CN105177076B - A kind of immobilised enzymes conversion DL-2- amino-△2The method of thiazoline -4- carboxylic acid synthesis L-cysteine - Google Patents
A kind of immobilised enzymes conversion DL-2- amino-△2The method of thiazoline -4- carboxylic acid synthesis L-cysteine Download PDFInfo
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- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 title claims abstract description 78
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 68
- 239000004201 L-cysteine Substances 0.000 title claims abstract description 55
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 51
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 49
- 235000013878 L-cysteine Nutrition 0.000 title claims abstract description 38
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 30
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 26
- CDMKLKAZVMTVHX-UHFFFAOYSA-N 4,5-dihydro-1,3-thiazol-3-ium-4-carboxylate Chemical compound OC(=O)C1CSC=N1 CDMKLKAZVMTVHX-UHFFFAOYSA-N 0.000 title abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
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- 239000000243 solution Substances 0.000 claims description 34
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 19
- 238000004132 cross linking Methods 0.000 claims description 14
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of methods of immobilised enzymes conversion DL-2- amino-△ 2- thiazoline -4- carboxylic acid synthesis L-cysteine.The present invention passed through using polyvinyl alcohol-sodium alginate complex carrier be crosslinked-racemase containing DL-ATC, the fusion protein (SEQ ID NO.1) of L-ATC hydrolase and nitrogen-carbamyl-L-cysteine hydrolase (SEQ ID NO.4) fixed and obtain co-immobilization enzyme system by embedded-cross-linked technique, DL-ATC generation L-cysteine can be converted.Encoding fusion protein, nitrogen-carbamyl-L-cysteine hydrolase gene sequence respectively as shown in SEQ ID NO.2,5, will be described gene constructed to obtaining high-purity target protein by expression and purity on pET-28a.Co-immobilization enzyme system transformation efficiency of the invention, stability are high, have important industrial application value in the synthesis of L-cysteine.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of immobilised enzymes conversion DL-2- amino-△2Thiazole
The method of quinoline -4- carboxylic acid (DL-ATC) synthesis L-cysteine.
Background technique
L-cysteine is in more than 20 kinds of amino acid of constitutive protein matter uniquely with the ammonia of reproducibility group sulfydryl (- SH)
Base acid, has important physiological function.It is widely applied in medicine, food additives and cosmetics at present.China is L- half
Cystine big producer, the 80% of Zhan Quanqiu total output, product has not only captured domestic market, but also it is each largely to export to the world
Ground.Currently, domestic production L-cysteine mainly relies on the keratin in the hair of human or animal to extract L- Guang through sour water solution
After propylhomoserin, L-cysteine is made using electroreduction.This method yield is low, and energy consumption is high, and hydrolytic process generates a large amount of stimulations
Property gas, liquid waste processing is difficult, and environmental pollution is serious.In recent years, with the development of L-cysteine production technology, in the world
L-cysteine is gradually prepared instead of hair-hydrolyzation with microbe transformation method.With DL-ATC enzymatic conversion method in microbe transformation method
Most advantage.DL-ATC enzymatic conversion method is to utilize conversion enzyme system contained in microbial cell (including DL-ATC racemase, L-
ATC hydrolase, sulphur-carbamyl-L-cysteine hydrolase, one carbamyl of nitrogen-L-cysteine hydrolase) DL-ATC is raw
Object is converted into L-cysteine.This method is that Japan Sano in 1977 et al. is proposed, the vacation that they screen from soil
Monad (Pseudomonas sp.) can be by DL-2- amino-△2Thiazoline -4- carboxylic acid (DL-ATC) is biologically converted into L- half
Cystine.This method has many advantages, such as that high specificity, simple production process, side reaction and by-product are few, product is uniform, easy extraction,
It is suitable for very much the amino acid synthesized with fermentation method comparision of production difficulty.
Although microbial enzyme method conversion DL-ATC synthesis this technology of L-cysteine has more mature answer in the world
With, but due to there are thallus service life it is not long (due to L-ATC hydrolase in the thermal stability of physiological temp not high, mutability,
Its half-life period only has 100 hours or so), the enzyme in enzymatic reaction approach is induced enzyme, wild mushroom long to the substrate response time
Enzyme amount is insufficient in strain, and the relatively low continuous production ability for making whole set process of vigor is limited significantly, and production cost occupies high
Under not, the general method is served only for the generation of medical grade high-purity L-cysteine.Especially there are cysteines in wild mushroom
Catabolic pathway, the L-cysteine accumulated in conversion process are decomposed, and release a large amount of H2S gas, seriously affects
Yield.And at present to strain gene group information shortage and it is some it is unknown due to, to the base of wild pseudomonad
Because engineered work difficulty is very big, before being in progress not always.
