金属离子对重组深渊滕黄单胞菌低温α-淀粉酶LamA的活性和稳定性影响

Effect of metal ions on the activity and stability of recombinant cold-adapted α-amylase LamA from Luteimonas abyssi

【背景】深渊藤黄单胞菌XH031 (Luteimonas abyssi XH031)是从深海分离到的一株具有很强淀粉降解能力的细菌,前期实验显示其α-淀粉酶LamA在低温环境下仍能保持较高酶活力。若能够提升其热稳定性,会有更好的应用前景。【目的】分析钙离子的存在对LamA热稳定性的影响,并通过钙离子结合位点的关键氨基酸的定点突变,初步明确其作用机制。【方法】在不同的离子条件下检测LamA的热稳定性,利用生物信息学方法预测可能影响钙离子结合及耐热性的氨基酸位点,对目的氨基酸进行定点突变,表达和纯化突变蛋白,并进行功能鉴定。【结果】钙离子明显提高了LamA的热稳定性:在未添加钙离子时,于65°C处理30 min已完全失活;而在5 mmol/L钙离子条件下,于65°C处理30 min后仍具有36%的酶活力。对预测位点进行定点突变后,突变蛋白D200R和H233D/M234C完全失活;N120D、Q185E和T224D活性降低。在未添加钙离子时,突变蛋白稳定性受高温影响程度与野生型差别不大;而在钙离子条件下,N120D在65°C时的酶活力仅为野生型的27.8%,推测位点Asn120与钙离子的结合能够稳定低温酶LamA在较高温度下的结构。【结论】初步明确了钙离子可提升低温α-淀粉酶LamA的热稳定性,为今后相关酶类的工程改造提供理论基础。

[Background]Luteimonas abyssi XH031 is one kind of marine bacteria with strong starch degradation ability.Previous studies showed that LamA,a cold-adaptedα-amylase identified from strain XH031,kept high activity under low temperature.LamA will have great application prospects if the high temperature tolerance is improved.[Objective]To determine the calcium ion-dependent thermo-stability enhancement mechanism of LamA,site-directed mutagenesis of key amino acids in the calcium ion binding region was constructed.[Methods]The thermo-stability of LamA was measured in the presence of different chemicals.The amino acid sites that affect calcium ion binding and thermal stability were searched by bioinformatics analysis.Furthermore,the mutant proteins were constructed by the site-directed mutagenesis method and then overexpressed and purified.[Results]Under the calcium-free conditions,LamA was completely inactivated after incubation at 65°C for 30 minutes.However,in the presence of 5 mmol/L calcium ion,LamA still had 36%of the activity after incubation at 65°C for 30 min.The result showed that calcium ions can significantly improve the thermo-stability of LamA.The D200R and H233D/M234C mutant proteins completely lost starch degradation activities.Moreover,the activities of N120D,Q185E and T224D mutant proteins were decreased.However,the mutant proteins kept similar stability compared with the wild-type enzyme under the high temperature and calcium-free conditions.N120D mutant protein preserved only 27.8%residual activity compared with the wild-type enzyme at 65°C supplemented with the calcium ion.Through molecular biology experiments and protein structure simulation,we speculated that the calcium ion binding to the Asn120 site stabilized the structure of LamA under high temperatures.[Conclusion]This study preliminarily clarified the mechanism of calcium ion-dependent thermo-stability enhancement of the cold-adaptedα-amylase LamA and provided a theoretical basis for the engineering transformation of related enzymes.