminoacid sequence of described fusion rotein is as shown in SEQIDNO.1; The aminoacid sequence of described nitrogen-carboxamide-Cys lytic enzyme is as shown in SEQIDNO.4;
(2) co-immobilization enzyme system transforms DL-ATC and generates Cys
The microsphere containing co-immobilization enzyme system step (1) obtained joins containing DL-ATC, KH
2pO
4, sorbyl alcohol system in carry out being obtained by reacting Cys.
2. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that: the fusion rotein described in step (1) is prepared by the method comprised the steps:
1) DNA fragmentation of sequence as shown in SEQIDNO.3 is passed through
ndei,
ecoRi is connected to structure on pET-28a (+) carrier and obtains expression vector pET-atcAB;
2) carry out abduction delivering after expression vector pET-atcAB being proceeded to e. coli bl21 (DE3), carry out cytoclasis after having expressed, use Ni
2+the method purifying of affinity chromatography obtains target protein.
3. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that: the nitrogen-carboxamide-Cys lytic enzyme described in step (1) is prepared by the method comprised the steps:
1) DNA fragmentation of sequence as shown in SEQIDNO.6 is passed through
ndei,
ecoRi is connected to structure on pET-28a (+) carrier and obtains expression vector pET-atcC;
2) carry out abduction delivering after expression vector pET-atcC being proceeded to e. coli bl21 (DE3), carry out cytoclasis after having expressed, use Ni
2+the method purifying of affinity chromatography obtains target protein.
4. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that:
The mass ratio of the fusion rotein described in step (1) and nitrogen-carboxamide-Cys lytic enzyme is 1:0.2-1:1;
The aqueous solution containing polyvinyl alcohol and sodium alginate described in step (1) is pH7.5, the aqueous solution containing 80g/L polyvinyl alcohol and 15g/L sodium alginate;
The concentration of the calcium chloride solution described in step (1) is 20g/L;
In step (1) the washing spherular solution that leaches be pH7.5, containing 15g/LKH
2pO
4the aqueous solution.
5. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that:
In step (1), first time is cross-linked the massfraction of glutaraldehyde solution used is 10%, and the volume of glutaraldehyde solution is the 3%-8% of crosslinking reaction system cumulative volume, and the time of crosslinking reaction is 3h;
In step (1), the massfraction of the crosslinked glutaraldehyde solution used of second time is 0.02%, and the time of crosslinking reaction is 3h.
6. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that: step (1) is: get fusion rotein and nitrogen-carboxamide-Cys lytic enzyme in mass ratio for the ratio of the amount of 1:0.2-1:1 joins pH7.5, containing in the aqueous solution of 80g/L polyvinyl alcohol and 15g/L sodium alginate, be 4-10mg/mL to total protein final concentration, after abundant mixing, add massfraction be 10% glutaraldehyde solution make the volume fraction of this solution in crosslinking reaction system reach 3%-8%, after abundant mixing at 4 DEG C crosslinking reaction 3h, reaction solution is added dropwise in the calcium chloride solution of 20g/L, leaches microsphere, microsphere is placed in the calcium chloride solution of 20g/L, then soaks sclerosis 1h, at 4 DEG C, be cross-linked 3h again with the glutaraldehyde solution that massfraction is 0.02% after leaching microsphere, then leach microsphere, with pH be 7.5 containing 15g/LKH
2pO
4solution washing 3 times, be placed in 4 DEG C for subsequent use.
7. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that: the reaction system of step (2) is: the microsphere concentration containing co-immobilization enzyme system is 50-200g/L, DL-ATC concentration is 5-20g/L, KH
2pO
4concentration is 0.5-2g/L, and sorbitol concentration is 10-200g/L, pH is 6-8.
8. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that: the condition of the reaction described in step (2) is: temperature of reaction is 28-37 DEG C, mixing speed is 100-200 rev/min, and the reaction times is 2-6h.
9. immobilized enzyme according to claim 1 transforms the method that DL-ATC synthesizes Cys, it is characterized in that: step (2) to contain the microsphere of co-immobilization enzyme system by filtered and recycled after having reacted, then use KH
2pO
4concentration is 2g/L, sorbitol concentration to be 200g/L, pH be 7.5 lavation buffer solution washing microsphere after be recycled and reused for step (2).
10. synthesizing a co-immobilization enzyme system for Cys for transforming DL-ATC, it is characterized in that: obtained by the step (1) in claim 1.
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| CN101348809A (en) * | 2007-07-18 | 2009-01-21 | 天津科技大学 | Method for producing L-aminothiopropionic acid by enzyme conversion |
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| CN101348809A (en) * | 2007-07-18 | 2009-01-21 | 天津科技大学 | Method for producing L-aminothiopropionic acid by enzyme conversion |
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