It can be seen that founding the new method of a set of efficient, high yield enzymatic conversion method DL-ATC synthesis L-cysteine just
Seem particularly significant.
Summary of the invention
It is an object of the invention to solve utilization wild type pseudomonad method of the existing technology to convert DL-2- amino-
△2The technical issues of encountering in thiazoline -4- carboxylic acid (DL-ATC) synthesis L-cysteine technique provides a kind of highly efficient
Immobilised enzymes conversion DL-ATC synthesis L-cysteine method.
The purpose of the invention is achieved by the following technical solution:
A kind of method of immobilised enzymes conversion DL-ATC synthesis L-cysteine, includes the following steps:
(1) preparation of co-immobilization enzyme system
The fusion protein AtcAB and nitrogen-ammonia that will be made of DL-ATC racemase, rigid connection peptide and L-ATC hydrolase
Formyl-L-cysteine hydrolase A tcC is added in the aqueous solution containing polyvinyl alcohol and sodium alginate, after mixing well,
Glutaraldehyde solution is added and carries out cross-linking reaction.Reaction solution is instilled in calcium chloride solution dropwise, filters out spherula.Spherula soaks again
Bubble carries out cure process in calcium chloride solution.It is crosslinked again with glutaraldehyde solution after filtering out spherula, then filters out spherula, washed
It is spare afterwards.
The amino acid sequence of the fusion protein AtcAB encodes the gene of the fusion protein as shown in SEQ ID NO.1
Nucleotide sequence as shown in SEQ ID NO.2；Nitrogen-the carbamyl-L-cysteine hydrolase A tcC amino acid sequence
Column are as shown in SEQ ID NO.4；Encode the nucleotide sequence such as SEQ of nitrogen-carbamyl-L-cysteine hydrolase gene
Shown in ID NO.5.
(2) co-immobilization enzyme system conversion DL-ATC generates L-cysteine
The spherula containing co-immobilization enzyme system that step (1) obtains is added to containing reactant DL-ATC, KH2PO4、
It is reacted to obtain L-cysteine in the system of sorbierite.
Fusion protein described in step (1) preferably passes through the method included the following steps and is prepared:
1) sequence DNA fragmentation as shown in SEQ ID NO.3 is connected to pET-28a (+) carrier by NdeI, EcoRI
Upper building obtains expression vector pET-atcAB.
2) expression vector pET-atcAB is transferred to e. coli bl21 (DE3) and carries out inducing expression afterwards, expression is completed laggard
Row clasmatosis, uses Ni2+The method of affinity chromatography purifies to obtain destination protein.
Nitrogen-carbamyl-L-cysteine hydrolase described in step (1) preferably pass through the method that includes the following steps into
Row preparation:
1) sequence DNA fragmentation as shown in SEQ ID NO.6 is connected to pET-28a (+) carrier by NdeI, EcoRI
Upper building obtains expression vector pET-atcC.
2) expression vector pET-atcC is transferred to e. coli bl21 (DE3) and carries out inducing expression afterwards, expression is completed laggard
Row clasmatosis, uses Ni2+The method of affinity chromatography purifies to obtain destination protein.
The expression vector pET-28a (+) is Novagen Products, can arbitrarily be bought from market；The large intestine bar
Bacterium BL21 (DE3) bacterial strain is Promega Products, also can arbitrarily be bought from market.
Fusion protein described in step (1) and nitrogen-carbamyl-L-cysteine hydrolase mass ratio are preferably 1:
0.2-1:1。
Aqueous solution containing polyvinyl alcohol and sodium alginate described in step (1) is preferably pH 7.5, the poly- second containing 80g/L
The aqueous solution of enol and 15g/L sodium alginate.
The concentration of calcium chloride solution described in step (1) is preferably 20g/L.
Glutaraldehyde solution used is crosslinked in step (1) for the first time, the mass fraction of glutaraldehyde is preferably 10%, addition
The volume of glutaraldehyde solution is preferably the 3%-8% of cross-linking reaction system total volume, and the time of cross-linking reaction is preferably 3h.
Glutaraldehyde solution used is crosslinked second in step (1), and the mass fraction of glutaraldehyde is preferably 0.02%, crosslinking
The time of reaction is preferably 3h.
