【作者】 崔永燕； 杨敏； 周德庆；

【Author】 Cui Yongyan;Yang Min;Zhou Deqing;Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences;College of Food Science,Ocean University of Shanghai;

【机构】 中国水产科学研究院黄海水产研究所； 上海海洋大学食品学院；

【摘要】 褐藻胶裂解酶通过β-消除反应降解褐藻胶,在非还原端C4和C5之间形成双键,生成具有多种生物活性的功能性褐藻胶寡糖(alginate oligosaccharides,AOS)。然而,目前褐藻胶裂解酶的热稳定性难以满足其在工业上的应用。本研究提出一种基于HotSpot Wizard 3.0分析和计算机辅助计算ΔΔGfold的有效合理设计策略,利用定点突变获得了12个突变体。在50℃条件下孵育3 h后,突变体Q246L和K249P可保留60%以上酶活,而Q246V和K249V可保留70%以上的酶活性,显著提高了褐藻胶裂解酶AlyMc(保留45%酶活)的热稳定性。TLC结果表明AlyMc降解褐藻胶的产物以DP2-4的寡糖为主,其中三糖含量最高。综合分析了酶的三维结构和分子动力学模拟,发现突变增强了突变位点处残基与周围残基之间的疏水相互作用。分子动力学模拟结果显示,突变体Q246V降低了176-178残基的自由度;K249V突变降低了188-192残基的自由度。突变引起疏水作用力的增强和结构灵活性的降低可能是酶热稳定性提高的原因。更多还原

【Abstract】 Alginate lyase degrades alginate by the β-elimination mechanism to form a double bond between 0.4 and C5 at the non-reducing end,whose degradation products are functional alginate oligosaccharides with various biological activities.However,the current thermal stability of alginate lyase is difficult to meet its industrial applications.In this study,an efficient rationally combined strategy based on HotSpot Wizard 3.0 and computer-aid ΔΔG_fold value calculation was proposed,and 12 mutants were carried out by site-directedmutagenesis.The mutants Q246 L,and K249 P retained more than 60% of the enzyme activity,and the mutants Q246 V and K249 V retained more than 70% of the enzyme activity by incubation at 50 ℃ for 3 h,which significantly improves the thermal stability of alginate lyase AlyMc（45% enzyme activity）.The TLC results indicated that the products of alginate degradation by AlyMc were mainly oligosaccharides of DP2-4,with the highest content of trisaccharides.The three-dimensional structure of the enzymes and molecular dynamics simulation were comprehensively analyzed,and we found that mutation enhanced the hydrophobic interaction between the residues at the mutation site and the surrounding residues.Molecular dynamics simulations showed that the mutant Q246 V reduced the degrees of freedom of residues 176-178,and the K249 V mutation reduced the degrees of freedom of residues 188-192.Mutation-induced enhancement of hydrophobic forces and reduction of structural flexibility may be responsible for the increased thermal stability of the enzymes.更多还原