CN104480100A - Method for preparing L-tertiary leucine by immobilized coupled bi-enzyme - Google Patents
Method for preparing L-tertiary leucine by immobilized coupled bi-enzyme Download PDFInfo
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Abstract
The invention discloses a method for preparing L-tertiary leucine by immobilized coupled bi-enzyme. The method comprises the following steps: (1) preparing a cellulose carrier; (2) preparing immobilized coupled bi-enzyme; (3) reacting the immobilized coupled bi-enzyme with a substrate and a coenzyme in an NH4Cl-NH3 buffer system which has pH of 6.0-11.0 to prepare L-tertiary leucine. According to the method, leucine dehydrogenase and hydrogenlyase which are coupled on the cellulose carrier in an immobilized manner are constructed by utilizing bioengineered strains to produce L-tertiary leucine. The method is high in product conversion rate, mild in reaction condition and simple to operate; and the expensive coenzyme is renewable, and the enzymes can be recycled, so that the enzyme utilization efficiency can be improved. The method is suitable for industrial production.
Description
Technical field
The invention belongs to S-Leucine preparing technical field, be specifically related to a kind of immobilization and be coupled the method that two enzyme prepares S-Leucine.
Background technology
S-Leucine is a kind of Non-natural chiral amino acid, because its hydrophobicity tertiary butyl has larger sterically hindered, molecular conformation is controlled than being easier in organic synthesis, be widely used in chemical and medicine industry industry, particularly S-Leucine is as a kind of chirality pharmaceutical intermediate compound, for the synthesis of biostats, anticancer, antiviral etc.Therefore S-Leucine has very high commercial application value, and anti-hiv drug A Zhatawei (Atazanavir) 2009 annual sales amount that such as Shi Guibao company utilizes S-Leucine to synthesize as pharmaceutical intermediate is 1,400,000,000 dollars.
S-Leucine obtains by chemical synthesis or biological synthesis process, the many yields of chemical synthesis step are low, strict to equipment requirements, and complex process, easy contaminate environment, and biotransformation method to have the gentle yield of reaction conditions high, low for equipment requirements, technique is simple, and environmental pollution is little, chiral selectivity advantages of higher, biotransformation method has been widely used in the manufacture of S-Leucine.The method that biological catalysis prepares S-Leucine can be divided into two large classes: utilize enzyme to split DL Terleu (as utilized lytic enzyme, racemase) and direct biocatalysis is synthesized (as with leucine dehydrogenase).But the theoretical yield of the former S-Leucine is lower than 50%, although the latter's theoretical yield height needs to utilize a large amount of expensive coenzyme NAD H.Krix etc. utilize hydrogenlyase to be coupled leucine dehydrogenase, achieve the regeneration of expensive coenzyme, but this method enzyme can not reuse, and coenzyme input amount is up to 2mM, and economy is poor.In Chinese patent CN102978251.A, the input amount of enzyme used accounts for 4% of substrate, and enzyme not recoverable, the consumption of enzyme is too large, and utilising efficiency is not high, economical not.
Summary of the invention
The object of the invention is to overcome prior art defect, a kind of method providing immobilization to be coupled two enzyme to prepare S-Leucine.
Concrete technical scheme of the present invention is as follows:
Immobilization is coupled the method that two enzyme prepares S-Leucine, comprises the steps:
(1) cellulose carrier is prepared;
(2) prepare immobilization and be coupled two enzyme: specifically comprise:
1) build the E.coli engineering bacteria of leucine dehydrogenase gene of docking module marks, the leucine dehydrogenase gene order of above-mentioned docking module marks as shown in SEQ ID 1,
2) build the E.coli engineering bacteria of formate dehydrogenase gene of docking module marks, the formate dehydrogenase gene sequence of above-mentioned docking module marks as shown in SEQ ID 2,
3) build the E.coli engineering bacteria of scaffolding protein gene, above-mentioned scaffolding protein gene order as shown in SEQ ID 3,
4) successively by cultivating, IPTG abduction delivering and ultrasonic broken born of the same parents, be separated respectively and obtain step 1), 2) and 3) leucine dehydrogenase of the docking module marks expressed in the E.coli engineering bacteria that builds, the hydrogenlyase docking module marks and scaffolding protein;
5) 10 ~ 40 DEG C of absorption 10 ~ 100min are incorporated in by mixed to the hydrogenlyase of the leucine dehydrogenase of the docking module marks of above-mentioned acquisition, docking module marks, scaffolding protein and cellulose carrier, centrifugally abandon supernatant liquor, the CaCl of precipitation containing 0.5 ~ 50mM
2with NaCl and pH be 6.0 ~ 11.0 HEPES buffer solution, obtain immobilization and be coupled two enzyme;
(3) by the two enzyme-to-substrate of above-mentioned immobilization lotus root connection and the NH of coenzyme in pH=6.0 ~ 11.0
4cl-NH
3react in buffer system, prepare S-Leucine.