It is preferably pH 7.5, KH containing 15g/L that washing, which filters out spherular solution, in step (1)2PO4Aqueous solution.
It is furthermore preferred that step (1) are as follows: take fusion protein and nitrogen-carbamyl-L-cysteine hydrolase is 1 in mass ratio:
The ratio of the amount of 0.2-1:1 is added in the aqueous solution of pH 7.5, polyvinyl alcohol containing 80g/L and 15g/L sodium alginate, until total
Final concentration of protein is 4-10mg/mL, and after mixing well, the glutaraldehyde solution that the mass fraction that certain volume is added is 10% makes
Volume fraction of the solution in cross-linking reaction system reaches 3%-8%, after mixing well at 4 DEG C cross-linking reaction 3h.It will be anti-
It answers liquid to be added dropwise in the calcium chloride solution of 20g/L dropwise, filters out spherula.Spherula is placed on to the calcium chloride solution of 20g/L
In, then impregnate hardening 1h.It is crosslinked 3h again with the glutaraldehyde solution that mass fraction is 0.02% at 4 DEG C after filtering out spherula, then
Filter out spherula.The KH containing 15g/L for being 7.5 with pH2PO4Aqueous solution wash 3 times, be placed in 4 DEG C it is spare.
The reaction system of step (2) is preferred are as follows: the bead bulk concentration containing co-immobilization enzyme system is 50-200g/L, reaction
Object DL-ATC concentration is 5-20g/L, KH2PO4Concentration is 0.5-2g/L, sorbitol concentration 10-200g/L, pH 6-8.
The condition of reaction described in step (2) is preferred are as follows: reaction temperature is 28-37 DEG C, speed of agitator 100-200
Rev/min, reaction time 2-6h.
Under the reaction condition described in step (2), the conversion ratio that DL-ATC is converted into L-cysteine reaches 75-90%.
After the completion of conversion reaction, the spherula containing co-immobilization enzyme system can be recycled by filtering, the bead of recycling
Body KH2PO4It, can be again after the washing buffer that concentration is 2g/L, sorbitol concentration 200g/L, pH are 7.5 is washed 2-3 times
Above-mentioned conversion reaction system is established with it, carries out conversion reaction.It recycles repeatedly, realizes co-immobilization enzyme system and repeatedly convert
DL-ATC synthesizes L-cysteine.Under the conditions of the present invention, co-immobilization enzyme system use eight periods when, still keep 70% with
On enzymatic conversion vigor, for realize L-cysteine industrialized production lay a good foundation.
A kind of co-immobilization enzyme system synthesizing L-cysteine for converting DL-ATC, (1) obtains through the above steps.
The present invention passes through DL-ATC racemase (atcA), L-ATC hydrolase (atcB) gene from pseudomonad close
One section of rigid connection peptide sequence is added in numeral optimization, two gene orders centre after optimization.Form the fusion table of atcA+atcB
Up to frame, passes through chemical synthesis full genome and be cloned into the expression and purification that expression vector pET-28a (+) carries out fusion protein afterwards；
Secondly, one carbamyl of nitrogen-L-cysteine hydrolase (atcC) gene is crossed after codon optimization through chemical synthesis full genome
And it is cloned into the expression and purification that fusion protein is also carried out after expression vector pET-28a (+)；Third, by above-mentioned two kinds of recombinations egg
Brix with polyvinyl alcohol-sodium alginate complex carrier by be crosslinked-embedded-cross-linked technique carries out the co-immobilization of enzyme, be made
The immobilized multienzyme system of DL-ATC synthesis L- cysteine can be converted.Finally, determining that immobilized multienzyme system catalyzed conversion is anti-
The condition answered, to form the new method of complete immobilised enzymes conversion DL-ATC synthesis L-cysteine.
