| Nitrile hydratase (NHase, E.C.4.2.1.84) and amidase (Amidase, EC 3.5.1.4) are the key enzymes in the nitrile compounds metabolic pathway of microbial. These two enzymes play an important role in the preparation of pharmaceutical intermediates and fine chemicals. However, there are still many problems in the practical industrial application, such as low expression level of enzyme, instability expression of enzyme and low product purity for the existence of nitrilase in the metabolic pathway. (S)-2-(4-chlorophenyl)-3-methylbutyric acid (S-CPIAC) is the acid moiety of esfenvalerate, which can be made by nitrile hydratase and amidase coupling catalysis from 2-(4-chlorophenyl)-3-methylbutyronitrile (CPIN). At present, not only the number of the enzyme is less but also the catalytic activity is depressed. Therefore, study of the recombinant expression of nitrile hydratase and amidase, and to further develop new enzyme with better catalytic performance have important theoretical significance and practical value. In this thesis, the synthesis of S-CPIAC from CPIN by nitrile hydratase and amidase coupling catalysis was set as a model reaction. A set of putative nitrile hydratase and amidase genes in the genetic information database were found using genome mining method and realized functional expression in E. coli. We constructed multi-enzyme co-expression system and explored the application of nitrile hydratase and amidase coupling catalysis in the preparation of 5-CPIAC. The main content of this thesis includes the following five parts:The first part is the building of nitrile hydratase library. So far, there are only three reported microbes which can be used to prepare 2-(4-Chlorophenyl)-3-methylbutyramide (CPIAm) by enzyme catalyzed hydrolysis of CPIN. Co-type NHase can catalyze aliphatic and aromatic nitrile compounds, so the conserved amino acid sequence of Co-type NHase was set as molecular probe and this molecular probe was used to search novel Co-type NHase genes in the genetic information database. Based on our target substrate, ten NHase genes were chosen and obtained by PCR amplification. The chosen NHase genes were inserted into the vector pET-30a(+) and achieved functional expression in E. coli. A small NHase library was built containing ten NHases from different microorganism. NHaseK showed the highest enzyme activity (71.2±7.37 U/g DCW) when using CPIN as substrate to screen enzyme in the constructed NHase library. Therefore, we selected NHasek to do further research.The second part is functional expression of NHaseK in E. coli. NHaseK protein (α- and β-subunits) and 17K consists of 202 amino acids (Mr.22 kDa),218 amino acids (Mr.24kDa), and 156 amino acids (Mr.17 kDa), respectively. Experiment indicated that 17K was the activator of NHaseK and was very important for functional expression of NHaseK in E. coli. When 17K gene and NHaseK structural genes were expressed as one unit in E. coli, a large amount of soluble NHase protein was expressed, the specific activity and total activity reached 0.48 U/mg proteins and 120.1 U/L, respectively, but the expression level of 17K was very low. To improve the expression level of 17K and NHaseK activity, we redesigned the expression cassette. The expression level of 17K improved obviously when an efficient SD sequence was introduced into the 5’region of 17K gene. The specific activity and total activity of NHaseK increased by 56% and 50%, respectively, reached 0.75 U/mg proteins and 180.6 U/L. Recombinant NHaseK was purified by Ni-NTA affinity chromatography and its biochemical characterization was studied. The optimum pH and temperature of NHaseK were 6.5 and 35℃, respectively, and NHaseK was remarkably stable below 35℃. NHaseK could hydrolyze a wide range of aliphatic, aromatic, and heterocyclic nitriles and could convert racemic nitriles to the corresponding S-amides, with E values ranging from 9 to 17. Such versatility and robustness indicated that NHaseK has important potential in commercial applications.The third part is functional expression of amidase in E. coli. High-level expression of amidase in E. coli has often been disappointing owing to the formation of inclusion bodies or inactive protein. Ten amidase genes were obtained by PCR amplification and expressed as a fusion protein in E. coli. Approximately half of the recombinant proteins were expressed in soluble form, but they had no amidase activity. KamH was coupled with NHaseK which had the highest enzyme activity, so we selected KamH to do further research. Inclusion body formation and a lack of enzyme activity were also observed when we used different expression vectors to express KamH as a fusion protein and adjusted the IPTG concentration. After cleavage of the peptide by enterokinase, the enzyme activity was partly restored, reaching 4.2±0.25 U/L. When KamH was expressed in its native form, almost no inclusion bodies were formed, and the recombinant protein had maximum activity (35.6±1.61 U/L). This indicated that the peptide at the N-terminus influenced not only soluble expression but also activity of KamH. Similar results were obtained with heterologously expressed amidases from Rhodococcus erythropolis MP50 (MamH) and Agrobacterium tumefaciens d3 (DamH). When MamH and DamH were expressed in their native form, recombinant enzyme activity reached 27.4±1.69 U/L and 22.9± 1.55 U/L, respectively. Expression of amidases in their native form is a powerful strategy and this strategy may be an alternative method for the efficient expression of amidases.The fourth part is optimizing the enzyme production conditions and enzymology characteristics research. Firstly, the media component and fermentation conditions were optimized. Enzyme activity of KamH increased by 84.8%, reached 65.8±1.82 U/L under the optimal fermentation condition. Then, we studied the enzymology characteristics of KamH. The optimum pH was 8.0, and the enzyme showed relatively high activity over a broad pH range from 6.5 to 9.0. KamH showed maximum activity at 40℃ and was remarkably stable below 30℃. KamH exhibited excellent activity toward a wide range of aliphatic, aromatic, and heterocyclic amides and displayed strict (S)-enantioselectivity to racemic amides (e.e. value>95%). When KamH was used to catalyze CPIAm, the e.e. value of CPIAC was 97.5% at the conversion rate of 50%. This indicated that KamH has important potential in the fine chemicals and pharmaceutical intermediates industries. In the evolutionary tree, KamH and AS family amidase were in the same branch, and had closer evolutionary distance with the enantioselective amidase. The amino acid sequence of KamH was compared with the amino acid sequence of the reported AS family amidase, and found that KamH contained AS family amidase conserved sequence (GGSSSGS) and the catalytic triad Ser-cis Ser-Lys. The position of the catalytic triad in KamH was K96-S171-S195. This suggests that KamH is a member of AS family amidase.The fifth part is the construction of multi-enzyme coexpression system. Two types multi-enzyme coexpression systems were constructed, and realized the coexpression of NHaseK and KamH. Firstly, three single-plasmid coexpression systems were constructed, pETDuet-N-K, pRSFDuet-N-K and pCDFDuet-N-K. Among of them, pETDuet-N-K has the best expression effect, and the activity of NHaseK and KamH were 46.4±2.52 U/L and 20.3±1.35 U/L, respectively. Then, three double-plasmid coexpression systems were constructed, pETDuet-N-A, pRSFDuet-N- A and pCDFDuet-N-A. Among of them, pETDuet-N-A has the best protein expression, and the actity of NHaseK and KamH were 52.8±2.64 U/L and 22.9±1.48 U/L, respectively. By contrast, the protein expression effect of double-plasmid coexpression system was superior to that of single-plasmid coexpression system. When the constructed coexpression systems were used to catalyze CPIN, optical purity of CPIAC was obtained (e.e. value 99%). |
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