In a preferred embodiment of the invention, described step (1) is: get 0.2g ~ 10g Microcrystalline Cellulose, after adding 0.5 ~ 10mL deionized water dissolving, phosphoric acid to the phosphoric acid final concentration slowly adding precooling is 20% ~ 85%, and ice bath 0.5h ~ 5h also stirs, then adds frozen water 20 ~ 200mL, centrifugally abandon supernatant, with frozen water washing, finally adjust pH to neutral, obtain described cellulose carrier.
In a preferred embodiment of the invention, the step 1 of described step (1)) comprising:
A is to contain the plasmid pUC18-ldh of leucine dehydrogenase gene for template, pcr amplification is carried out with primer LDHCt-F1 and LDHCt-R1, obtain the leucine dehydrogenase gene fragment as shown in SEQ ID 4, wherein LDHCt-F1 and LDHCt-R1 is respectively as shown in SEQ ID 5 and SEQ ID 6;
B with plasmid pET20b-ctdoc (see Self-Assembly of Synthetic Metabolons through SyntheticProtein Scaffolds:One-Step Purification, Co-immobilization, and Substrate Channeling.ACSSynth.Biol.2013,2,102-110; Annexation of a High-Activity Enzyme in a SyntheticThree-Enzyme Complex Greatly Decreases the Degree ofSubstrate Channeling.ACS Synth.Biol.2014,3,380-386) be template, be that primer carries out use pcr amplification with LDHCt-F2 and LDHCt-R2, obtain the first docking module gene fragment as shown in SEQ ID 7, wherein LDHCt-F2 and LDHCt-R2 is respectively as shown in SEQ ID 8 and SEQ ID 9;
C, with above-mentioned leucine dehydrogenase gene fragment and the first docking module gene fragment for template, carry out overlap pcr amplification with primer LDHCt-F1 and LDHCt-R2, obtain the leucine dehydrogenase gene docking module marks;
D, the leucine dehydrogenase gene using Nde I and Sal I difference double digestion docking module marks and pET20b plasmid, transformation of E. coli E.coli DH5 α after connecting with T4 DNA ligase, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coliBL21 (DE3) builds the leucine dehydrogenase gene of docking module marks.
In a preferred embodiment of the invention, the step 2 of described step (1)) comprising:
A, with containing the plasmid pUC18-fdh of formate dehydrogenase gene for template, pcr amplification is carried out with primers F DHCc-F1 and FDHCc-R1, obtain the leucine dehydrogenase gene fragment as shown in SEQ ID 10, wherein FDHCc-F1 and FDHCc-R1 is respectively as shown in SEQ ID 11 and SEQ ID 12;
B, with plasmid pET20b-ccdoc (see Self-Assembly of Synthetic Metabolons through SyntheticProtein Scaffolds:One-Step Purification, Co-immobilization, and Substrate Channeling.ACSSynth.Biol.2013,2,102-110; Annexation of a High-Activity Enzyme in a SyntheticThree-Enzyme Complex Greatly Decreases the Degree of Substrate Channeling.ACS Synth.Biol.2014,3,380-386) be template, be that primer carries out use pcr amplification with FDHCc-F2 and FDHCc-R2, obtain the second docking module gene fragment as shown in SEQ ID 13, wherein FDHCc-F2 and FDHCc-R2 is respectively as shown in SEQ ID 14 and SEQ ID 15;
C, with above-mentioned formate dehydrogenase gene fragment and the second docking module gene fragment for template, carry out overlap pcr amplification with primers F DHCc-F1 and FDHCc-R2, obtain the formate dehydrogenase gene docking module marks;
D, the formate dehydrogenase gene using Nde I and Sal I difference double digestion docking module marks and pET20b plasmid, transformation of E. coli E.coli DH5 α after connecting with T4 DNA ligase, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coliBL21 (DE3) builds the formate dehydrogenase gene of docking module marks.
