链霉菌M-Z18膜蛋白ε-聚赖氨酸降解酶的分离纯化、酶学性质及应用

Purification，characterization and application of ε-poly-Llysine-degrading enzyme from Streptomyces sp．M-Z18

摘要:

摘要:【目的】研究链霉菌Streptomyces sp. M-Z18 ε-聚赖氨酸降解酶(Pld)的分离纯化及其生理生化特性，并利用该酶制备低聚合度ε-聚赖氨酸(ε-PL)。【方法】菌体细胞经超声破碎、NaSCN溶解和HiTrapTM Butyl HP疏水层析制备到Pld，随后研究了其酶学性质、动力学和降解ε-PL过程，最后利用常量稀释法比较了不同聚合度范围ε-PL 的最小抑菌浓度。【结果】从Streptomyces sp．M-Z18细胞膜上分离纯化到Pld，纯化倍数为80.4倍，回收率达到59.3%。以L-赖氨酰对硝基苯胺为底物，酶促反应的最适温度为37℃，最适pH为7.0，动力学常数Km为0.621 mmol/L，Vmax为701.16 nmol/min·mg;酶活在pH 7.0-10.0和50℃以下稳定。降解ε-PL实验发现，纯化到的Pld以内切方式降解ε-PL。抑菌实验表明，高聚合度ε-PL(30-35)对细菌的抑制效果较好，而低聚合度ε-PL(8－20)更有利于抑制酵母菌的生长，各种聚合度ε-PL对霉菌的生长抑制均较差。【结论】从ε-PL产生菌中分离纯化到内切型ε-PL降解酶，发现不同聚合度范围ε-PL对微生物的抑制能力存在显著差异。

Abstract:

Abstract:［Objective］The ε-poly-L-lysine-degrading enzyme (Pld) derived from Streptomyces sp．M-Z18 was purified and characterized． Furthermore，Pld was used to produce the low polymerization of ε-poly-L-lysine (ε-PL)．［Methods］ Pld was purified to electrophoretical homogeneity through HiTrapTM Butyl HP hydrophobic chromatography after pretreated by ultrasonic and NaSCN dissolving． Subsequently，enzymatic characteristics，kinetic parameters and the time profile of ε-PL degradation by the purified Pld were studied． Meanwhile，we examined the effect of ε-PL with different degrees of polymerization on the minimal inhibitory concentration of bacteria and fungi．［Results］Pld was purified to homogeneity with a final fold of 80.4 and an overall yield of 59.3%. The optimal temperature and pH for the purified Pld were 37°C and 7.0，respectively．Moreover，the Km with L-lysyl-p-nitroanilide as substrate was calculated to be 0. 621 mmol/L，and the Vmax was 701.16 nmol /min·mg．Pld was stable in the range of pH 7.0－10.0，and temperature up to 50°C，respectively． Time profile of ε-PL degradation by the purified Pld indicated that Pld catalyzed endo-type degradation of ε-PL. The experiments of minimal inhibitory showed that ε-PL with high degree of polymerization (30－35) had a superior antibacterial effect on bacteria and the low degree of polymerization ε-PL (8－20) had a better antibacterial effect on yeasts．However，ε-PL with various degrees of polymerization had a poor antibacterial effect on mould． ［Conclusion］The present result showed that an endo-type Pld from ε-PL-producing strain was purified． Meanwhile，it is proved that ε-PL with different degrees of polymerization have exhibited significant different antibacterial effects on microorganism.