浸麻类芽孢杆菌葡甘露聚糖降解酶的异源表达及功能研究

Heterologous expression and characterization of novel glucomannan-degrading enzymes from Paenibacillus macerans

摘要:

【目的】克隆表达浸麻类芽孢杆菌(Paenibacillus macerans)的葡甘露聚糖降解酶，研究其性质和功能，丰富葡甘露聚糖降解酶资源，了解浸麻类芽孢杆菌降解葡甘露聚糖机制。【方法】检索浸麻类芽孢杆菌的葡甘露聚糖降解酶基因，构建重组菌株，表达纯化重组酶，系统研究其功能及在降解葡甘露聚糖中的作用。【结果】克隆表达了5个葡甘露聚糖降解酶组分。结果显示PmMan1和PmMan2为内切β-甘露聚糖酶，PmGlc1、PmGlc2和PmGlc3为外切β-葡萄糖苷酶。其中PmGlc1只能水解pNPβGlc，PmGlc2能水解二糖和人参皂苷的β-1,6-葡萄糖苷键，而PmGlc3对β-葡萄糖苷键的选择性较为广泛。PmMan1、PmMan2、PmGlc2和PmGlc3能够降解葡甘露寡糖，PmMan1和PmMan2可以降解葡甘露聚糖。共同降解葡甘露聚糖时，PmGlc2和PmGlc3与PmMan2具有协同效应，且PmGlc3与PmMan2的协同作用更为显著。【结论】从浸麻类芽孢杆菌中获得了4种葡甘露聚糖降解酶，阐明了该菌葡甘露聚糖降解酶系成员的作用，丰富了酶资源和理论研究成果的同时，为酶法制备活性葡甘露寡糖提供了有效工具。

Abstract:

[Objective] We cloned, expressed, and characterized the glucomannan-degrading enzymes from Paenibacillus macerans, aiming to enrich the glucomannan-degrading enzyme resources and decipher the mechanism of glucomannan degradation by P. macerans. [Methods] We retrieved the glucomannan-degrading enzyme genes and constructed recombinant strains to express the recombinant enzymes. The roles of the purified proteins in the degradation of glucomannan were then studied. [Results] We cloned and expressed five glucomannan-degrading enzymes, among which PmMan1 and PmMan2 were endo-β-mannanases, and PmGlc1, PmGlc2, and PmGlc3 were exo-β-glucosidases. PmGlc1 only hydrolyzed pNPβGlc, and PmGlc2 hydrolyzed β-1,6-glucosidic bonds in disaccharides and ginsenosides. PmGlc3 hydrolyzed a wide range of β-glucosidic bonds. PmMan1, PmMan2, PmGlc2, and PmGlc3 degraded glucomannan oligosaccharides, and PmMan1 and PmMan2 degraded glucomannan. In the degradation of glucomannan, PmGlc2 and PmGlc3 had synergistic effects with PmMan2, and the synergistic effect was more significant between PmGlc3 and PmMan2. [Conclusion] We obtained four glucomannan-degrading enzymes from P. macerans and clarified the role of the enzymes in glucomannan degradation. The findings enrich the enzyme resources and theoretical research achievements and provide an effective tool for enzymatic preparation of active glucomannan oligosaccharides.