In a preferred embodiment of the invention, the step 3 of described step (1)) comprising:
A, with plasmid pET20b-scaf3 (see Self-Assembly of Synthetic Metabolons through SyntheticProtein Scaffolds:One-Step Purification, Co-immobilization, and Substrate Channeling.ACSSynth.Biol.2013,2,102-110; Annexation of a High-Activity Enzyme in a SyntheticThree-Enzyme Complex Greatly Decreases the Degree of Substrate Channeling.ACS Synth.Biol.2014,3,380386) be template, with primer Scaf2-F and Scaf2-R for carrying out pcr amplification, obtain the scaffolding protein gene fragment as shown in SEQID 3, wherein Scaf2-F and Scaf2-R is respectively as shown in SEQ ID 16 and SEQ ID 17;
B, use the Nde I and Sal I above-mentioned scaffolding protein gene fragment of double digestion and pET20b plasmid respectively, transformation of E. coli E.coli DH5 α after connecting with T4 DNA ligase, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coli BL21 (DE3) builds scaffolding protein gene.
6, a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the step 4 of described step (1)) be: be separated respectively and obtain step 1), 2) and 3) the E.coli engineering bacteria that builds is cultured to OD at 37 DEG C
600reach 0.2 ~ 1.0, add the IPTG that final concentration is 0.01 ~ 5mM, in 18 DEG C ~ 37 DEG C, cultivate 2 ~ 20h, centrifugally obtain cell, then with the CaCl containing 0.5 ~ 50mM
2with the HEPES buffer solution that the pH of NaCl is 6.0 ~ 11.0, after ultrasonic broken born of the same parents, obtain the leucine dehydrogenase of the docking module marks expressed in engineering bacteria, the hydrogenlyase docking module marks and scaffolding protein respectively.
Preferred further, the step 5 of described step (1)) be: by step 4) in the leucine dehydrogenase of docking module marks of preparation, the docking hydrogenlyase of module marks and scaffolding protein be that 1:1 ~ 3:1 ~ 4 mix with volume ratio, and add described cellulose carrier, at 10 ~ 40 DEG C of absorption 10 ~ 100min, centrifugally abandon supernatant liquor, the CaCl of precipitation containing 0.5 ~ 50mM
2with the HEPES buffer solution that the pH of NaCl is 6.0 ~ 11.0, being fixed is coupled two enzyme.
In a preferred embodiment of the invention, the described substrate in described step (3) is trimethylammonium pyruvic acid and ammonium formiate, and described coenzyme is NADH or NAD
+.
Preferred further, described NH
4cl-NH
3in buffer system, the final concentration that described immobilization is coupled two enzyme is 0.01 ~ 5.0g/L, and the final concentration of described trimethylammonium pyruvic acid is 32 ~ 260g/L, and the final concentration of described ammonium formiate is 48 ~ 158g/L, and the final concentration of described coenzyme is 0.01 ~ 0.2mM.
Preferred further, the reaction conditions of described step (3) is as follows: temperature of reaction 15 ~ 50 DEG C, stirs or intermittent stirring.
The invention has the beneficial effects as follows:
1, method of the present invention utilizes bioengineered strain to build immobilization and is coupled leucine dehydrogenase on cellulose carrier and hydrogenlyase to produce S-Leucine, conversion rate of products is high, reaction conditions is gentle, simple to operate, expensive coenzyme is renewable, enzyme recoverable, improves the utilising efficiency of enzyme, is applicable to suitability for industrialized production.
2, the present invention decreases the consumption of enzyme and coenzyme while the high yield ensureing S-Leucine, and efficiency is high and with low cost.
Accompanying drawing explanation
Fig. 1 is reaction process schematic diagram of the present invention.
Fig. 2 is the step 4 of the embodiment of the present invention 1) SDS-PAGE detect figure, wherein 1 is the E.coli engineering bacteria of formate dehydrogenase gene of docking module marks, 2 be docking module marks the E.coli engineering bacterias of leucine dehydrogenase gene, 3 is the E.coli engineering bacteria of scaffolding protein gene.
Fig. 3 is the step 5 of the embodiment of the present invention 1) SDS-PAGE detect figure, wherein M is marker, 1,3 for scaffolding protein adsorb leucine dehydrogenase, 2 are coupled two enzyme for immobilization, 4 is scaffolding protein absorption leucine dehydrogenase, and 5 scaffolding proteins, 6 without the control group of scaffolding protein.
Fig. 4 is the step 5 of the embodiment of the present invention 1) obtained immobilization is coupled the scanning electron microscope (SEM) photograph of two enzyme.
Fig. 5 is that the HPLC-UV of standard substance S-Leucine (13min) and D-Terleu (20min) analyzes collection of illustrative plates
Fig. 6 is that the HPLC-UV of catalysate S-Leucine analyzes collection of illustrative plates
Fig. 7 is the transformation efficiency figure of substrate trimethylammonium pyruvic acid in the embodiment of the present invention 3
Embodiment
Be further detailed below by way of embodiment technical scheme of the present invention and describe.
