WO2017016442A1 - 一种辅酶再生系统及其制备方法 - Google Patents

一种辅酶再生系统及其制备方法 Download PDF

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WO2017016442A1
WO2017016442A1 PCT/CN2016/090981 CN2016090981W WO2017016442A1 WO 2017016442 A1 WO2017016442 A1 WO 2017016442A1 CN 2016090981 W CN2016090981 W CN 2016090981W WO 2017016442 A1 WO2017016442 A1 WO 2017016442A1
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fdh
ldh
regeneration system
coenzyme regeneration
expression
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PCT/CN2016/090981
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English (en)
French (fr)
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李航
徐军
阙利民
江岳恒
蔡彤�
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雅本化学股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes

Definitions

  • the invention relates to the field of biotechnology, in particular to a coenzyme regeneration system and a preparation method thereof.
  • Oxidoreductases selectively catalyze the inclusion of carbonyls, aldehydes and ketones.
  • the oxidoreductase is used as a catalyst to prepare a chiral compound, and a certain amount of coenzyme is consumed while synthesizing the product.
  • Li Shuting et al. used L-tert-leucine to prepare L-tert-leucine.
  • the synthesis used leucine and formate dehydrogenase coupling system. Formate dehydrogenase can be coenzyme I regeneration, and the conversion rate of the product can be Up to 82%.
  • Coenzyme is a consumable compound. It is not reusable like oxidoreductase. As a donor of hydrogen in the reaction, the coenzyme exists in an oxidized state after the reaction. These coenzymes are often more expensive than the products produced, have poor stability, and are difficult to reuse, resulting in high reaction costs in industrial production, thereby limiting the use of oxidoreductases. In industrial production, it is impossible to add a large amount of coenzyme, so the construction of a highly efficient coenzyme regeneration system and repeated use of coenzymes are issues that must be solved at present.
  • the object of the present invention is to provide a coenzyme regeneration system in view of the deficiencies of the prior art.
  • a coenzyme regeneration system including formate dehydrogenase (FDH), bright Leucine dehydrogenase (LDH) and ammonium formate.
  • FDH formate dehydrogenase
  • LDH bright Leucine dehydrogenase
  • ammonium formate ammonium formate
  • the coenzyme regeneration system further comprises NAD + and / or NADH.
  • the coenzyme regeneration system further comprises pyridoxal phosphate.
  • the formate dehydrogenase and the leucine dehydrogenase in the coenzyme regeneration system are derived from a genetically engineered fermentation broth, and the genetically engineered bacteria simultaneously express formate dehydrogenase and leucine. Dehydrogenase.
  • the genetically engineered bacteria is an E. coli BL21 (DE3) strain that simultaneously expresses formate dehydrogenase and leucine dehydrogenase.
  • Another aspect of the present invention provides a method of preparing a coenzyme regeneration system, the method comprising the steps of:
  • a coenzyme regeneration system is prepared in a buffer, the coenzyme regeneration system comprising: the FDH and LDH-containing enzyme solution, ammonium formate, NAD + , and pyridoxal phosphate.
  • the step (1) includes the following steps:
  • the FDH gene was ligated into the pET28a vector digested with the endonuclease Nde I and Xho I to construct the plasmid pFDH-pET28a recombinantly expressing FDH;
  • the LDH gene was ligated into the pET21a vector digested with the endonuclease Xho I and Nde I to construct the recombinant expression plasmid pLDH-pET21a;
  • the plasmid pFDH-pET28a and plasmid pLDH-pET21a were co-transferred into E. coli BL21 (DE3).
  • E. coli BL21-FDH/LDH co-expressing FDH and LDH was obtained.
  • the method further comprises the steps of:
  • the co-expressing strain E.coli BL21-FDH/LDH was cultured in LB liquid medium containing Amp and Kan, and IPTG was added to a final concentration of 0.01-0.05 mmol/L, and the expression was induced at 16-27 °C for 12-20 h. After the cells are broken, the supernatant is centrifuged to obtain the FDH/LDH co-expressing enzyme solution.
  • the total DNA of Candida is used as a template, and the upstream and downstream primers are respectively subjected to PCR amplification to obtain the FDH gene, wherein:
  • the upstream primer was 5'-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3', wherein the nick was the NdeI cleavage site; the downstream primer was 5'-AAACTCGAGTGCGACCTTTTTGTCATTAC-3', wherein the nick was the XhoI cleavage site.
  • IPTG is added to a final concentration of 0.02 mmol/L, the induction temperature is 22 ° C, and the induction time is 16 h.
  • the fermentation broth is centrifuged to remove the supernatant, and the supernatant is obtained to obtain the FDH/ LDH co-expresses the enzyme solution.
  • the invention has the advantages that the coenzyme regeneration system of the invention has high regeneration efficiency of the coenzyme, simple and convenient preparation method, greatly reduces the cost of the coenzyme reaction, and is suitable for large-scale industrial application.
  • Figure 1 shows a schematic diagram of the construction of plasmid pFDH-pET28a
  • Figure 2 shows the restriction endonuclease map of pTD-pET28a, PCR amplification product of lane 1TD, and restriction enzyme digestion of lane 2pTD-pET28a recombinant plasmid;
  • Figure 3 shows the restriction endonuclease map of pFDH-pET28a, PCR amplification product of lane 1FDH, and restriction enzyme digestion of lane 2pFDH-pET28a;
  • Figure 4 shows the restriction endonuclease map of pLDH-pET21a, and the LDH fragment obtained by lane digestion of the p1HLD-pET21a recombinant plasmid.
  • LB liquid medium/g ⁇ L -1 peptone 10, yeast extract 5, sodium chloride 10, pH 7.0.
  • LB solid medium/g ⁇ L -1 peptone 10, yeast extract 5, sodium chloride 10, agar strip 20, pH 7.0.
  • Solution I 0.2973 g of glucose, 0.091 g of Tris, 0.112 g of EDTA to a volume of 30 mL.
  • Solution II fresh configuration: 0.2 mol/L NaOH, 1% SDS.
  • Solution III 44.163 g of potassium acetate, 17.25 mL of glacial acetic acid to a volume of 150 mL.
  • Example 1 PCR amplification of the gene of interest
  • Primers were designed based on the formate dehydrogenase (FDH) threonine deaminase (TD) gene sequence (Genbank number: XM_001525495 and AAB18593) and its context. After screening, the optimal primer for the formate dehydrogenase (FDH) was 5'-AAA CATATG AAAATCGTTCTCGTTTTGTACTCC-3' (NdeI restriction site at the scribe); the downstream primer was 5'-AAA CTCGAG TGCGACCTTTTTGTCATTAC-3' (The XhoI restriction site is located at the scribe). The upstream and downstream primers were designed according to the gene sequence of threonine deaminase (AAB18593).
  • FDH formate dehydrogenase
  • TD threonine deaminase
  • the best upstream primer was 5'-AAA AAGCTT GTTAGCAGCCGGATCTCAG-3' (HindIII cleavage site at the scribe); downstream The primer was 5'-AAA CATATG GTATTAAAACAAATTCTTC-3' (NdeI restriction site at the scribe).
