Microbial Enzymatic Preparation Of Chiral Biotin Intermediate Lactone In Organic Solvent

D-biotin intermediate lactone is an important chiral building block of D-biotin. Preparation of the chiral lactone is a long-standing problem in D-biotin synthesis.In this paper, microbial enzyme-catalyzed resolution of racemic biotin intermediate lactone by enantioselective lactonization in organic solvent was studied. The resolution route was schemed as below:A strain for producing high lactonization activity and enantioselectivy lipase with preference for the formation of d-biotin intermediate lactone was isolated from our laboratory. The strain was elementary identified as Aspergillus oryzae WZ007.The conditions for enzyme production of Aspergillus oryzae WZ007 were also studied. The appropriate culture medium ingredient was soluble starch 0.5%, sucrose 1.5%, olive oil 1%, peptone 1.5%, yeast extract 0.5%, K₂HPO₄ 0.2%, MgSO₄ 0.1%, NaCl 0.5%, tween-20 0.5%, initial pH value 6.5. Optimized fermenting conditions were temperature 28℃, culture medium quantity 80ml, rotate speed 200rpm. The maximal enzyme activity obtained in above condition was 51.6U/L, increased 114% as compared with previously condition.After the dryness technique of Aspergillus oryzae WZ007 mycelium was investigated, we chose the mycelium lyophilized in 0.1M pH7.2 barbital sodium-chlorhydric acid buffer. We selected the anhydrous organic phase as the biocatalysis reaction system.With the optimization of conditions for enzymatic enantioselective latonization, we obtained an optimal reaction condition which enzyme reaction solvent, reaction temperature, water added quantity, water activity, substrate quantity, enzyme quantity, reaction time, conversion ratio were dichloromethane, 25℃, 0.2%, 0.84, 2%, 1.5%, 24h respectively. In the optimal reaction condition, conversion ratio was 43.5% with e.e.p of 97.9% and E value of 212. By the kinetics study of the enzyme catalyzed lactonization, we got K_m 1.36 mol/L, V_max 4.53 mmol/min. When the product lactones was separated and purified, its specific rotation was identified above 60, met the needs of practical application purposes already.