It is compared with the existing conventional method using pseudomonad conversion DL-ATC preparation L-cysteine, the method for the present invention
The advantages of and effect it is as follows:
(1) after the key gene atcA in path for transformation being carried out codon optimization with atcB gene, connected using rigidity
It connects peptide this two gene is joined end to end and carries out amalgamation and expression, expression efficiency is high, and (destination protein accounts for total protein of cell
25% or so)；Fusion protein exists with soluble form, and there are two types of enzyme activities for tool, and being equivalent to primary expression can be obtained two kinds
Target enzyme；Ni is carried out using histidine tag2+Affinity chromatography, purification efficiency is high, and one time affinitive layer purification can reach 90%
Purity；
(2) full genome synthesis is carried out after the third key gene atcC in path for transformation being carried out codon optimization,
High expression (destination protein accounts for 35% of total protein of cell or so) is successfully realized in Escherichia coli, destination protein is with solvable
Property active form there are in cell, utilize histidine tag to carry out Ni2+Affinity chromatography, purification efficiency is high, and one time affinity chromatography is pure
Changing is the purity that can reach 90% or more；
(3) sodium alginate-polyvinyl alcohol complex carrier is utilized, it is right using the co-immobilization technology of glutaraldehyde secondary cross-linking
Above-mentioned recombinant protein has carried out co-immobilization, efficiently solves the problems, such as that native enzyme activity is low, stability is poor, while overcoming general
Immobilised enzymes is easy the defect of leakage in logical immobilization technology, improves operational stability, and co-immobilization enzyme uses eight periods
When still keep 70% or more conversion ratio.
It applies the invention in the industrialized production of L-cysteine, can greatly improve production efficiency and be produced into
This, with important industrial application value.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
The preparation of embodiment 1DL-ATC racemase/L-ATC hydrolysis enzyme fusion proteins
1, material
Strain: e. coli bl21 (DE3) is purchased from Promega company.
Plasmid: plasmid pET28a (+) is purchased from Wuhan Miao Ling Biotechnology Co., Ltd.
LB liquid medium: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L.
Kalamycin resistance plate: the LB solid medium containing 30mg/L kanamycins, 1.5% agar powder.
Kalamycin resistance LB culture medium: the LB liquid medium containing 30mg/L kanamycins.
2, method
(1) building of atcAB expression vector
The DL-ATC racemase gene (atcA) of pseudomonad Pseudomonas sp.BS pnca gene group will be derived from
(GenBanK accession number are as follows: BAD15357) and L-ATC hydrolase gene (atcB) (its sequence information is present in one section
One section of size that GenBanK accession number is AB176845 is in the DNA sequence dna of 10Kb) codon optimization is carried out, after optimization
The sequence of one section of coding rigid connection peptide of addition, forms the amalgamation and expression frame of atcA/atcB, the fusion table among two gene orders
Up to frame nucleotide sequence as shown in SEQ ID NO.2, this section of amalgamation and expression frame coding protein be then named as AtcAB,
Amino acid sequence is as shown in SEQ ID NO.1.The expression cassette 5 ' end and 3 ' end respectively introduce restriction enzyme site NdeI and
This sequence designations is opt-atcAB by chemical synthesis complete genome sequence as shown in SEQ ID NO.3 after EcoRI.opt-
The synthesis of atcAB complete genome sequence transfers to Jin Sirui Biotechnology Co., Ltd to complete, when delivery artificial synthesized genetic fragment
Opt-atcAB is connected on carrier pUC57.Carrier pUC57 containing opt-atcAB segment is subjected to double enzymes with NdeI and EcoRI
Target fragment is recycled after cutting, it is spare.Double digestion is carried out to expression vector pET28a (+) using NdeI and EcoRI simultaneously, and will
The opt-atcAB gene obtained after double digestion is connected into pET28a (+) carrier, converts Escherichia coli TOP10, building expression
Carrier pET-atcAB.It is spare that plasmid is extracted after digestion and sequencing confirm that expression vector establishment is errorless.
(2) preparation of BL21 (DE3) competent cell
1) the coli strain BL21 (DE3) on picking LB plate, overnight incubation.
2) bacterium solution of overnight incubation is transferred to according to the inoculum concentration of 1% (V/V) equipped with 50mL LB liquid medium
It is cultivated in the triangular flask of 300mL, OD600Stop culture when to 0.4 or so, set 20min on ice, 4 DEG C, 4000g be centrifuged 10min.It abandons
The CaCl of ice-cold 100mM is added in supernatant2Solution suspension stands 30min on ice.Centrifugal concentrating obtains BL21 (DE3) impression
State cell is put in -70 DEG C of preservations.
(3) conversion of pET-atcAB expression vector
1) that the expression vector pET-atcAB of 50ng is transferred to BL21 (DE3) competence prepared by 100 μ L steps (2) is thin
In born of the same parents, mixes, set 30min on ice, 42 DEG C of heat shock 90s stand 2min on ice.
2) LB liquid medium of 900 μ L, 37 DEG C, 100 turns/min culture 1h is added.