  • the FDH and TD gene fragments were amplified by the above and lower primers, respectively.
  • the PrimeSTAR HS DNA polymerase PCR reaction system is shown in Table 5.
  • PCR reaction procedure a total of 30 cycles, the FDH gene fragment size is about 1300 bp, and the TD gene fragment size is about 1500 bp.
  • Step2 65 ° C, 10 s annealing
  • the plasmid construction process is shown in Figure 1 (using FDH as an example), using Candida total DNA as a template, using the specific endonuclease NdeI and XhoI double-digested gene fragments or genes linked to the T vector.
  • the fragment was simultaneously digested with the vector plasmid, purified and recovered, and ligated at 16 ° C for 5 h.
  • the ligated product was transformed into competent cell E. coli DH5 ⁇ , and a single colony transformed on LB solid plate was picked and connected to 5 mL LB.
  • the liquid medium (addition of the corresponding antibiotic according to the resistance of the plasmid) was shake-cultured overnight at 37 ° C in a 220 r/min shaker.
  • the plasmid was extracted, and the plasmid was picked for double enzyme digestion verification. After the verification was correct, the constructed plasmid pFDH-pET28a was used for DNA sequence determination.
  • the total DNA of Escherichia coli was used as a template, and PCR amplification was performed on the above and lower primers respectively. As shown in Fig. 2, a specific DNA band was obtained, and the relative molecular size was about 1.5 kb, which was consistent with the theoretical value.
  • the constructed plasmid pTD-pET28a was digested with HindIII and NdeI, and the products were electrophoretically digested. As shown in Fig. 2, the bands were as expected, and bands appeared at 5.4 kb and 1.5 kb, respectively. It is expected that the expression plasmid is constructed correctly.
  • the constructed plasmid pTD-pET28a was subjected to DNA sequence alignment, and after sequencing, no mutation was observed. Transformation into E. coli BL21 (DE3) gave the TD expression strain E. coli BL21-TD for subsequent protein expression.
  • PCR amplification was performed on the above and lower primers respectively.
  • a specific DNA band was obtained with a relative molecular size of about 1.3 kb, which was consistent with the theoretical value.
  • the constructed plasmid pFDH-pET28a was digested with NdeI and XhoI, and the products were electrophoretically digested.
  • the bands were as expected, and bands appeared at 5.4 kb and 1.3 kb, respectively, and the expression plasmid was constructed correctly. After sequencing, there was no mutation.
  • the constructed plasmid can be transformed into E. coli BL21 for subsequent protein expression.
  • LDH leucine dehydrogenase
  • E. coli BL21 DE3
  • LB Amp and Kan 100 ⁇ g/ml each
  • solid plates in LB Amp and Kan 100 ⁇ g/ml each
  • the single colony that grew up was a strain containing both formate dehydrogenase and leucine dehydrogenase genes, and the genetically engineered strain was named E. coli BL21-FDH/LDH. This strain can simultaneously express formate dehydrogenase and leucine dehydrogenase.
  • a single colony of the engineered bacteria carrying the recombinant plasmid was inoculated into 5 mL of LB medium, and cultured overnight at 37 ° C, 220 r / min.
  • the culture was carried out at 37 ° C in 100 mL of LB liquid medium (addition of the corresponding antibiotic according to the resistance of the plasmid) with an inoculation amount of 5 ⁇ .
  • the OD 600 value reached 0.6-0.8, it was induced at the optimal concentration of IPTG and the induction temperature for 16 h.
  • the constructed recombinant plasmid was transformed into competent cells of Escherichia coli E. coli BL21 by heat shock method, thereby obtaining an engineered bacteria which can overexpress the exogenous enzyme.
  • the engineered bacteria carrying the recombinant plasmid were picked and inoculated into 3 mL of LB medium, and cultured overnight at 37 °C.
  • 1 mL of the bacterial solution was prepared as a negative control, and another 1 mL was reserved for inoculation.
  • the remaining 1 mL of the bacterial solution was induced by adding IPTG (1 mmol/L) and expressed at 37 ° C for 3 hours.
  • the negative control and the induced bacterial liquid were subjected to SDS-PAGE electrophoresis.
  • Expression of the recombinase protein in E. coli exists in two forms: soluble proteins and inclusion bodies.
  • the inclusion body protein does not have enzymatic activity, and only soluble protein has enzyme catalytic activity. Due to the high expression level of the selected vector pET28a, even if a large amount of protein of interest is present in the form of inclusion bodies, there are quite a few protein of interest present as soluble proteins.
  • Factors affecting the soluble expression of the protein are: induction time, concentration of the IPTG inducer, and induction temperature. To optimize the conditions of protein expression, the soluble expression of E. coli BL21-FDH and E. coli BL21-GDH was investigated under different induction durations, different IPTG induced final concentrations and different induction temperatures.
  • the longer the induction time the higher the expression level of the target protein, until it reaches a maximum at a certain time point, and the expression level of the protein tends to stop as the induction time prolongs.
  • the heteroprotein is also expressed more and affects the activity of the target protein.
  • the soluble expression levels of proteins at 4h, 8h, 12h, 16h and 20h were investigated.
  • a single colony of the engineered bacteria carrying the recombinant plasmid was inoculated into 5 mL of LB medium, and cultured overnight at 37 ° C, 220 r / min. Each of them was cultured at 37 ° C in 30 mL of LB liquid medium (containing Kan 100 ⁇ g/ml) in an inoculation amount of 5 ⁇ .
  • the OD 600 value reached 0.6-0.8, the expression was induced at the optimal concentration of IPTG induction and induction temperature (4h, 8h, 12h, 16h and 20h), the cells were collected by centrifugation, and the cells were treated for SDS-PAGE electrophoresis. The soluble expression of the protein is detected to determine the optimal induction time.
  • the strain E.coli BL21-TD was induced at 16 °C, and the soluble expression of the protein was observed at 4h, 8h, 12h, 16h and 20h, respectively.
  • the results showed that the target protein had a small amount of protein before the induction agent was added. Local expression. As the induction time increases, the expression level of the target protein increases, and the protein expression level tends to a maximum at 16h, and then the expression level of the protein tends to stop. Therefore, it was determined that the optimal induction time after the addition of IPTG was 16 h.
  • the strain E.coliB L21-FDH was induced at 22 °C, and the soluble expression of the protein was observed at 4h, 8h, 12h, 16h and 20h, respectively.
  • the results showed that the target protein had a small amount of protein before the induction agent was added. Local expression. As the induction time increases, the expression level of the target protein increases, and the protein expression level tends to a maximum at 16h, and then the expression level of the protein tends to stop. Therefore, it was determined that the optimal induction time after the addition of IPTG was 16 h.
  • the strain E.coli BL21-LDH was induced at 22 °C, and the soluble expression of the protein was observed at 4h, 8h, 12h, 16h and 20h, respectively.
  • the results showed that the target protein had only a small amount of locality before the inducing agent was added. expression.
  • the optimal induction time after the addition of IPTG was determined to be 16 h.
