Study On Cross-linked Aggregates Of (r)-oxynitrilase In Synthesis Of Chiral Cyanohydrins

The enantiomeric purity of chiral cyanohydrins is an important criterion in the synthesis of valuable structural moieties which are building blocks for pharmaceutical products. The synthesis of chiral cyanohydrins has theoretical and applied significance in drug intermediate development. The oxynitrilases are demonstrated to be employed as the key enzyme in the asymmetric synthesis of chiral cyanohydrins and the immobilization of oxynitrilases deserve further research because of its limitations in stability and recycling.The (R)-oxynitrilase from almonds were immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and subsequent cross-linking using glutaraldehyde. The resulting preparation was an effective hydrocyanation catalyst compared to the free enzyme, which accept 46 substrates for the addition of HCN to produce the corresponding cyanohydrins in better optical and chemical yields. The e.e. of benzaldehyde cyanohydrin product was 94 %, furanaldehyde and thiophenealdehyde cyanohydrin products were more than 85%, methylketone cyanohydrin product exceeded 65%, the cyclopentylaldehyde and cyclohexylaldehyde cyanohydrin products were increased by 53% and 73%, respectively.The optimum pH and temperature to obtain high e.e. were pH 4.5 and 10℃, respectively. Furthermore, the effects of buffer pH, temperature, organic solvent, aqueous content, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the CLEA were studied. The best solvent was diethyl ether with an optimum aqueous phase content of 20% (v/v). The optimum substrate concentration and HCN concentration were 0.2mol/ and 0.6mol/L, respectively, besides, the optimum concentration of CLEA was 10U/ml. Under the optimized conditions, the initial velocity and e.e. of cyanhydrin products were 60μmol/ h and 96%, respectively. The aqueous phase containing the CLEA also showed considerably efficient reusability for 5 batch reactions. It was demonstrated that the CLEA is an easy and efficient biocatalysts in recycling.