3) it is coated with kalamycin resistance plate, overnight incubation extracts plasmid and carries out digestion verification after picking single colonie culture,
Obtain engineered strain BL21 (DE3)+atcAB, bacterial strain inducible expression recombination fusion protein AtcAB.
(4) expression and purification of recombinant protein
The single bacterium colony of picking engineered strain BL (DE3)+atcAB and inoculation enters 100mL kalamycin resistance LB culture medium
In, in 37 DEG C of overnight incubations.After taking out bacterium solution, it is inoculated in 100mL kalamycin resistance LB culture medium by 1:100 (V/V),
In 37 DEG C of cultures to OD600When=0.6,1mol/L IPTG to final concentration of 1mmol/L is added, bacterium culture, induction are shaken in 37 DEG C
Fusion protein AtcAB expression.10min collection thallus is centrifuged under 8000r/min after inducing 4h.By this thallus 20mL phosphoric acid
Salt buffer (8g/L NaCL, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, pH=7.4) and washing 3 times
And with 10mL sample-loading buffer (20mM Na3PO4, 0.5M NaCl；10mM imidazoles, pH7.4) be resuspended after carry out ultrasonication, grasp
Make condition are as follows: 50HZ, 200W, ultrasonic 3S, interval 5S work 100 times.After the completion of ultrasound, 12000g centrifugation 15min is collected respectively
Electrophoresis detection is carried out after precipitating and supernatant.It was found that recombination fusion protein AtcAB is present in thallus in a manner of solubility expression, swash
Light thin layer scanning analysis shows that, target protein accounts for 25% of bacterial protein or so.
His Trap affinity is used after the ultrasonication supernatant of above-mentioned acquisition is filtered with 0.45 μm of filter membrane
Columns (GE healthcare Products), method to specifications carry out the purifying of recombination fusion protein.Specific side
Method is as follows:
1) it is filled distilled water with 5mL syringe, unscrews the plug of column, connected column with syringe with the connector of offer,
Column is washed with 1mL/min flow velocity.
2) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity.
3) by fusion protein (filtered ultrasonication supernatant) loading, 1mL/min flow velocity.
4) 10mL sample-loading buffer is used, column is washed with 1mL/min flow velocity.
5) 20mL washing buffer (20mM Na is used3PO4, 0.5M NaCl, 40mM imidazoles, pH7.4), it is flowed with 1mL/min
Speed washes column.
6) 10mL elution buffer (20mM Na is used3PO4, 0.5M NaCl, 300mM imidazoles, pH7.4), it is flowed with 1mL/min
Speed elution, is in charge of collection, and every pipe 1mL, 12%SDS-PAGE detection merge the sample containing destination protein in elution fraction, freezes
It is dry to save backup.After purification through this affinity chromatography, SDS-PAGE shows its purity 90% or more to recombinant protein.
Embodiment 2 recombinates the preparation of one carbamyl of nitrogen-L-cysteine hydrolase
1, material
Strain: e. coli bl21 (DE3) is purchased from Promega company.
Plasmid: plasmid pET28a (+) is purchased from Wuhan Miao Ling Biotechnology Co., Ltd.
LB liquid medium are as follows: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L.
Kalamycin resistance plate: the LB solid medium containing 30mg/L kanamycins, 1.5% agar powder.
Kalamycin resistance LB culture medium: the LB liquid medium containing 30mg/L kanamycins.
2, method
(1) building of atcC expression vector；
It will be from one carbamyl of nitrogen of pseudomonad Pseudomonas sp.BS pnca gene group-L-cysteine hydrolysis
(it is 10Kb's that its sequence information is present in one section of size that one section of GenBanK accession number is AB176845 to enzyme gene (atcC)
In DNA sequence dna) carry out codon optimization, the gene order after optimization as shown in SEQ ID NO.5, this section of gene order coding
Protein is then named as AtcC, and amino acid sequence is as shown in SEQ ID NO.4.5 ' the ends and 3 ' ends point of gene after optimization
Not Yin Ru chemical synthesis complete genome sequence as shown in SEQ ID NO.6 after restriction enzyme site NdeI and EcoRI, by this sequence designations
For opt-atcC.The synthesis of opt-atcC complete genome sequence transfers to Jin Sirui Biotechnology Co., Ltd to complete, and when delivery is artificial
The genetic fragment opt-atcC of synthesis is connected on carrier pUC57.By the carrier pUC57 containing opt-atcC segment with NdeI and
EcoRI recycles target fragment after carrying out double digestion, spare.Expression vector pET28a (+) is carried out using NdeI and EcoRI simultaneously
The opt-atcC gene obtained after double digestion is connected into pET28a (+) carrier, and converts Escherichia coli TOP10 by double digestion,
Construction of expression vector pET-atcC.It is spare that plasmid is extracted after digestion and sequencing confirm that expression vector establishment is errorless.