  • the level of protein expression can be altered by altering the concentration of IPTG, and expression can be adjusted from very low levels to very high levels until the level at which the pET vector is fully induced. Therefore, the soluble expression levels of protein at the final concentration of protein induction were 0.01, 0.02, 0.05, 0.1, 0.2 mmol/L and 0.5 mmol/L, respectively.
  • the final concentrations of 6 different IPTGs were determined (0.01, 0.02, 0.05, 0.1, 0.2 mmol/L and 0.5 mmol/L), and the expression was induced at the optimal induction temperature for 16 h.
  • the cells were collected, and the cells were treated, and the soluble expression of the protein was detected by SDS-PAGE electrophoresis to determine the optimal concentration of IPTG.
  • TD 50kDa
  • the expression level of the protein was approximately the same, so the final concentration of IPTG was selected to be 0.02 mmol/L for protein expression.
  • FDH FDH (47kDa) showed significant bands after IPTG was added, The expected size matches.
  • the target protein was expressed in a large amount.
  • the target protein was also expressed in a small amount. The results showed that protein expression could be induced at a low concentration of IPTG, so the final concentration of IPTG was selected to be 0.02 mmol/L for protein expression.
  • LDH (42kDa) showed a significant expression band after IPTG addition, which was consistent with the expected size.
  • the target protein was expressed in a large amount.
  • the target protein was also expressed in a small amount. Therefore, the final concentration of IPTG was selected to be 0.01 mmol/L for induced expression of the protein.
  • the culture temperature of the bacteria has an important influence on the expression level of the target protein.
  • the soluble expression levels of proteins at 16 °C, 22 °C, 28 °C, 32 °C and 37 °C were investigated.
  • the cells were centrifuged at 5 different temperatures (16 ° C, 22 ° C, 28 ° C, 32 ° C and 37 ° C) for 16 h, and the cells were collected by centrifugation.
  • the soluble expression of the protein was detected by SDS-PAGE electrophoresis, and the optimal induction temperature was determined.
  • the soluble expression of protease was high at 16 °C, 22 °C, 28 °C, 32 °C and 37 °C, there was no significant difference, but at 22 °C
  • the soluble expression of the protein is higher than that of other temperatures, considering the economical and high soluble protein expression, so the induction temperature is selected at 16-28 ° C (preferably 22 ° C) for protein expression.
  • the induction temperature was selected at an induction temperature of 22 °C.
  • the induction temperature was selected at an induction temperature of 22 °C.
  • the optimal protein expression conditions of recombinant enzyme E.coli BL21-FDH were as follows: the optimal induction temperature was 22 °C, the optimal induction concentration was 0.02 mmol/L, and the optimal induction time was 16 h.
  • the relative concentrations of the recombinant enzyme E. coli BL21-LDH were: 0.01, 0.02, 0.2 mmol/L and 0.5 mmol/L, and the induction of the recombinant enzyme E. coli BL21-FDH.
  • the relatively high concentrations were: 0.02 mmol/L, 0.2 mmol/L and 0.5 mmol/L, so the optimal induction concentration of co-expressing strain E. coli BL21-FDH/LDH was determined to be 0.02 mmol/L.
  • the optimal protein expression conditions of co-expressing strain E.coli BL21-FDH/LDH were determined as follows: the optimal induction temperature was 22 °C, the optimal induction concentration was 0.02 mmol/L, and the optimal induction time was 16 h.
  • the enzyme labeler is used herein to determine the enzymatic activity of formate dehydrogenase.
  • An enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 ⁇ mol of product per minute under the assay conditions.
  • the activity of FDH was measured by using ammonium formate as the substrate.
  • the total reaction volume was 1 mL.
  • the reaction mixture was incubated at 30 ° C, and a certain amount of FDH crude enzyme solution was added to start timing.
  • the oxidation reaction is as follows:
  • the calculation method of enzyme activity is:
  • ⁇ A represents the change in absorbance at 340 nm in 1 min
  • Vt represents the volume (ml) of the reaction solution
  • Vs represents the volume (ml) of the sample
  • d represents the cuvette light path (0.625 cm)
  • 6220 represents the molar absorption coefficient ( L/mol -1 ⁇ cm -1 )
  • c represents the concentration of the protein (mg/ml).
  • the enzyme activity in the supernatant of the FDH cell suspension was measured using HCOONH 4 as a substrate. The results showed that the activity of the FDH cell suspension supernatant to catalyze the production of NADH was about 10 U/ml. Based on the total protein concentration measured by the Bradford method, the specific enzyme activity of the crude enzyme solution of FDH was calculated to be 13.51 U/mg.
  • the threonine as the substrate was used to measure the activity of threonine deaminase.
  • the total reaction volume was 1 mL, and 0.1 g of threonine, 0.03 mg of PLP, a certain amount of crude enzyme solution and Na 2 HPO 4 (0.1 mol) were added.
  • the enzymatic activity of leucine dehydrogenase in the coupling reaction was determined by using carbonyl butyric acid as the substrate.
  • the total reaction volume was 1 mL, and 400 ⁇ mol of carbonylbutyric acid, 0.2 mg of NAD + and 65 mg of HCOONH 4 were added respectively to a certain amount of crude enzyme.
  • Solution and Na 2 HPO 4 (0.1 mol/L, pH 8.0) buffer.
  • the reaction mixture was reacted at 30 ° C, 150 r / min shaker for 2 h, an equal volume of acetonitrile was added, and the mixture was diluted by 5 times for derivatization. After centrifugation, the amount of product formed was analyzed by HPLC to calculate the enzyme activity.
  • An enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 ⁇ mol of product per minute under the assay conditions.
  • the enzyme activity of TD was measured by using threonine as a substrate. The results showed that TD conversion of threonine to carbonylbutyric acid The capacity of the enzyme was 6.67 U/ml, and the specific enzyme activity of the crude enzyme solution of TD was calculated to be 4.156 U/mg.
  • the activity of LDH in the coupling reaction of E.coli BL21-FDH/LDH was measured by using carbonylbutyric acid as the substrate. The results showed that the ability of LDH to convert carbonylbutyric acid to L-aminobutyric acid was 7.38 U/ml, and LDH was calculated.
  • the specific enzyme activity of the crude enzyme solution was 6.43 U/mg.
  • the concentration of the protein was determined by the Bradford method, and the absorbance of the known bovine serum albumin solution was measured to obtain a standard curve of absorbance and protein concentration.
  • the sample to be tested was uniformly mixed with the Bradford solution, and allowed to stand at room temperature for 5 min. Using a 96-well plate, each mixture was placed in parallel in 3 wells, and then the absorbance of the solution at a wavelength of 595 nm was measured, and the average of the absorbances of the three wells was taken as the absorbance of the sample to be tested. The concentration of the protein was calculated from the standard curve.
  • the protein content was calculated to be 0.0642 mg/ml, and the protein content calculated according to the diluted concentration of the protein was 1.605 mg/ml.
  • the L-aminobutyric acid content was determined by a 2,4-dinitrofluorobenzene derivatization method, and the amount of product produced was measured by HPLC.
  • E. coli BL21-TD or E. coli BL21-FDH/LDH was fermented in 100 mL of LB (containing 100 ⁇ g/ml of Amp and Kan), and a crude enzyme solution was prepared for the synthesis of L-aminobutyric acid.
