Biocatalytic Asymmetric Synthesis Of Unnatural Amino Acid (S)-2-cyclopropylglycine

Unnatural chiral amino acid（S）-2-cyclopropylglycine has been widely used in the synthesis of polypeptide analogues and the development of new drugs,such as macrocyclic hunger hormone receptor antagonists,bradykinin B1 receptor antagonists,spindle kinesin inhibitors.It can be used in the treatment of antibacterial,analgesic,anticancer,anticonvulsant and neurodegenerative diseases.Therefore,the development of（S）-2-cyclopropylglycine has very important academic value and application prospect.At present,the main methods for asymmetric synthesis of（S）-2-cyclopropylglycine are chemical method,resolution method and biological method.Compared with the traditional chemical synthesis method,the biocatalytic synthesis route is environmentally friendly and has great potential.Asymmetric biocatalytic synthesis of（S）-2-cyclopropylglycine is mainly realized by enzymatic coupling,and carbonyl reductive aminase and cofactor regeneration enzyme are expressed in different bacteria.The products and cofactors must pass through the cell membrane for material diffusion in and out of the cell as well as the intercellular material exchange to ensure the bioconversion process.In view of this situation,the subject plans to use genetic engineering technology,express carbonyl reductive aminase gene and express cofactor regeneration enzyme gene linked by flexible linker to form a single chain polypeptide with carbonyl reductive amination activity and cofactor regeneration activity,so as to achieve the expression of a double functions fusion enzyme with double active centers in single cells.Asymmetric reductive amination of carbonyl group and in-situ regeneration of cofactor were carried out circularly and simultaneously to improve the bioconversion efficiency.A green and efficient new method for asymmetric biocatalytic synthesis of（S）-2-cyclopropylglycine was developed.The specific contents are as follows:1.Construction of recombinant enzyme expression vector,induction of recombinant enzyme expression and optimization of expression conditions.Leucine dehydrogenase（Bc-LDH and Ti-LDH）from Bacillus cereus and Thermoactinomyces intermedius were selected as carbonyl reductive aminase.Formate dehydrogenase（Kp-FDH）from Komagatael-la pastoris was selected as cofactor regeneration enzyme.The target genetic sequence was synthesized and ligated with pET28a to obtain three recombinant plasmids pET28a-kpfdh,pET28a-bcldh and pET28a-tildh.Four recombinant plasmids pET28a-kpfdh-linker-bcldh（PET28a-klb）、pET28a-bcldh-linker-kpfdh（PET28a-blk）、pET28a-kpfdh-linker-tildh（pET28a-klt）and pET28a-tildh-linker-kpfdh（pET28a-tlk）were constructed by linking the C-terminus or N-terminus of the express carbonyl reductive aminase gene and express cofactor regeneration enzyme gene with flexible linker,respectively.DNA sequencing and agarose gel electrophoresis analysis showed that the recombinant plasmids were successfully constructed.The recombinant plasmids were transformed into competent cells,and the target proteins were expressed under the induction of IPTG.The optimized expression conditions were obtained by SDS-PAGE analysis and enzyme activity assay.Kp-FDH:25℃、0.2 mM IPTG、48 h;Bc-LDH:25℃、0.2 mM IPTG、48 h;Ti-LDH:25℃、0.4 mM IPTG、24 h;KLB:16℃、0.6 mM IPTG、48 h;BLK:16℃、1.0 mM IPTG、48 h;KLT:16℃、1.0 mM IPTG、72 h;TLK:16℃、0.4 mM IPTG、48 h。