(2) preparation of competent cell
1) the coli strain BL21 (DE3) in picking LB plate, overnight incubation.
2) bacterium solution of overnight incubation is transferred to according to the inoculum concentration of 1% (V/V) equipped with 50mL LB liquid medium
It is cultivated in the triangular flask of 300mL, OD600Stop culture when to 0.4 or so, set 20min on ice, 4 DEG C, 4000g be centrifuged 10min.It abandons
The CaCl of ice-cold 100mM is added in supernatant2Solution suspension stands 30min on ice.Centrifugal concentrating obtains BL21 (DE3) impression
State cell is put in -70 DEG C of preservations.
(3) conversion of pET-atcC expression vector
1) the expression vector pET-atcC of 50ng is transferred to BL21 (DE3) competent cell prepared by 100 μ L steps (2)
In, it mixes, sets 30min on ice, 42 DEG C of heat shock 90s stand 2min on ice.
2) LB liquid medium of 900 μ L, 37 DEG C, 100 turns/min culture 1h is added.
3) it is coated with kalamycin resistance plate, overnight incubation extracts plasmid and carries out digestion verification after picking single colonie culture,
Obtain engineered strain BL21 (DE3)+atcC, bacterial strain inducible expression recombinant protein A tcC.
(4) expression and purification of recombinant protein
The single bacterium colony of picking engineered strain BL (DE3)+atcC and inoculation enters 100mL kalamycin resistance LB culture medium
In, in 37 DEG C of overnight incubations.After taking out bacterium solution, it is inoculated in 100mL kalamycin resistance LB culture medium by 1:100 (V/V),
In 37 DEG C of cultures to OD600When=0.6,1mol/L IPTG to final concentration of 1mmol/L is added, bacterium culture, induction are shaken in 37 DEG C
Recombinant protein expression.10min collection thallus is centrifuged under 8000r/min after inducing 4h.By this thallus 20mL phosphate-buffered
Liquid (8g/L NaCL, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, pH=7.4) and it washs 3 times and uses 10mL
Sample-loading buffer (20mM Na3PO4, 0.5M NaCl；10mM imidazoles, pH7.4) be resuspended after carry out ultrasonication, operating condition are as follows:
50HZ, 200W, ultrasonic 3S, interval 5S work 100 times.After the completion of ultrasound, 12000g centrifugation 15min collects respectively precipitate and on
Electrophoresis detection is carried out after clear.It was found that recombinant protein A tcC is present in thallus in a manner of solubility expression, laser thin layer scanning point
Analysis display, target protein account for 35% of bacterial protein or so.
His Trap affinity is used after the ultrasonication supernatant of above-mentioned acquisition is filtered with 0.45 μm of filter membrane
Columns (GE healthcare Products), method to specifications carry out the purifying of recombinant protein.Specific method is such as
Under:
1) it is filled distilled water with 5mL syringe, unscrews the plug of column, connected column with syringe with the connector of offer,
Column is washed with 1mL/min flow velocity.
2) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity.
3) by recombinant protein (filtered ultrasonication supernatant) loading, 1mL/min flow velocity.
4) 20mL sample-loading buffer is used, column is washed with 1mL/min flow velocity.
5) 20mL washing buffer (20mM Na is used3PO4, 0.5M NaCl, 40mM imidazoles, pH7.4), it is flowed with 1mL/min
Speed washes column.
6) 10mL elution buffer (20mM Na is used3PO4, 0.5M NaCl, 100mM imidazoles, pH7.4), it is flowed with 1mL/min
Speed elution, is in charge of collection, and every pipe 1mL, 12%SDS-PAGE detection merge the sample containing destination protein in elution fraction, freezes
It is dry to save backup.After purification through this affinity chromatography, SDS-PAGE shows its purity 90% or more to recombinant protein.