  • the optimum conditions are: temperature 30 ° C; pH 8.0; initial concentration of NAD + is 0.25 mg / ml;
  • the maximum substrate feeding amount is 70g/L; in the reaction system of 10mL, the amount of threonine deaminase added is 8mL, and the formic acid dehydrogenase is co-expressed.
  • the amount of leucine dehydrogenase added was 10 mL of cells; the final reaction time was 16 h. Conversion to prepare L-aminobutyric acid. With a feed rate of 70 g/L, the yield can reach 95%.
  • the optimal expression temperature of recombinant protease E.coli BL21-FDH/LDH was 22 °C
  • the optimal induction concentration was 0.02 mmol/L
  • the optimal induction time was 16 h.
  • the enzyme activity of LDH in this coupling reaction was determined to be 7.38 U/ml.
  • the optimum conditions are: temperature 30 ° C; pH 8.0; initial concentration of NAD + is 0.25 mg / ml; substrate feed amount is 70 g / L
  • the ratio of enzyme addition is: in a 10 mL reaction system, the amount of threonine deaminase added is 8 mL of cells, and the amount of co-expressed formate dehydrogenase and leucine dehydrogenase is 10 mL of cells;
  • the final reaction time is 16 h and the yield is up to 95%.
  • the coenzyme regeneration system of the invention has high regeneration efficiency of coenzyme, and the preparation method is simple and convenient, and greatly Reduces the cost of coenzyme reactions and is suitable for large-scale industrial applications.

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Abstract

提供了一种辅酶再生系统，其包括：甲酸脱氢酶（FDH）、亮氨酸脱氢酶（LDH）和甲酸铵。还提供了该辅酶再生系统的制备方法，包括步骤（1）甲酸脱氢酶和亮氨酸脱氢酶的异源表达；（2）配制辅酶再生系统。

Description

一种辅酶再生系统及其制备方法 技术领域
本发明涉及生物技术领域，尤其涉及一种辅酶再生系统及其制备方法。
背景技术
相较于传统的化学催化，生物催化因其酶催化反应的高度的化学选择性，区域选择性和立体选择性等优势而备受关注和研究。其中由于氧化还原酶的高效性和特异性等，它们在生物合成中占据相当重要的地位。氧化还原酶能选择性的催化含羰基，醛类及酮类化合物。氧化还原酶作为催化剂制备手性化合物，合成产物的同时会消耗掉一定量的辅酶。例如:黎舒婷等用三甲基丙酮酸制备L-叔亮氨酸，合成中使用亮氨酸酶与甲酸脱氢酶偶联体系，甲酸脱氢酶可以是辅酶Ⅰ再生，产物的转化率可以达到82％。
不像其它的酶类，在生物转化反应中，辅酶与底物的关系只是化学计量的关系。辅酶是一种消耗性的化合物，不像氧化还原酶一样可以重复利用，在反应中作为氢的供体，反应结束后辅酶以氧化态形式存在。而这些辅酶往往比所制备的产品的价格还要昂贵，稳定性比较差，难以重复利用，导致了工业化生产中反应成本的高昂，从而限制了氧化还原酶的使用。在工业化生产中，不可能投加大量的辅酶，所以高效的辅酶再生系统的构建和辅酶的反复使用是当下必须解决的问题。
因此，本领域的技术人员致力于开发一种简单、高效的辅酶再生技术。
发明内容
本发明的目的是针对现有技术的不足，提供一种辅酶再生系统。
本发明的上述目的通过以下技术方案实现：
一种辅酶再生系统，包括甲酸脱氢酶(formate dehydrogenase，FDH)、亮 氨酸脱氢酶(leucine dehydrogenase，LDH)和甲酸铵。
根据本发明的一个实施例，所述的辅酶再生系统还包括NAD+和/或NADH。
根据本发明的一个实施例，所述的辅酶再生系统还包括磷酸吡哆醛。
根据本发明的一个实施例，所述的辅酶再生系统中的甲酸脱氢酶和亮氨酸脱氢酶来自基因工程菌发酵液，并且所述基因工程菌同时表达甲酸脱氢酶和亮氨酸脱氢酶。
根据本发明的一个实施例，所述基因工程菌为同时表达甲酸脱氢酶和亮氨酸脱氢酶的E.coli BL21(DE3)菌株。
本发明的另一方面是提供一种辅酶再生系统的制备方法，该方法包括以下步骤：
(1)甲酸脱氢酶和亮氨酸脱氢酶的异源表达
构建同时表达FDH和LDH的基因工程菌，经发酵制得含FDH和LDH的酶液；
(2)配制辅酶再生系统
在缓冲液中配制辅酶再生系统，所述辅酶再生系统包括：所述含FDH和LDH的酶液、甲酸铵、NAD+、和磷酸吡哆醛。
根据本发明的一个实施例，其中步骤(1)中包括以下步骤：
(a)构建FDH载体
将FDH基因连入使用核酸内切酶Nde Ⅰ和Xho Ⅰ双酶切的pET28a载体，构建得到重组表达FDH的质粒pFDH-pET28a；
(b)构建LDH载体
将LDH基因连入使用核酸内切酶Xho Ⅰ和Nde Ⅰ双酶切的pET21a载体，构建得到重组表达质粒pLDH-pET21a；
(c)构建同时表达FDH和LDH的基因工程菌
将质粒pFDH-pET28a和质粒pLDH-pET21a共转入E.coli BL21(DE3)， 得到共表达FDH与LDH的菌株E.coli BL21-FDH/LDH。
根据本发明的一个实施例，该方法还包括以下步骤：
