磷脂酶D交联聚集体的制备及其酶学性能研究

Preparation and characterization of cross-linked enzyme aggregates of phospholipase D

磷脂酶D(phospholipase D,PLD)可以催化磷脂的磷酸二酯键水解和转磷脂酰基反应,目前已广泛应用于化工和医药等行业。该研究利用交联酶聚集体(cross-linked enzyme aggregates, CLEAs)技术开发了一种新型的PLD交联聚集体固定化酶(PLD-CLEAs)。以酶活性回收率为指标,确定了PLD-CLEAs的最佳制备条件:饱和度60%的硫酸铵为沉淀剂,质量分数0.125%的戊二醛为交联剂,交联反应时间为1.5 h,此时PLD-CLEAs的酶活力回收率为118.8%。与游离酶相比,PLD-CLEAs最适pH值向中性偏移,由8.0变为7.0;且PLD-CLEAs在较宽的温度和pH范围内均保持较高的酶活性,在高温(50℃)下的热稳定性明显提高。另外,在重复使用10次后,PLD-CLEAs仍能保持50.4%的酶活性。研究结果表明,PLD-CLEAs具有比游离酶更优异的热稳定性、pH耐受性和重复使用稳定性,有良好的工业应用前景。

Phospholipase D can catalyze the hydrolysis of phosphodiester bond of phospholipids, and the conversion of phospholipid acyl reaction. Phospholipase D is widely used in chemical and pharmaceutical industries. In this study, a novel cross-linked phospholipase D aggregates(PLD-CLEAs) was developed. Using the recovery rate of enzyme activity as the main indicator, the optimal preparation conditions of PLD-CLEAs were determined as follows: the precipitant was ammonium sulfate with 60% saturation, the crosslinking agent was glutaraldehyde, and its mass fraction was 0.125%, and the crosslinking time was 1.5 h. The enzyme activity recovery of PLD-CLEAs prepared under the optimum conditions was 118.8%. Compared with free PLD, the optimal pH of PLD-CLEAs shifted towards neutral direction(from 8.0 to 7.0). And PLD-CLEAs were maintained a relatively high enzyme activity in wide temperature and pH ranges. Moreover, the thermal stability of PLD-CLEAs was improved obviously at high temperature(50 ℃). In addition, PLD-CLEAs still had 50.4% of initial enzyme activity after 10 repeated uses. The above research results showed that PLD-CLEAs had superior thermal and pH stabilities and reusability in comparison with the free ones, thus has a good prospect of industrial application.