The building of 3 co-immobilization multi-enzyme system of embodiment
The obtained recombination fusion protein AtcAB 50mg of the Example 1 and obtained recombinant protein A tcC of embodiment 2
25mg be added to simultaneously 12.5mL polyvinyl alcohol containing 80g/L and 15g/L sodium alginate with 0.1N NaOH tune pH to 7.5
In aqueous solution, until the final concentration of 6mg/mL of total protein, after mixing well, it is molten to be added the glutaraldehyde that 750 μ L mass fractions are 10%
Liquid, after mixing well at 4 DEG C cross-linking reaction 3h.Reaction solution is added dropwise to the calcium chloride of 20g/L dropwise with No. 6 needle applicators
In solution, spherula is filtered out.Spherula is placed in the calcium chloride solution of 20g/L, then impregnates hardening 1h.After filtering out spherula
It is crosslinked 3h again with the glutaraldehyde solution that 400mL mass fraction is 0.02% at 4 DEG C, then filters out spherula.It is 7.5 with pH
KH containing 15g/L2PO4Cleaning solution wash 3 times, be placed in 4 DEG C it is spare.
The catalytic conversion reaction of 4 co-immobilization enzyme system of embodiment
The conversion reaction system that volume is 1L is established, conversion reaction system and reaction condition are as follows: containing implementation in system
Spherula 150g obtained in example 3 containing co-immobilization enzyme system, reactant DL-ATC 15g, KH2PO41.5g, sorbierite
100g, conversion reaction system pH are 7.5；Reaction temperature is 30 DEG C, and speed of agitator is 150 revs/min, reaction time 1h.
After the reaction was completed, the content of detection L-cysteine is extracted reaction solution, specific detection method sees reference document:
Gaitonde, M.K.,A spectrophotometric method for the direct determination of
cysteine in the presence of other naturally occurring amino
Acids.Biochemistry Journal, 1967,104,627-633. are according to product L-cysteine with substrate DL-ATC's
Molecule molar ratio calculates conversion ratio.At this point in the reaction, DL-ATC is converted into the conversion ratio of L-cysteine up to 90%.
After the completion of conversion reaction, spherula can be recycled by filtering, the spherula KH of recycling2PO4Concentration is 2g/
L, sorbitol concentration is that the washing buffer that 200g/L, pH are 7.5 is washed 3 times, establishes above-mentioned conversion reaction system with it again,
Carry out conversion reaction.It recycles repeatedly, realizes that co-immobilization enzyme system repeatedly converts DL-ATC synthesis L-cysteine.
With this condition, when immobilization enzyme system uses eight periods, the conversion ratio of reaction system is each still 70% or more
The conversion ratio in period is as follows:
| Service life | Conversion ratio (%) |
| 1 | 90.4 |
| 2 | 88.6 |
| 3 | 86.5 |
| 4 | 84.3 |
| 5 | 82.1 |
| 6 | 77.2 |
| 7 | 74.2 |
| 8 | 70.3 |
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method of immobilised enzymes conversion DL-ATC synthesis L-cysteine, it is characterised in that include the following steps:
(1) preparation of co-immobilization enzyme system
By the fusion protein being made of DL-ATC racemase, rigid connection peptide and L-ATC hydrolase and nitrogen-carbamyl-L- half
Cystine hydrolase is added in the aqueous solution containing polyvinyl alcohol and sodium alginate, and after mixing well, glutaraldehyde solution is added
Carry out cross-linking reaction；Reaction solution is instilled in calcium chloride solution, spherula is filtered out；Spherula be immersed in calcium chloride solution again into
Row cure process；It is crosslinked again with glutaraldehyde solution after filtering out spherula, then filters out spherula, it is spare after washing；
The amino acid sequence of the fusion protein is as shown in SEQ ID NO.1；Nitrogen-carbamyl-L-cysteine the water
The amino acid sequence of enzyme is solved as shown in SEQ ID NO.4；
(2) co-immobilization enzyme system conversion DL-ATC generates L-cysteine
The spherula containing co-immobilization enzyme system that step (1) obtains is added to containing DL-ATC, KH2PO4, sorbierite body
It is reacted to obtain L-cysteine in system.
2. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that: step
Suddenly fusion protein described in (1) passes through the method included the following steps and is prepared:
1) sequence DNA fragmentation as shown in SEQ ID NO.3 is passed throughNdeI、EcoRI is connected to structure on pET-28a (+) carrier
It builds to obtain expression vector pET-atcAB；
2) expression vector pET-atcAB is transferred to e. coli bl21 (DE3) and carries out inducing expression afterwards, carried out after the completion of expression thin
Born of the same parents are broken, use Ni2+The method of affinity chromatography purifies to obtain destination protein.
3. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that: step
Suddenly nitrogen-carbamyl-L-cysteine hydrolase described in (1) passes through the method included the following steps and is prepared:
1) sequence DNA fragmentation as shown in SEQ ID NO.6 is passed throughNdeI、EcoRI is connected to structure on pET-28a (+) carrier
It builds to obtain expression vector pET-atcC；
2) expression vector pET-atcC is transferred to e. coli bl21 (DE3) and carries out inducing expression afterwards, carried out after the completion of expression thin
Born of the same parents are broken, use Ni2+The method of affinity chromatography purifies to obtain destination protein.
4. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that:
Fusion protein as described in step (1) and nitrogen-carbamyl-L-cysteine hydrolase mass ratio are 1:0.2-1:1；
Aqueous solution as described in step (1) containing polyvinyl alcohol and sodium alginate be pH 7.5, polyvinyl alcohol containing 80g/L and
The aqueous solution of 15g/L sodium alginate；
The concentration of calcium chloride solution as described in step (1) is 20g/L；
It is pH 7.5, KH containing 15g/L that washing, which filters out spherular solution, in step (1)2PO4Aqueous solution.
5. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that:
The mass fraction for being crosslinked glutaraldehyde solution used in step (1) for the first time is 10%, and the volume of glutaraldehyde solution is crosslinking
The 3%-8% of reaction system total volume, the time of cross-linking reaction are 3h；
The mass fraction that second is crosslinked glutaraldehyde solution used in step (1) is 0.02%, and the time of cross-linking reaction is 3h.
6. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that: step
Suddenly (1) are as follows: take fusion protein and nitrogen-carbamyl-L-cysteine hydrolase is that the ratio of amount of 1:0.2-1:1 adds in mass ratio
Enter into the aqueous solution of pH 7.5, polyvinyl alcohol containing 80g/L and 15g/L sodium alginate, until the final concentration of 4-10mg/ of total protein
ML, after mixing well, the glutaraldehyde solution that mass fraction is 10%, which is added, makes volume fraction of the solution in cross-linking reaction system
Reach 3%-8%, after mixing well at 4 DEG C cross-linking reaction 3h；Reaction solution is added dropwise in the calcium chloride solution of 20g/L, is filtered out
Spherula；Spherula is placed in the calcium chloride solution of 20g/L, then impregnates hardening 1h；Matter is used at 4 DEG C after filtering out spherula
The glutaraldehyde solution that amount score is 0.02% is crosslinked 3h again, then filters out spherula；The KH containing 15g/L for being 7.5 with pH2PO4It is water-soluble
Liquid wash 3 times, be placed in 4 DEG C it is spare.
7. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that: step
Suddenly the reaction system of (2) are as follows: the bead bulk concentration containing co-immobilization enzyme system is 50-200g/L, and DL-ATC concentration is 5-20g/
L, KH2PO4Concentration is 0.5-2g/L, sorbitol concentration 10-200g/L, pH 6-8.
8. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that: step
Suddenly the condition of reaction described in (2) are as follows: reaction temperature is 28-37 DEG C, and speed of agitator is 100-200 revs/min, the reaction time
For 2-6h.
9. the method for immobilised enzymes conversion DL-ATC synthesis L-cysteine according to claim 1, it is characterised in that: step
Suddenly the spherula containing co-immobilization enzyme system is recovered by filtration in (2) after the reaction was completed, then uses KH2PO4Concentration is 2g/L, sorb
Determining alcohol is to be recycled and reused for step (2) after the washing buffer that 200g/L, pH are 7.5 washs spherula.
10. a kind of for converting the co-immobilization enzyme system of DL-ATC synthesis L-cysteine, it is characterised in that: pass through claim
Step (1) in 1 obtains.
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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 |
| CN102417900A (en) * | 2011-11-17 | 2012-04-18 | 天津启仁医药科技有限公司 | ATC racemase and coding gene thereof, and application of recombinant expression protein thereof |
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| CN102417900A (en) * | 2011-11-17 | 2012-04-18 | 天津启仁医药科技有限公司 | ATC racemase and coding gene thereof, and application of recombinant expression protein thereof |
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| 假单胞菌F-12转化合成半胱氨酸的发酵优化及基因工程菌构建;赵婧楠;《中国优秀硕士学位论文全文数据库工程科技I辑》;20150515;B018-61 |
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