(d)发酵所述菌株E.coli BL21-FDH/LDH，制备所述含FDH和LDH的酶液
将共表达菌株E.coli BL21-FDH/LDH在含Amp和Kan的LB液体培养基中培养，加入IPTG至终浓度为0.01～0.05mmol/L，16～27℃诱导表达12～20h，发酵液经菌体破壁后离心取上清即得所述FDH/LDH共表达酶液。
根据本发明的一个实施例，其中步骤(a)中以假丝酵母总DNA为模板，分别以上下游引物进行PCR扩增，从而获得所述FDH基因，其中：
上游引物为5’-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3’，其中，划线处为NdeI酶切位点；下游引物为5’-AAACTCGAGTGCGACCTTTTTGTCATTAC-3’，其中，划线处为XhoI酶切位点。
根据本发明的一个实施例，其中加入IPTG至终浓度为0.02mmol/L，诱导温度为22℃，诱导表达时间为16h，发酵液经菌体破壁后离心取上清即得所述FDH/LDH共表达酶液。
本发明的优点是：本发明的辅酶再生系统，辅酶再生效率高，制备方法简单方便，极大地降低了辅酶反应的成本，适用于大规模的工业化应用。
附图说明
图1显示了构建质粒pFDH-pET28a的示意图；
图2显示了pTD-pET28a的酶切验证图，泳道1TD的PCR扩增产物，泳道2pTD-pET28a重组质粒酶切验证；
图3显示了pFDH-pET28a的酶切验证图，泳道1FDH的PCR扩增产物，泳道2pFDH-pET28a重组质粒酶切验证；
图4显示了pLDH-pET21a的酶切验证图，泳道1pLDH-pET21a重组质粒酶切验证，泳道2酶切获得的LDH片段。
具体实施方式
以下实施例是对本发明的详细说明。应当理解，以下所述仅为本发明的优选实施例，在不脱离本发明的精神和范围的情况下，对本发明进行修改和等同替换均应涵盖在本发明的范围之内。
在下文中列出本发明所用的实验原料。
表1实验质粒
Figure PCTCN2016090981-appb-000001
表2实验菌株
Figure PCTCN2016090981-appb-000002
表3实验中所用到的PCR引物
Figure PCTCN2016090981-appb-000003
表4实验试剂
Figure PCTCN2016090981-appb-000004
培养基
LB液体培养基/g·L-1：蛋白胨10，酵母提取物5，氯化钠10，pH 7.0。
LB固体培养基/g·L-1：蛋白胨10，酵母提取物5，氯化钠10，琼脂条20，pH 7.0。
主要溶液和缓冲液
(1)质粒抽提相关溶液：
Solution Ⅰ：0.2973g葡萄糖，0.091g Tris，0.112g EDTA定容至30mL。
Solution Ⅱ(新鲜配置)：0.2mol/L NaOH，1％SDS。
SolutionⅢ：44.163g乙酸钾，17.25mL冰醋酸定容至150mL。
(2)50×TAE(电泳缓冲液)(/L)
242g Tris 57.1mL醋酸，100mL EDTA(0.05mol/L)。
实施例1：PCR扩增目的基因
(1)PCR扩增引物
根据甲酸脱氢酶(FDH)苏氨酸脱氨酶(TD)基因序列(Genbank number：XM_001525495和AAB18593)及其前后序列设计引物。经过筛选验证，最佳的甲酸脱氢酶(FDH)上游引物为5’-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3’(划线处为NdeI酶切位点)；下游引物为5’-AAACTCGAGTGCGACCTTTTTGTCATTAC-3’(划 线处为XhoI酶切位点)。根据苏氨酸脱氨酶的基因序列(AAB18593)设计上下游引物，经过筛选验证，最佳的上游引物为5’-AAAAAGCTTGTTAGCAGCCGGATCTCAG-3’(划线处为HindIII酶切位点)；下游引物为5’-AAACATATGGTATTAAAACAAATTCTTC-3’(划线处为NdeI酶切位点)。
以假丝酵母和大肠杆菌总DNA为模板，分别以上下游引物扩增得到FDH和TD基因片段。
(2)PCR反应体系
PrimeSTAR HS DNA聚合酶PCR反应体系如表5所示。
表5PCR反应体系
Figure PCTCN2016090981-appb-000005
(3)PCR反应程序
PCR反应程序：共30个循环，FDH基因片段大小约为1300bp，TD基因片段大小约为1500bp。
Step1:98℃，10s  变性
Step2:65℃，10s  退火
Step3:72℃，1.5min延伸
实施例2：重组质粒的构建
质粒构建流程如图1所示(以FDH构建为例)，以假丝酵母总DNA为模板，使用特异性内切酶NdeI和XhoI双酶切扩增到的基因片段或与T载体相连的基因片段，同时双酶切载体质粒，纯化回收后，在16℃条件下连接5h，转化连接产物于感受态细胞E.coli DH5α中，挑取转化于LB固体平板上的单菌落，接到5mL LB液体培养基中(根据质粒的抗性添加相应的抗生素)，在37℃条件下，220r/min摇床中振荡培养过夜。抽提质粒，挑取质粒进行双酶切验证，验证正确后，将构建好的质粒pFDH-pET28a进行DNA序列的测定。
(1)苏氨酸脱氨酶表达质粒的构建
以大肠杆菌总DNA为模板，分别以上下游引物进行PCR扩增，图2所示，得到一条特异性的DNA条带，相对分子大小约为1.5kb，与理论值相符。将构建好的质粒pTD-pET28a使用HindIII和NdeI双酶切验证，电泳酶切产物，图2所示，条带与预期相符，分别在5.4kb和1.5kb处出现条带，所有条带大小符合预期，表达质粒构建正确。将构建好的质粒pTD-pET28a进行DNA序列比对，经测序后比对无突变。转化入E.coli BL21(DE3)，得到TD表达菌株E.coli BL21-TD，进行后续的蛋白表达。
(2)甲酸脱氢酶表达质粒的构建
以假丝酵母总DNA为模板，分别以上下游引物进行PCR扩增，图3所示，得到一条特异性的DNA条带，相对分子大小约为1.3kb，与理论值相符。将构建好的质粒pFDH-pET28a使用NdeI和XhoI双酶切验证，电泳酶切产物，图3所示，条带与预期相符，分别在5.4kb和1.3kb处出现条带，表达质粒构建正确。经测序后比对无突变。构建好的质粒可以转化入E.coli BL21，进行后续的蛋白表达。
(3)亮氨酸脱氢酶表达质粒的构建
根据亮氨酸脱氢酶(LDH)的基因序列(Genbank number：WP_016086354)，委托英潍捷基生物公司合成基因序列片段。利用特异性内 切酶NdeI和XhoI双酶切连接有LDH片段的T载体，图4所示，得到3条DNA条带，相对分子大小分别为2.5kb，1.1kb，0.25kb，其中2.5kb为T载体的线性条带，1.1kb为LDH的基因片段，大小与理论值相符。将构建好的质粒pLDH-pET21a使用NdeI和XhoI双酶切验证，图4所示，电泳酶切产物，条带与预期相符，分别在5.4kb和1.1kb处出现条带，表达质粒构建正确。将构建好的质粒pLDH-pET28a进行DNA序列比对，经测序后比对无突变。转化入E.coli BL21(DE3)，得到LDH表达菌株E.coli BL21-LDH，进行后续的蛋白表达。