2.To characterize the enzymatic properties of the recombinant enzymes.Firstly,the effect of temperature on enzyme activity was investigated to obtain the optimal temperature of recombinant enzymes.The optimal temperatures of Kp-FDH,Bc-LDH and Ti-LDH were 70℃,60℃and 70℃,respectively;the optimal temperatures of cofactor regeneration enzyme subunits in KLB,BLK,KLT and TLK were 70℃,70℃,60℃and 70℃,respectively;the optimal temperatures of carbonyl reductiv aminase subunits were 70℃,60℃,40℃and 40℃,respectively.Secondly,the effect of pH on enzyme activity was investigated to determine the optimal pH of recombinant enzymes.The optimal pH of Kp-FDH,Bc-LDH and Ti-LDH were 6.0-9.0,9.0 and 9.0respectively;the optimal pH of cofactor regeneration enzyme subunits in KLB,BLK,KLT and TLK were 8.0,9.0,9.0 and 9.0 respectively;the optimal pH of carbonyl reductive aminase subunits were 9.0,9.0,7.0 and6.0,respectively.Then,the thermostability of the recombinases was investigated.Compared with monofunctional enzymes,bifunctional enzymes showed lower thermostability at higher temperature,but its thermostability did not change significantly at 4℃and 25℃.The results showed that the ligand did not change the thermostability of the two enzymes in their low-temperature state.Finally,the kinetic parameters of recombinant enzymes were determined.The K_m,k_cat and k_cat/K_m of Kp-FDH were 10.15,75.38 and 7.42,respectively;the K_m,k_cat and k_cat/K_m of Bc-LDH were 0.62,1204.56 and 1953.11,respectively;the K_m,k_cat and k_cat/K_m of Ti-LDH were 2.35,920.93 and 391.39,respectively.The K_m,k_cat and k_cat/K_m of cofactor regeneration enzyme subunit in KLB were 9.65,14.61 and 1.51,respectively;the K_m,k_cat and k_cat/K_m of carbonyl reductive aminase subunit were 0.88,653.85 and 740.53,respectively.The K_m,k_catat and k_cat/K_m of cofactor regeneration enzyme subunit in BLK were 13.99,7.16 and 0.51,respectively;the K_m,k_cat and k_cat/K_m of carbonyl reductive aminase subunit were 1.28,350.11 and 272.75,respectively.The K_m,k_catand k_cat/K_m of cofactor regeneration enzyme subunit in KLT were11.97,4.46 and 0.37,respectively;the K_m,k_cat and k_cat/K_m of carbonyl reductive aminase subunit were 1.24,83.12 and 67.09,respectively.The K_m,k_cat and k_cat/K_m of cofactor regeneration enzyme subunit in TLK were34.00,56.18 and 1.65,respectively;the K_m,k_cat and k_cat/K_m of carbonyl reductive aminase subunit were 0.33,95.76 and 290.11,respectively.The k_cat/K_m of the first group of recombinase fusion were significantly reduced,the k_cat/K_m of the second group of recombinase fusion were reduced,but the reduction was not obvious.Due to the k_cat/K_m did not significantly decreased,the linker had no significant effect on the catalytic activity of recombinant bifunctional enzyme,so it can be used in biocatalysis.3.The screening and optimization of biocatalysts for asymmetric synthesis of（S）-2-cyclopropylglycine.Firstly,screen out the E.coli-TLK from six groups of catalysts of E.coli-Kp-FDH+E.coli-Bc-LDH,E.coli-Kp-FDH+E.coli-Ti-LDH,E.coli-KLB,E.coli-BLK,E.coli-KLT and E.coli-TLK,which has the best catalytic activity.Based on the effects of temperature,pH,ammonium formate concentration and coenzyme concentration on the conversion,the optimum conditions for the asymmetric synthesis of（S）-2-cyclopropylglycine catalyzed by E.coli-TLK were obtained as follows:40℃,pH 8,molar ratio of cosubstrate ammonium formate to substrate cyclopropylglyoxylate 10∶1,coenzyme concentration 0.2 mM.A green and efficient bifunctional enzyme for asymmetric biocataly-tic synthesis of（S）-2-cyclopropylglycine was constructed by gene recom-bination technique and the process of carbonyl reductive amination and cofactor regeneration was accomplished in one step.