Embodiment 1
Immobilization is coupled the method that two enzyme prepares S-Leucine, and its reaction principle as shown in Figure 1, specifically comprises the steps:
(1) cellulose carrier is prepared: get 0.2g ~ 10g Microcrystalline Cellulose, after adding 0.5 ~ 10mL deionized water dissolving, phosphoric acid to the phosphoric acid final concentration slowly adding precooling is 20% ~ 85%, ice bath 0.5h ~ 5h also stirs, add frozen water 20 ~ 200mL again, centrifugally abandon supernatant, wash with frozen water, finally adjust pH to neutral, obtain described cellulose carrier.
(2) prepare immobilization and be coupled two enzyme: specifically comprise:
1) build the E.coli engineering bacteria of the leucine dehydrogenase gene of docking module marks, the leucine dehydrogenase gene order of above-mentioned docking module marks is as shown in SEQ ID 1, and this step is specific as follows:
A, with containing the plasmid pUC18-ldh (synthesis of Sheng Gong bio-engineering corporation) of leucine dehydrogenase gene (from Bacillus cereus) for template, pcr amplification is carried out with primer LDHCt-F1 and LDHCt-R1, obtain the leucine dehydrogenase gene fragment as shown in SEQ ID 4, wherein LDHCt-F1 and LDHCt-R1 is respectively as shown in SEQ ID 5 and SEQ ID 6; PCR reaction system is: 22 μ L H
2o, 1 μ L LDHCt-F1,1 μ L LDHCt-R1,1 μ L pUC18-ldh, 25 μ L PrimeSTAR Max Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 57 DEG C of renaturation 10s, and 72 DEG C extend 6s, circulate 35 times, and 72 DEG C extend 10min;
B, with plasmid pET20b-ctdoc (see ACS Synth.Biol.2013,2,102-110; ACS Synth.Biol.2014,3,380-386) be template, be that primer carries out use pcr amplification with LDHCt-F2 and LDHCt-R2, obtain the first docking module gene fragment as shown in SEQ ID 7, wherein LDHCt-F2 and LDHCt-R2 is respectively as shown in SEQ ID 8 and SEQ ID 9; PCR reaction system is: 22 μ L H
2o, 1 μ L LDHCt-F2,1 μ L LDHCt-R2,1 μ LpET20b-ctdoc, 25 μ L PrimeSTAR Max Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 57 DEG C of renaturation 10s, and 72 DEG C extend 6s, circulate 35 times, and 72 DEG C extend 10min;
C, with above-mentioned leucine dehydrogenase gene fragment and the first docking module gene fragment for template, carry out overlap pcr amplification with primer LDHCt-F1 and LDHCt-R2, obtain the leucine dehydrogenase gene docking module marks; PCR reaction system is: 21 μ L H
2o, 1 μ L LDHCt-F1,1 μ L LDHCt-R2,1 μ L ctdoc, 1 μ L ldh, 25 μ L PrimeSTARMax Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 57 DEG C of renaturation 10s, and 72 DEG C extend 8s, circulate 35 times, and 72 DEG C extend 10min;
D, the leucine dehydrogenase gene using Nde I and Sal I difference double digestion docking module marks and pET20b plasmid, transformation of E. coli E.coli DH5 α after connecting with T4 DNA ligase, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coliBL21 (DE3) builds the leucine dehydrogenase gene of docking module marks.