实施例3：共表达重组菌株的构建
将pFDH-pET28a和pLDH-pET21a共转化入E.coli BL21(DE3)，并涂布于LB(Amp和Kan各100μg/ml)固体平板上，在LB(Amp和Kan各100μg/ml)固体平板上长出的单菌落，就是同时含有甲酸脱氢酶和亮氨酸脱氢酶基因的菌株，将得到基因工程菌株命名为E.coli BL21-FDH/LDH。该菌株可以同时表达甲酸脱氢酶与亮氨酸脱氢酶。
实施例4：粗酶液的制备
挑取带有重组质粒的工程菌的单菌落接种到5mL LB培养液中，在37℃，220r/min条件下过夜培养。分别以5‰的接种量接入100mL LB液体培养基(根据质粒的抗性添加相应的抗生素)中37℃培养。当OD600值达到0.6～0.8，在IPTG最优诱导终浓度和诱导温度下诱导16h。培养后的发酵液在3700r/min冷冻离心15min后弃去上清液，将菌泥用适量的预冷的Na2HPO4(0.1mol/L，pH＝8.0)缓冲液洗涤后再次离心。最后用20mL Na2HPO4(0.1mol/L，pH＝8.0)缓冲液重悬后超声破碎，超声功率360W，10s+20s，20min，破碎过程菌液始终保持在冰水浴中。细胞破碎液经3700r/min，4℃离心15min，所得上清液即为粗酶液，可进行酶活测定，SDS-PAGE电泳检测及转化反应。
实施例5：重组酶蛋白的试表达
将构建的重组质粒通过热激法转入到大肠杆菌E.coilBL21的感受态细胞中，从而得到可以过量表达外源酶的工程菌。挑取带有重组质粒的工程菌接种到3mL LB培养液中，在37℃条件下过夜培养。
取1mL菌液备作阴性对照，另取1mL留作接种用，剩下的1mL菌液中加入IPTG(1mmol/L)诱导，在37℃表达3h。将阴性对照和诱导后的菌液进行SDS-PAGE电泳检测。
挑选E.coli BL21-FDH，E.coli BL21-TD和E.coli BL21-FDH/LDH的单菌落，在37℃条件下过夜培养，在诱导浓度为1mmol/L，诱导温度为37℃的条件下诱导表达3h，将诱导后的菌液进行SDS-PAGE电泳检测，结果显示，分别在47kDa，500kDa，42kDa处有明显表达条带。诱导前，目的蛋白仅有微量的本底表达，诱导后，可溶性蛋白的表达量明显增多，说明目标蛋白在重组菌中能够很好的表达。
实施例6：重组酶蛋白表达条件的优化
重组酶蛋白在大肠杆菌中的表达以两种形式存在：可溶性蛋白和包涵体。而包涵体蛋白不具有酶催化活性，只有可溶性蛋白具有酶催化活性。由于选择的载体pET28a具有表达量高的特点，即使大量目的蛋白聚集以包涵体形式存在，但是也有相当多的目的蛋白以可溶性蛋白存在。影响蛋白的可溶性表达的因素有：诱导时间，IPTG诱导剂的浓度以及诱导温度。为优化蛋白表达的条件，分别考察了不同诱导时长，不同IPTG诱导终浓度和不同的诱导温度下，E.coli BL21-FDH和E.coli BL21-GDH的可溶性表达。
(1)诱导时长的优化
一般情况下，诱导时间越长，目的蛋白的表达量越高，直到某一时间点达到最大，以后随着诱导时间的延长，蛋白的表达量趋于停止。但是诱导时间太长的话，杂蛋白也表达较多，影响目的蛋白的活性。本文考察了诱导时长分别为4h，8h，12h，16h和20h时蛋白的可溶性表达量。
挑取带有重组质粒的工程菌的单菌落接种到5mL LB培养液中，在37℃，220r/min条件下过夜培养。分别以5‰的接种量接入30mL LB液体培养基(含Kan 100μg/ml)中37℃培养。当OD600值达到0.6～0.8时，在最优IPTG诱导终浓度和诱导温度下，诱导表达(4h，8h，12h，16h和20h)，离心收集菌体，处理菌体后进行SDS-PAGE电泳检测蛋白的可溶性表达，确定最优的诱导时长。
在16℃条件下诱导菌株E.coli BL21-TD，对诱导时长分别为4h，8h，12h，16h和20h时蛋白的可溶性表达量进行考察，结果表明，加入诱导剂前，目的蛋白有少量的本地表达。随着诱导时长的增加，目的蛋白的表达量随之增加，直到16h蛋白表达量趋于一个最大值，随后蛋白的表达量趋于停止。因此确定加入IPTG后的最佳诱导时间长为16h。
在22℃条件下诱导菌株E.coliB L21-FDH，对诱导时长分别为4h，8h，12h，16h和20h时蛋白的可溶性表达量进行考察，结果表明，加入诱导剂前，目的蛋白有少量的本地表达。随着诱导时长的增加，目的蛋白的表达量随之增加，直到16h蛋白表达量趋于一个最大值，随后蛋白的表达量趋于停止。因此确定加入IPTG后的最优诱导时长为16h。
在22℃条件下诱导菌株E.coli BL21-LDH，对诱导时长分别为4h，8h，12h，16h和20h时蛋白的可溶性表达量进行考察，结果表明，加入诱导剂前，目的蛋白只有少量本地表达。随着诱导时长的增加，目的蛋白的表达量随之增加，直到16h蛋白表达量趋于一个最大值，随后蛋白的表达量趋于停止。因此确定加入IPTG后的最佳诱导时间为16h。
(2)IPTG诱导浓度的优化
在DE3宿主菌中，通过改变IPTG的浓度可以改变蛋白表达的水平，表达可以从很低的水平调节至很高的水平，直至pET载体完全诱导的水平。故本文考察了蛋白诱导终浓度分别为0.01﹑0.02﹑0.05﹑0.1﹑0.2mmol/L和0.5mmol/L时蛋白的可溶性表达量。
当OD600值达到0.6～0.8，考察6种不同IPTG的诱导终浓度(0.01﹑0.02﹑0.05﹑0.1﹑0.2mmol/L和0.5mmol/L)，在最优诱导温度下诱导表达16h后，离心收集菌体，处理菌体后进行SDS-PAGE电泳检测蛋白的可溶性表达，确定最优的IPTG诱导浓度。
与未加诱导剂相比，IPTG加入后，TD(50kDa)有明显表达条带，与预期大小相符。但是在诱导浓度为0.02-0.5mmol/L的范围内，蛋白的表达量大致相同，故选择IPTG的终浓度为0.02mmol/L进行蛋白的诱导表达。
与未加诱导剂相比，IPTG加入后，FDH(47kDa)有明显表达条带，与 预期大小相符。当IPTG终浓度达到0.02mmol/L，0.2mmol/L和0.5mmol/L时，目的蛋白均有大量的表达。当IPTG终浓度达到0.01，0.05mmol/L和0.1mmol/L时，目的蛋白也有少许量的表达。结果表明：在IPTG低浓度下就能诱导蛋白表达，故选择IPTG的终浓度为0.02mmol/L进行蛋白的诱导表达。
与未加诱导剂相比，IPTG加入后，LDH(42kDa)有明显表达条带，与预期大小相符。当IPTG终浓度达到0.01，0.02，0.2mmol/L和0.5mmol/L时，目的蛋白均有大量的表达。当IPTG终浓度达到0.05mmol/L和0.1mmol/L时，目的蛋白也有少许量的表达。故选择IPTG的终浓度为0.01mmol/L进行蛋白的诱导表达。
