Identification And Expression Of Delftia Tsuruhatensis R-amidase Gene And Its Application In Cilastatin Synthesis

R-amidase [EC 3.5.1.4] catalyze the resolution of R/S-2,2-dimethylcyclopropane- carboxamide. S-2,2-dimethylcyclopropanecarboxamide is the key chiral building block of cilastatin. So the study on R-amidase and its industrial application is very important to the greening process of cilastain/imipenem manufacturing. This work studied the proficient manufacturing, enzymology, and application of R-amidase, and finaly the bioprocess was established for the manufacturing of S-2,2-dimethylcyclopropanecarboxamide.Through optimization of medium and conditions, the R-amidase activity of D. tsuruhatensis CCTCC M 205114 in 5 L fermentor was improved to 192.7 U/L. Through N+ implantation, one new strain of D. tsuruhatensis CCTCC M was obtained with 38.7% improvement of R-amidase activity in flask level. Enzyme activity of 358.2 U/L was obtained in 5 L fermentor culture, which was 86.9% higher than that of the old strain. The new strain had much higher basic expression level (103.6 U/L) before induction than the that of the original strain (4.6 U/L), this implied the part break of the control mechanism. But even higher enzyme activity was achieved with genetic engineering technique. 1.4 kb fragment was amplified with Pyrobest DNA polymerase. The final recombinant strain was E. coli BL21(DE3)/pET-dam. The engineered R-amidase under T7 promotor gave the the expression level of 2963 U/L in 5 L fermentor. So the fermentation volume needed for resolution of 10kg R/S-2,2-dimethylcyclopropanecarboxamide was reduced from 2327.9 L to 77.8 L, which can be operated in lab scale for harvest and purification.The cloned R-amidase gene (Genbank No: HM165184.1) was analyzed. The result showed that the monomer of R-amidase contained 467 aa, its MW was 49.9 kDa, pI was 6.73. This R-amidase belonged to Amidase Signature family, had the characteristic GASSSG sequence. The monomer was consisted of three domains, N-terminal domain, small domain and big domain. The catalytic triad was deduced to be 83K, 156S, 180S. A preliminary docking result of the R-amidase with R/S-2,2-dimethylcyclopropanecarboxamide hint that the R-isomer was more prone to enter the catalytic pocket, this perhaps is the main reason for the high enantioselectivity of the R-amidase toward R-isomer. The distance between R-DMCPCA and 180S is 7.61 A. Ramachandran plot analysis also showed that more accurate R-amidase crystal data was needed for accurate protein-substrate analysis.To carry out the research work efficiently, a simple HPLC method was developed to quickly analyze 2,2-dimethylcyclopropanecarboxamide and 2,2-dimethylcyclopropane- carboxylic acid. This HPLC method was suitable for the fast enzyme activity analysis and chiral resolution reaction control. Enzyme activity analysis showed that the R-amidase activity of D. tsuruhatensis CCTCC M is inducible. The induction is under stringent control by the host. The amount of induced enzyme activity equals to the amount needed to hydrolyze the amide in about 1 hour. But 2,2-dimethylcyclopropanecarboxamide concentration higher than 1.2 g/L can result in the inhition of R-amidase expression. It was concluded that break through of the expression control mechanism is the most important thing to obtain high expression level. A new immobilization process with chitosan was developed in the study of enzyme application. In this immobilization process, the carrier preparation, activation and immobilization steps were accomplished in one step. The immobilized R-amidase was used as catalyst for R/S-2,2-dimethylcyclopropanecarboxamide resolution. The reaction solution was filtered, and then purified with adsorbent resin. The total yield of S-DMCPCA was 30.88%. but the immobilization yield of R-amidase was still to be improved before industrial application.Experiments showed that Km of the R-amidase was 19.7μmol/L, and Kcat was 198.7/s. According to the model established in this work, enzyme utilization efficiency (U), conversion, and the start x (Km/[S]) have below relationship:U = (1-c)/( X0+1-c). If the U should be greater than 80% while conversion reaches 99.9%, the start substrate concentration should be higher than 400 Km (1.78 g/L). This also means that if the substrate is higher than 1.78 g/L, the enzyme utilization efficiency would always be higher than 80% even if the reaction has nearly reached end. So in the actual substrate concentration of 6 g/L, enzyme efficency will always keep high, and more enzyme means short reaction time and low enzyme utilization efficiency. Since the R-amidase is stable for more than 48 h in neutral condition, less enzyme and more substrate means better enzyme utilization efficiency, which is equal to batch reactions catalyzed by immobilizaed enzyme. In the actual reaction, enzyme amount was cut to a very low level so that the reaction solution can be directly put forward to downstream purification. In the end, 20.5 kg of S-2,2-dimethylcyclopropanecarboxamide was obtained from 60 kg R/S-2,2-dimethylcyclopropanecarboxamide with ee value of 100% and total yield of 34.2%.Based on the experience accumulated in this and other work, some stages of industrial enzyme engineering process were also discussed, which were route design stage, technique development stage, process development stage, industrial scale-up stage and commercial stage.