Abstract:

The free nitrilase enzymes are usually easy to be inactivated in the biotransformation reaction of nitriles; and they showed poor stability and low reuse ratio. All of those would lead to high production costs. In this study, the combination immobilization of resting cells was employed to improve the application performance of nitrilase. Chitosan and polyvinyl alcohol were selected for encapsulation experiments, and the moderate residual activity and mechanical strength were observed. The immobilization conditions include the concentration of polyvinyl alcohol and chitosan, as well as immobilization reagents, which were preliminarily optimized. Results showed that 80g/L of polyvinyl alcohol, 40g/L of chitosan, and saturated boric acid solution containing 60g/L of sodium tripolyphosphate were the optimal immobilization conditions. Compared with free cells, the thermal stability and storage stability of immobilized cells were improved significantly. The 3- cyanopyridine bioconversion was carried out by feeding batch reaction with immobilized cells. Finally, 208g/L of nicotinic acid was obtained through 525min of conversion, and the results laid the foundation for the practical application of nicotinic acid bioproduction.

Key words: immobilized cells, biotransformation, nitrilase, nitrile compounds

摘要：

传统的腈水解酶游离催化剂在腈类化合物生物转化过程中存在易失活、稳定性低、重复利用率不高等特点，本研究采用复合固定方式对基因工程腈水解酶进行固定化以期提升其应用性能。通过固定化壳聚糖和聚乙烯醇（PVA）作为包埋材料，获得了具有高机械强度和酶活性的固定化细胞。初步探究了固定化聚乙烯醇和壳聚糖的浓度及固定剂的优选条件，分别为80g/L的PVA、40g/L的壳聚糖及60g/L三聚磷酸钠的饱和硼酸溶液；与游离细胞相比，固定化细胞的热稳定性和贮藏稳定性均显著提高，可增加约2倍；在525min的转化时间内，采用底物流加模式可转化合成烟酸208g/L。本文为复合固定化催化剂在腈化合物生物转化及烟酸合成中的应用奠定了坚实基础。

关键词: 固定化细胞, 生物转化, 腈水解酶, 腈化合物