(3)诱导温度的优化
细菌的培养温度对目的蛋白的表达量有重要影响。本文考察了蛋白的诱导温度分别为16℃，22℃，28℃，32℃和37℃时蛋白的可溶性表达量。
当OD600值达到0.6～0.8，在最优IPTG诱导终浓度，考察在5个不同温度(16℃，22℃，28℃，32℃和37℃)下诱导16h后，离心收集菌体，处理菌体后进行SDS-PAGE电泳检测蛋白的可溶性表达，确定最优的诱导温度。
在E.coli BL21-TD的表达中，在16℃，22℃，28℃，32℃和37℃这5个温度下，蛋白酶的可溶性表达均较高，没有显著性的差异，但是在22℃的诱导温度下，蛋白的可溶性表达较之于其他温度表达较高，综合考虑经济和高的可溶性蛋白表达，故选择诱导温度为16-28℃(优选为22℃)进行蛋白的诱导表达。
在E.coli BL21-FDH的表达中，在16℃，22℃，28℃，32℃和37℃这5个温度下，当诱导温度为16℃，22℃，28℃这3个诱导温度下，蛋白酶的可溶性表达均较高，没有显著性的差异，但是，28℃条件下可溶性蛋白量相对于包涵体较之于16℃和22℃少。当诱导温度升高为32℃及以上温度时，可溶性表达量显著降低，而大部分目的蛋白都以包涵体的形式存在于沉淀中，这可能是由于虽然高温有利于细菌的生长繁殖，蛋白表达量也高，但蛋白表达过多过快就形成了包涵体。综合考虑经济和高的可溶性蛋白表达，故选择诱导温度为22℃进行蛋白的诱导表达。
在E.coli BL21-LDH的表达中，在16℃，22℃，28℃，32℃和37℃这5个温度下，当诱导温度为16℃，22℃，28℃这3个诱导温度下，蛋白酶的可溶性表达均较高，但是，28℃条件下可溶性蛋白量相对于包涵体较之于16℃和22℃少。当诱导温度升高为32℃及以上温度时，可溶性表达量显著降低，而大部分目的蛋白都以包涵体的形式存在于沉淀中，这可能是由于虽然高温有利于细菌的生长繁殖，蛋白表达量也高，但蛋白表达过多过快就形成了包涵体。综合考虑经济和高的可溶性蛋白表达，故选择诱导温度为22℃进行蛋白的诱导表达。
(4)共表达重组菌株的蛋白条件优化
重组酶E.coli BL21-FDH的最优蛋白表达条件为：最优诱导温度为22℃，最优诱导浓度为0.02mmol/L，最优诱导时长为16h。
结合蛋白SDS-PAGE图可知，由于重组酶E.coli BL21-LDH的诱导相对较高浓度为：0.01，0.02，0.2mmol/L和0.5mmol/L，而重组酶E.coli BL21-FDH的诱导相对较高浓度为：0.02mmol/L，0.2mmol/L和0.5mmol/L，故确定共表达菌株E.coli BL21-FDH/LDH的最优诱导浓度为0.02mmol/L。故最终确定共表达菌株E.coli BL21-FDH/LDH的最佳蛋白表达条件为：最佳诱导温度为22℃，最佳诱导浓度为0.02mmol/L，最佳诱导时长为16h。
在最优条件下诱导，进行SDS-PAGE电泳，诱导FDH和LDH的表达，分别在47kDa和42kDa位置有明显的表达条带，与预期大小相符。诱导后的重组菌E.coil菌体经过超声破壁处理后离心，E.coil共表达的FDH和LDH大部分存在于上清液中，为可溶性的蛋白。
实施例7：酶活的测定
(1)甲酸脱氢酶酶活的测定
本文中使用酶标仪来测定甲酸脱氢酶的酶活。一个酶活力单位(U)定义为在测定条件下，每分钟产生1μmol产物所需的酶量。
以甲酸铵为底物测FDH的酶活，总反应体积为1mL，反应液组成为：Na2HPO4(0.1mol/L，pH＝8.0)缓冲液中加入1.67mmol/L NAD+，167mmol/L HCOONH4。反应混合物于30℃保温，加入一定量的FDH粗酶液后开始计时， 每隔60s检测NADH在340nm吸光度的增加量，根据它在340nm的摩尔消光系数ε＝6220L/(mol-1·cm-1)来计算甲酸脱氢酶的酶活。氧化反应如下：
Figure PCTCN2016090981-appb-000006
酶活力的计算方法为：
Figure PCTCN2016090981-appb-000007
Figure PCTCN2016090981-appb-000008
式中△A表示1min内340nm处吸光度的变化，Vt表示反应液的体积(ml)，Vs表示样品的体积(ml)，d表示比色池光径(0.625cm)，6220表示摩尔吸收系数(L/mol-1·cm-1)，c表示蛋白质的浓度(mg/ml)。
以HCOONH4为底物，测定FDH破菌液上清中的酶活，结果表明，FDH破菌液上清催化产生NADH的活性约为10U/ml。根据Bradford法测出的蛋白总浓度，计算出FDH的粗酶液的比酶活为13.51U/mg。
(2)苏氨酸脱氨酶和亮氨酸脱氢酶酶活的测定
以苏氨酸为底物测苏氨酸脱氨酶的酶活，总反应体积为1mL，分别加入0.1g苏氨酸，0.03mg PLP，一定量的粗酶液和Na2HPO4(0.1mol/L pH＝8.0)缓冲液，在30℃，150r/min摇床中反应2h，加入等体积的乙腈，再用水稀释500倍，12000r/min，离心5min后用HPLC分析产物生成量，计算酶活。
分别以羰基丁酸为底物测偶联反应中亮氨酸脱氢酶的酶活，总反应体积为1mL，分别加入400μmol羰基丁酸，0.2mg NAD+，65mg HCOONH4，一定量的粗酶液和Na2HPO4(0.1mol/L，pH＝8.0)缓冲液。反应混合物在30℃，150r/min的摇床中反应2h，加入等体积的乙腈，再稀释5倍进行衍生，离心后用HPLC分析产物生成量，计算酶活。
一个酶活力单位(U)定义为在测定条件下，每分钟产生1μmol产物所需的酶量。
以苏氨酸为底物测TD的酶活，结果表明，TD转化苏氨酸生成羰基丁酸 的能力为6.67U/ml，计算出TD的粗酶液的比酶活为4.156U/mg。以羰基丁酸为底物测E.coli BL21-FDH/LDH偶联反应中LDH的酶活，结果表明，LDH转化羰基丁酸生成L-氨基丁酸的能力为7.38U/ml，计算出LDH的粗酶液的比酶活为6.43U/mg。
实施例8：粗酶液蛋白质含量的测定
采用Bradford法测定蛋白质的浓度，测定已知的牛血清蛋白溶液的吸光度，得到吸光度与蛋白质浓度的标准曲线。将待测样品与Bradford溶液混合均匀，在室温静置反应5min。使用96孔板，每个混合液平行放置3个孔中，随后测定595nm波长下溶液的吸光度，以这3个孔的吸光度的平均值作为待测样品的吸光度。根据标准曲线计算蛋白质的浓度。
采用Bradford法测定的吸光度与蛋白质浓度的标准曲线。
(1)苏氨酸脱氨酶粗酶液的蛋白质含量的确定
测得的样品的吸光度的平均值为A＝0.643。根据标准曲线y＝4.8618x+0.3310(R2＝0.9906)，计算得出蛋白质含量为0.0642mg/ml，根据蛋白的稀释浓度算得的蛋白质含量为1.605mg/ml。