2) build the E.coli engineering bacteria of the formate dehydrogenase gene of docking module marks, the formate dehydrogenase gene sequence of above-mentioned docking module marks is as shown in SEQ ID 2, and this step is specific as follows:
A, with containing the plasmid pUC18-fdh (synthesis of Sheng Gong bio-engineering corporation) of formate dehydrogenase gene (from Candida boidinii) for template, pcr amplification is carried out with primers F DHCc-F1 and FDHCc-R1, obtain the leucine dehydrogenase gene fragment as shown in SEQ ID 10, wherein FDHCc-F1 and FDHCc-R1 is respectively as shown in SEQ ID 11 and SEQ ID12; PCR reaction system is: 22 μ L H
2o, 1 μ L FDHCc-F1,1 μ L FDHCc-R1,1 μ L pUC18-fdh, 25 μ L PrimeSTAR Max Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 51.6 DEG C of renaturation 10s, and 72 DEG C extend 6s, circulate 35 times, and 72 DEG C extend 10min;
B, with plasmid pET20b-ccdoc (see ACS Synth.Biol.2013,2,102-110; ACS Synth.Biol.2014,3,380-386) be template, be that primer carries out use pcr amplification with FDHCc-F2 and FDHCc-R2, obtain the second docking module gene fragment as shown in SEQ ID 13, wherein FDHCc-F2 and FDHCc-R2 is respectively as shown in SEQ ID 14 and SEQ ID 15; PCR reaction system is: 22 μ L H
2o, 1 μ L FDHCc-F2,1 μ L FDHCc-R2,1 μ LpET20b-ccdoc, 25 μ L PrimeSTAR Max Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 51.6 DEG C of renaturation 10s, and 72 DEG C extend 6s, circulate 35 times, and 72 DEG C extend 10min;
C, with above-mentioned formate dehydrogenase gene fragment and the second docking module gene fragment for template, carry out overlap pcr amplification with primers F DHCc-F1 and FDHCc-R2, obtain the formate dehydrogenase gene docking module marks; PCR reaction system is: 21 μ L H
2o, 1 μ L L FDHCc-F1,1 μ L FDHCc-R2,1 μ L ccdoc, 1 μ L fdh, 25 μ LPrimeSTAR Max Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 51.6 DEG C of renaturation 10s, and 72 DEG C extend 8s, circulate 35 times, and 72 DEG C extend 10min;
D, the formate dehydrogenase gene using Nde I and Sal I difference double digestion docking module marks and pET20b plasmid, transformation of E. coli E.coli DH5 α after connecting with T4 DNA ligase, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coliBL21 (DE3) builds the formate dehydrogenase gene of docking module marks.
3) build the E.coli engineering bacteria of scaffolding protein gene, above-mentioned scaffolding protein gene order is as shown in SEQ ID 3, and this step is specific as follows:
A, with plasmid pET20b-scaf3 (see ACS Synth.Biol.2013,2,102-110; ACS Synth.Biol.2014,3,380-386) be template, with primer Scaf2-F and Scaf2-R for carrying out pcr amplification, obtain the scaffolding protein gene fragment as shown in SEQ ID 3, wherein Scaf2-F and Scaf2-R is respectively as shown in SEQ ID 16 and SEQ ID 17; PCR reaction system is: 22 μ L H
2o, 1 μ L Scaf2-F, 1 μ L Scaf2-R, 1 μ L pET20b-scaf3,25 μ LPrimeSTAR Max Premix; PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 10s, 61.7 DEG C of renaturation 10s, and 72 DEG C extend 8s, circulate 35 times, and 72 DEG C extend 10min;
B, use the Nde I and Sal I above-mentioned scaffolding protein gene fragment of double digestion and pET20b plasmid respectively, transformation of E. coli E.coli DH5 α after connecting with T4 DNA ligase, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coli BL21 (DE3) builds scaffolding protein gene.
4) be separated respectively obtain step 1), 2) and 3) the E.coli engineering bacteria that builds is cultured to OD at 37 DEG C
600reach 0.2 ~ 1.0, add the IPTG that final concentration is 0.01 ~ 5mM, in 18 DEG C ~ 37 DEG C, cultivate 2 ~ 20h, centrifugally obtain cell, then with the CaCl containing 0.5 ~ 50mM
2with the HEPES buffer solution that the pH of NaCl is 6.0 ~ 11.0, the leucine dehydrogenase of the docking module marks expressed in engineering bacteria, the hydrogenlyase docking module marks and scaffolding protein is obtained respectively after ultrasonic broken born of the same parents, its SDS-PAGE detected result as shown in Figure 2, wherein distinguishes the leucine dehydrogenase of above-mentioned docking module marks, the docking hydrogenlyase of module marks and the band at scaffolding protein place shown in arrow.
5) by step 4) in the leucine dehydrogenase of docking module marks of preparation, the docking hydrogenlyase of module marks and scaffolding protein be that 1:1 ~ 3:1 ~ 4 mix with volume ratio, and add described cellulose carrier, at 10 ~ 40 DEG C of absorption 10 ~ 100min, centrifugally abandon supernatant liquor, the CaCl of precipitation containing 0.5 ~ 50mM
2with the HEPES buffer solution that the pH of NaCl is 6.0 ~ 11.0, being fixed is coupled two enzyme, and its SDS-PAGE detected result is as shown in swimming lane in Fig. 32, and its scanning electron microscope (SEM) photograph as shown in Figure 4.
