基因筛选克隆表达原花青素降解酶及其酶解条件优化的初步研究

针对化学法制备低聚原花青素存在环保压力和副产物多等问题，研究了通过生物酶法降解高聚原花青素来制备低聚原花青素的方法。首先通过基因筛选方法筛选出SananA和EcnanA基因用于酶法降解高聚原花青素，将SananA和EcnanA基因连接在不同质粒载体表达蛋白的结果表明，SananA基因连接在pETDuet-1载体上时的蛋白表达效果较优。进而，利用纯化的SananA蛋白优化高聚原花青素降解条件，结果显示较优的酶解条件为温度50℃、pH=10、反应时间8 h，在此条件下原花青素单体积累量为35.67 mg/g，二聚体积累量为14.49 mg/g。

Cloning and expression of proanthocyanidin degrading enzymes based on gene screening and the optimization of their enzymatic catalysis conditions

The chemical preparation of oligomeric procyanidins suffers from potential environmental problems and excessive byproduct formation. In order to overcome these drawbacks, this work attempts to identify an enzyme which can degrade polymeric procyanidins to form oligomeric procyanidins. After gene screening in a database, the genes of N-acetylneuraminate lyase from S. aureus and E. coli were selected as target genes. After the nanA genes from S. aureus and E. coli were ligated into the plasmids of pETDuet-1, pMAL and pZE, SDS-PAGE indicated that SananA has the highest soluble expression in the pETDuet-1 plasmid. Subsequent experiments for enzymatic hydrolysis indicated that a temperature of 50 ℃, at pH=10, for 8 h afforded the best degradation conditions, resulting in monomer yields of 35.67 mg/g and dimer yields of 14.49 mg/g from the polymeric procyanidins.