(2)甲酸脱氢酶粗酶液的蛋白质含量的确定
测得的样品的吸光度的平均值为A＝0.475，计算得出蛋白质含量为0.0296mg/ml，根据蛋白的稀释浓度算得的蛋白质含量为0.74mg/ml。
(3)亮氨酸脱氢酶与甲酸脱氢酶共表达的蛋白质含量的测定
测得的样品的吸光度的平均值为A＝0.554，计算得出蛋白质含量为0.0459mg/ml，根据蛋白的稀释浓度算得的蛋白质蛋白质含量为1.148mg/ml。实施例9：产物含量的测定
使用2，4-二硝基氟苯衍生法测定L-氨基丁酸含量，产物生成量通过HPLC来检测。生成L-氨基丁酸的色谱条件为：色谱柱为C18柱(Φ4.6mm×250mm，5μm)；柱温为30℃；流动相A：Na2HPO4(0.02mol/L，pH＝7.2)缓冲液，B：乙腈，等度洗脱，V(A)∶V(B)＝70∶30；流速为1.0ml/min；360nm紫外检测。使用标准品，制作标准曲线定量测定。生成羰基丁 酸的液相条件为：柱温为25℃；流动相A：NaH2PO4(0.02mol/L，pH＝3.0)缓冲液，B：乙腈，等度洗脱，V(A)∶V(B)＝90∶10；流速为1.0mL/min；205nm紫外检测。
实施例10：共表达体系催化制备L-氨基丁酸
使用TD，FDH和LDH共表达酶液在同一体系，催化底物苏氨酸生成L-氨基丁酸。在100mL LB(含Amp和Kan各100μg/ml)中发酵培养E.coli BL21-TD或E.coli BL21-FDH/LDH，并制备粗酶液，用于L-氨基丁酸的合成。
在250mL三角瓶中依次加入2.1g苏氨酸，3.0g HCOONH4，19.2mL Na2HPO4(0.1mol/L)缓冲液，再加入4.8mL TD粗酶液，6mL FDH与LDH共表达粗酶液，10mg NAD+和3.215mg磷酸吡哆醛(PLP)。置于恒温箱中，磁力搅拌，自动电位滴定仪检测pH值，用0.2mol/L NaOH溶液滴定。反应至20h，用HPLC测定产物生成量。
由L-苏氨酸制备L-氨基丁酸的转化反应中，经过大量实验，获得的最适条件为：温度为30℃；pH为8.0；NAD+的初始浓度为0.25mg/ml；采用分批加料的方式，最大底物投料量为70g/L；酶加量配比为，在10mL的反应体系中，苏氨酸脱氨酶的加入量为8mL菌体，共表达甲酸脱氢酶与亮氨酸脱氢酶的加入量为10mL菌体；最终反应时长为16h。转化制备L-氨基丁酸。投料量在70g/L的情况下，产率可达95％。
总结
对重组蛋白酶E.coli BL21-FDH/LDH进行表达条件的优化，得到最佳诱导温度为22℃，最佳诱导浓度为0.02mmol/L，最佳诱导时长为16h。在最佳表达条件下，测得此偶联反应中LDH的酶活为7.38U/ml。
由L-苏氨酸制备L-氨基丁酸的转化反应中，最适条件为：温度为30℃；pH为8.0；NAD+的初始浓度为0.25mg/ml；底物投料量为70g/L；酶加量配比为，在10mL的反应体系中，苏氨酸脱氨酶的加入量为8mL菌体，共表达甲酸脱氢酶与亮氨酸脱氢酶的加入量为10mL菌体；最终反应时长为16h，产率可达95％。
本发明的辅酶再生系统，辅酶再生效率高，制备方法简单方便，极大地 降低了辅酶反应的成本，适用于大规模的工业化应用。
在对本发明的具体实施例进行了详细的介绍的同时，还可以发现与本发明相关的本领域内相似的多种可替代设计和由权利要求限定的实施例。
Figure PCTCN2016090981-appb-000009
Figure PCTCN2016090981-appb-000010

Claims (10)

  1. 一种辅酶再生系统，其特征在于，所述辅酶再生系统包括甲酸脱氢酶(formate dehydrogenase，FDH)、亮氨酸脱氢酶(leucine dehydrogenase，LDH)和甲酸铵。
  2. 根据权利要求1所述的辅酶再生系统，其特征在于，所述的辅酶再生系统还包括NAD+和/或NADH。
  3. 根据权利要求1所述的辅酶再生系统，其特征在于，所述的辅酶再生系统还包括磷酸吡哆醛。
  4. 根据权利要求1所述的辅酶再生系统，其特征在于，所述的辅酶再生系统中的甲酸脱氢酶和亮氨酸脱氢酶来自基因工程菌发酵液，并且所述基因工程菌同时表达甲酸脱氢酶和亮氨酸脱氢酶。
  5. 根据权利要求1所述的辅酶再生系统，其特征在于，所述基因工程菌为同时表达甲酸脱氢酶和亮氨酸脱氢酶的E.coli BL21(DE3)菌株。
  6. 一种权利要求1-5所述的辅酶再生系统的制备方法，其中，所述方法包括以下步骤：
    (1)甲酸脱氢酶和亮氨酸脱氢酶的异源表达
    构建同时表达FDH和LDH的基因工程菌，经发酵制得含FDH和LDH的酶液；
    (2)配制辅酶再生系统
    在缓冲液中配制辅酶再生系统，所述辅酶再生系统包括：所述含FDH和LDH的酶液、甲酸铵、NAD+、和磷酸吡哆醛。
  7. 根据权利要求6所述的方法，其特征在于，所述步骤(1)中包括以下步骤：
    (a)构建FDH载体
    将FDH基因连入使用核酸内切酶Nde Ⅰ和Xho Ⅰ双酶切的pET28a载体，构建得到重组表达FDH的质粒pFDH-pET28a；
    (b)构建LDH载体
    将LDH基因连入使用核酸内切酶Xho Ⅰ和Nde Ⅰ双酶切的pET21a载体，构建得到重组表达质粒pLDH-pET21a；
    (c)构建同时表达FDH和LDH的基因工程菌
    将质粒pFDH-pET28a和质粒pLDH-pET21a共转入E.coli BL21(DE3)，得到共表达FDH与LDH的菌株E.coli BL21-FDH/LDH。
  8. 根据权利要求7所述的方法，其特征在于，所述方法还包括以下步骤：
    (d)发酵所述菌株E.coli BL21-FDH/LDH，制备所述含FDH和LDH的酶液
    将共表达菌株E.coli BL21-FDH/LDH在含Amp和Kan的LB液体培养基中培养，加入IPTG至终浓度为0.01～0.05mmol/L，16～27℃诱导表达12～20h，发酵液经菌体破壁后离心取上清即得所述FDH/LDH共表达酶液。
  9. 根据权利要求7所述的方法，其特征在于，所述步骤(a)中以假丝酵母总DNA为模板，分别以上下游引物进行PCR扩增，从而获得所述FDH基因，其中：
    上游引物为5’-AAACATATGAAAATCGTTCTCGTTTTGTACTCC-3’，其中，划线处为NdeI酶切位点；下游引物为5’-AAACTCGAGTGCGACCTTTTTGTCATTAC-3’，其中，划线处为XhoI酶切位点。
  10. 根据权利要求8所述的方法，其特征在于，加入IPTG至终浓度为0.02mmol/L，22℃诱导表达16h，发酵液经菌体破壁后离心取上清即得所述FDH/LDH共表达酶液。
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