(3) by the two enzyme-to-substrate of above-mentioned immobilization lotus root connection and the NH of coenzyme in pH=6.0 ~ 11.0
4cl-NH
3react in buffer system, prepare S-Leucine, wherein said substrate is trimethylammonium pyruvic acid and ammonium formiate, and described coenzyme is NADH or NAD
+, described NH
4cl-NH
3in buffer system, the final concentration that described immobilization is coupled two enzyme is 0.01 ~ 5.0g/L, the final concentration of described trimethylammonium pyruvic acid is 32 ~ 260g/L, the final concentration of described ammonium formiate is 48 ~ 158g/L, the final concentration of described coenzyme is 0.01 ~ 0.2mM, this step reaction condition is as follows: temperature of reaction 15 ~ 50 DEG C, stirs or intermittent stirring.
Embodiment 2
Step (1) is to step (2) with embodiment 1, and step (3) is as follows:
With the ammoniacal liquor of 5%, the trimethylammonium pyruvic acid of preparation 130g/L, the ammonium formiate of 95g/L, the mother liquor pH being mixed with 2 times is about 9; With the NADH of deionized water preparation 4mM; In 1mL reaction system, add 0.5mL mother liquor, the NADH of 0.05mL4mM, to add 0.45mL total protein concentration be that 0.38mg/mL is immobilized is coupled enzyme, 25 DEG C of reactions, and interval concussion.And sample in 0.5,1,2,3,4,5,8 and 24h, then dilute 5 times, boiling water bath 15min, centrifuging and taking supernatant does efficient liquid phase chromatographic analysis.
Analysis condition is: standard substance S-Leucine, D-Terleu and product S-Leucine all use Agilent 1200 high performance liquid chromatography, the analysis of Chirex 3126 chiral chromatographic column, moving phase or wash-out are 2mM copper sulfate mutually, 5% Virahol, flow velocity is 1mL/min, column temperature 35 DEG C, detector is UV-detector (254nm), and appearance time is 13min.
Wherein the HPLC-UV of standard substance S-Leucine and D-Terleu analyzes collection of illustrative plates as shown in Figure 5, and the HPLC-UV of product S-Leucine analyzes collection of illustrative plates as shown in Figure 6,
Front 8h production concentration and reaction times linear (linear regression analysis R
2=0.9978) 24h transformation efficiency is 65%, throw leucine dehydrogenase or hydrogenlyase total mass accounts for 0.09% of substrate total mass, coenzyme NAD H drops into quality and accounts for 0.23% of substrate total mass, and the intensity of the leucine dehydrogenase production S-Leucine of unit mass is production intensity 492g/L/d; E.e. value is greater than 99%.
Embodiment 3
Step (1) and step (2) are with embodiment 1, and step (3) is as follows:
With the ammoniacal liquor of 5%, the trimethylammonium pyruvic acid of preparation 182g/L, the ammonium formiate of 95g/L, the mother liquor pH being mixed with 2 times is about 9; With the NADH of deionized water preparation 4mM; In 1mL reaction system, add 0.5mL mother liquor, the NADH of 0.05mL4mM, to add 0.36mL total protein concentration be that 3mg/mL is immobilized is coupled enzyme, 25 DEG C of reactions, shakes continuously.And sample in 2,5,8,11 and 24h, then dilute 10 times, boiling water bath 15min, centrifuging and taking supernatant does efficient liquid phase chromatographic analysis.
Analysis condition is as embodiment 2, and result shows at front 11h production concentration and reaction times linear (linear regression analysis R
2=0.9979), show under high concentration substrate condition, be coupled two enzyme and can not produce substrate suppression.24h transformation efficiency is 81%, wherein the transformation efficiency of trimethylammonium pyruvic acid as shown in Figure 7, throw leucine dehydrogenase or hydrogenlyase total mass accounts for 0.4% of substrate total mass, coenzyme NAD H drops into quality and accounts for 0.23% of substrate total mass, and the production intensity of the leucine dehydrogenase production S-Leucine of unit mass is 207g/L/d; E.e. value is greater than 99%.
Embodiment 4
Step (1) and (2) are with embodiment 3, and additionally adding final concentration in step (3) is 1mM CaCl
2, result 24h transformation efficiency is 91%, and the production intensity of the leucine dehydrogenase production S-Leucine of unit mass is 253g/L/d; E.e. value is greater than 99%.
Embodiment 5
Step (1) and (2) are with embodiment 3, and additionally adding final concentration in step (3) is 1mM CaCl
2, allow centrifuging and taking supernatant liquor efficient liquid phase chromatographic analysis after its reaction 24h, then add identical concentration of substrate and coenzyme in precipitation, react 24h under similarity condition, centrifuging and taking supernatant liquor efficient liquid phase chromatographic analysis, so repeat to reclaim enzyme 3 times.Result shows that enzyme recycles three times, and substrate conversion efficiency is respectively 91%, 66%, 40%, and relative catalytic efficiency is respectively 100%, 73%, 43%.The utilising efficiency of enzyme is doubled.
