基于壳聚糖、甲壳素载体的荧光假单胞菌脂肪酶固定化及其水解性能研究

Immobilization of Pseudomonas fluorescens Lipase onto Chitosan and Chitin-based Carriers and Evaluation of Its Hydrolytic Performance

摘要:

【目的】构建具有分级多孔片层结构的壳聚糖和甲壳素载体，系统评估壳聚糖和甲壳素载体对荧光假单胞菌脂肪酶（Pseudomonas fluorescens lipase，PFL）的固定化效果，以及该固定化酶催化水解鱼油的性能。【方法】基于溶胶-凝胶法对壳聚糖、甲壳素进行改性，以制备载体，使其与游离PFL结合，再检测其酶学性质，并分析其水解凤尾鱼油的产物组成成分。【结果】经化学修饰后的载体呈片层堆叠结构，表面含有丰富的游离氨基，有利于PFL的共价固定化。壳聚糖载体的固定化效率达89.26%，酶负载量为2.21 g/mg，壳聚糖载体固定化PFL的酶活力为221.33 U/mg，是游离PFL的2.54倍；甲壳素载体的固定化效率为71.62%，酶负载量为1.84 g/mg，甲壳素载体固定化PFL的酶活力为185.34 U/mg，是游离PFL的2.12倍。壳聚糖、甲壳素载体固定化PFL的最适反应条件均为55 ℃和pH 8.0，均表现出良好的有机溶剂（甲醇、乙醇和丙三醇）耐受性及储藏稳定性，4 ℃存放11 d后酶活力分别为115.11 U/mg与91.43 U/mg。在凤尾鱼油水解反应中，用壳聚糖、甲壳素载体制备的固定化PFL水解度分别为23.1%和19.7%，且壳聚糖载体固定化PFL中的EPA和DHA占比分别提高至25.34%和18.51%，甲壳素载体固定化PFL中的EPA和DHA占比分别提高至25.96%和18.89%。【结论】该研究展示了分级壳聚糖、甲壳素载体在增强脂肪酶催化性能方面的应用潜力，为开发新型固定化酶催化体系用于深海鱼油中ω-3脂肪酸的高效富集提供了理论基础与实践支持。

Abstract:

[Objective]This study aims to construct chitosan and chitin-based carriers with hierarchical porous lamellar structures, systematically evaluate the immobilization effects of these carriers for Pseudomonas fluorescens lipase (PFL) and the performance of the immobilized enzyme in catalyzing the hydrolysis of fish oil.[Method]Chitin/chitosan were modified via the sol-gel method to prepare the carriers for the binding with free PFL. The enzymatic properties of the immobilized PFL were then evaluated, and then analysed the hydrolysate composition of anchovy oil catalyzed by the immobilized PFL.[Result]The modified carriers exhibited lamellar structures with abundant free amino groups on the surface, which facilitated covalent immobilization of the enzyme. The chitosan-based carrier achieved the immobilization efficiency of 89.26%, the enzyme loading of 2.21 g/mg, and the PFL enzyme activity of 221.33 U/mg (2.54 folds that of the free enzyme). The chitin-based carrier showed the immobilization efficiency of 71.62%, the enzyme loading of 1.84 g/mg, and the PFL enzyme activity of 185.34 U/mg (2.12 folds that of the free enzyme). The chitosan and chitin-immobilized PFL exhibited optimal catalytic activity at 55 ℃ and pH 8.0, along with significant tolerance to organic solvents such as methanol, ethanol, and glycerol and storage stability. After 11 days of storage at 4 ℃, the enzyme activity reached 115.11 U/mg and 91.43 U/mg, respectively. In the hydrolysis of anchovy oil, the hydrolysis degrees of the chitosan and chitin-immobilized PFL were 23.1% and 19.7%, respectively. The proportions of EPA and DHA in the chitosan-immobilized PFL increased to 25.34% and 18.51%, respectively, while the proportions of EPA and DHA in the chitin-immobilized PFL increased to 25.96% and 18.89%, respectively.[Conclusion]This study demonstrates the potential of hierarchical chitosan and chitin-based carriers in enhancing the catalytic performance of lipase and provides a theoretical basis and practical support for the development of immobilized enzymatic systems for the efficient enrichment of ω-3 fatty acids from deep-sea fish oils.