Embodiment 6
Step (1) and (2) are with embodiment 3, and difference is that step (3) reaction sampled after three days, and the transformation efficiency recording substrate trimethylammonium pyruvic acid is 99.8%, and product S-Leucine final concentration is 91.8g/L.Two enzymes of the Microcrystalline Cellulose adsorption support protein coupling that result shows by phosphoric acid expansionization have very high substrate conversion efficiency.
Embodiment 7
Step (1) and (2) are with embodiment 3, difference is that step (3) NADH final concentration is sample after 0.02mM reacts four days, the transformation efficiency recording substrate trimethylammonium pyruvic acid is 58.8%, and product S-Leucine final concentration is 53.5g/L.Two enzymes of the Microcrystalline Cellulose adsorption support protein coupling that result shows by phosphoric acid expansionization also have good catalytic effect when NADH concentration is very low.
The above, be only preferred embodiment of the present invention, therefore can not limit scope of the invention process according to this, the equivalence change namely done according to the scope of the claims of the present invention and description with modify, all should still belong in scope that the present invention contains.
Claims (10)
1. immobilization is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: comprise the steps:
(1) cellulose carrier is prepared;
(2) prepare immobilization and be coupled two enzyme: specifically comprise:
1) build the E.coli engineering bacteria of leucine dehydrogenase gene of docking module marks, the leucine dehydrogenase gene order of above-mentioned docking module marks as shown in SEQ ID 1,
2) build the E.coli engineering bacteria of formate dehydrogenase gene of docking module marks, the formate dehydrogenase gene sequence of above-mentioned docking module marks as shown in SEQ ID 2,
3) build the E.coli engineering bacteria of scaffolding protein gene, above-mentioned scaffolding protein gene order as shown in SEQ ID 3,
4) successively by cultivating, IPTG abduction delivering and ultrasonic broken born of the same parents, be separated respectively and obtain step 1), 2) and 3) leucine dehydrogenase of the docking module marks expressed in the E.coli engineering bacteria that builds, the hydrogenlyase docking module marks and scaffolding protein;
5) 10 ~ 40 DEG C of absorption 10 ~ 100min are incorporated in by mixed to the hydrogenlyase of the leucine dehydrogenase of the docking module marks of above-mentioned acquisition, docking module marks, scaffolding protein and cellulose carrier, centrifugally abandon supernatant liquor, the CaCl of precipitation containing 0.5 ~ 50mM
2with NaCl and pH be 6.0 ~ 11.0 HEPES buffer solution, obtain immobilization and be coupled two enzyme;
(3) by the two enzyme-to-substrate of above-mentioned immobilization lotus root connection and the NH of coenzyme in pH=6.0 ~ 11.0
4cl-NH
3react in buffer system, prepare S-Leucine.
2. a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: described step (1) is: get 0.2g ~ 10g Microcrystalline Cellulose, after adding 0.5 ~ 10mL deionized water dissolving, phosphoric acid to the phosphoric acid final concentration slowly adding precooling is 20% ~ 85%, and ice bath 0.5h ~ 5h also stirs, then adds frozen water 20 ~ 200mL, centrifugally abandon supernatant, with frozen water washing, finally adjust pH to neutral, obtain described cellulose carrier.
3. a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the step 1 of described step (1)) comprising:
A, with containing the plasmid pUC18-ldh of leucine dehydrogenase gene for template, pcr amplification is carried out with primer LDHCt-F1 and LDHCt-R1, obtain the leucine dehydrogenase gene fragment as shown in SEQ ID 4, wherein LDHCt-F1 and LDHCt-R1 is respectively as shown in SEQ ID 5 and SEQ ID 6;
B, with plasmid pET20b-ctdoc for template, be that primer carries out use pcr amplification with LDHCt-F2 and LDHCt-R2, obtain the first docking module gene fragment as shown in SEQ ID 7, wherein LDHCt-F2 and LDHCt-R2 is respectively as shown in SEQ ID 8 and SEQ ID 9;
C, with above-mentioned leucine dehydrogenase gene fragment and the first docking module gene fragment for template, carry out overlap pcr amplification with primer LDHCt-F1 and LDHCt-R2, obtain the leucine dehydrogenase gene docking module marks;
D, the leucine dehydrogenase gene using Nde I and Sal I difference double digestion docking module marks and pET20b plasmid, transformation of E. coli E.coli DH5 α after connecting with T4DNA ligase enzyme, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coliBL21 (DE3) builds the leucine dehydrogenase gene of docking module marks.
4. a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the step 2 of described step (1)) comprising:
A, with containing the plasmid pUC18-fdh of formate dehydrogenase gene for template, pcr amplification is carried out with primers F DHCc-F1 and FDHCc-R1, obtain the leucine dehydrogenase gene fragment as shown in SEQ ID 10, wherein FDHCc-F1 and FDHCc-R1 is respectively as shown in SEQ ID 11 and SEQ ID 12;
B, with plasmid pET20b-ccdoc for template, be that primer carries out use pcr amplification with FDHCc-F2 and FDHCc-R2, obtain the second docking module gene fragment as shown in SEQ ID 13, wherein FDHCc-F2 and FDHCc-R2 is respectively as shown in SEQ ID 14 and SEQ ID 15;
C, with above-mentioned formate dehydrogenase gene fragment and the second docking module gene fragment for template, carry out overlap pcr amplification with primers F DHCc-F1 and FDHCc-R2, obtain the formate dehydrogenase gene docking module marks;
D, the formate dehydrogenase gene using Nde I and Sal I difference double digestion docking module marks and pET20b plasmid, transformation of E. coli E.coli DH5 α after connecting with T4DNA ligase enzyme, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coliBL21 (DE3) builds the formate dehydrogenase gene of docking module marks.
5. a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the step 3 of described step (1)) comprising:
A, with plasmid pET20b-scaf3 for template, with primer Scaf2-F and Scaf2-R for carrying out pcr amplification, obtain the scaffolding protein gene fragment as shown in SEQ ID 3, wherein Scaf2-F and Scaf2-R is respectively as shown in SEQ ID 16 and SEQ ID 17;
B, use the Nde I and Sal I above-mentioned scaffolding protein gene fragment of double digestion and pET20b plasmid respectively, transformation of E. coli E.coli DH5 α after connecting with T4DNA ligase enzyme, then extracts the E.coli engineering bacteria that plasmid transformation escherichia coli E.coli BL21 (DE3) builds scaffolding protein gene.
6. a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the step 4 of described step (1)) be: be separated respectively and obtain step 1), 2) and 3) the E.coli engineering bacteria that builds is cultured to OD at 37 DEG C
600reach 0.2 ~ 1.0, add the IPTG that final concentration is 0.01 ~ 5mM, in 18 DEG C ~ 37 DEG C, cultivate 2 ~ 20h, centrifugally obtain cell, then with the CaCl containing 0.5 ~ 50mM
2with the HEPES buffer solution that the pH of NaCl is 6.0 ~ 11.0, after ultrasonic broken born of the same parents, obtain the leucine dehydrogenase of the docking module marks expressed in engineering bacteria, the hydrogenlyase docking module marks and scaffolding protein respectively.
7. a kind of immobilization as claimed in claim 6 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the step 5 of described step (1)) be: by step 4) in the leucine dehydrogenase of docking module marks of preparation, the docking hydrogenlyase of module marks and scaffolding protein be that 1:1 ~ 3:1 ~ 4 mix with volume ratio, and add described cellulose carrier, at 10 ~ 40 DEG C of absorption 10 ~ 100min, centrifugally abandon supernatant liquor, the CaCl of precipitation containing 0.5 ~ 50mM
2with the HEPES buffer solution that the pH of NaCl is 6.0 ~ 11.0, being fixed is coupled two enzyme.
8. a kind of immobilization as claimed in claim 1 is coupled the method that two enzyme prepares S-Leucine, and it is characterized in that: the described substrate in described step (3) is trimethylammonium pyruvic acid and ammonium formiate, described coenzyme is NADH or NAD
+.
9. a kind of immobilization as claimed in claim 8 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: described NH
4cl-NH
3in buffer system, the final concentration that described immobilization is coupled two enzyme is 0.01 ~ 5.0g/L, and the final concentration of described trimethylammonium pyruvic acid is 32 ~ 260g/L, and the final concentration of described ammonium formiate is 48 ~ 158g/L, and the final concentration of described coenzyme is 0.01 ~ 0.2mM.
10. a kind of immobilization as claimed in claim 8 or 9 is coupled the method that two enzyme prepares S-Leucine, it is characterized in that: the reaction conditions of described step (3) is as follows: temperature of reaction 15 ~ 50 DEG C, stirs or intermittent stirring.
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| CN107858384A (en) * | 2017-11-08 | 2018-03-30 | 厦门大学 | A kind of method that optical voidness L Terleus are prepared using inactive inclusion body |
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