低温几丁质酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征

Recombinant expression and characterization of cold-adapted chitinase genes in Kluyveromyces lactis

【目的】通过构建假交替单胞菌(Pseudoalteromonassp.DL-6)低温几丁质酶(chitinaseA,chi A;chitinase C,chi C)的重组乳酸克鲁维酵母菌株、纯化重组蛋白并对其进行酶学性质表征,为低温几丁质酶潜在工业化生产几丁寡糖奠定理论基础。【方法】人工合成密码子优化的几丁质酶基因,构建重组乳酸克鲁维酵母表达质粒(p KLAC1-chi A、p KLAC1-chi C)并用电脉冲法转化到乳酸克鲁维酵母中,实现低温几丁质酶的可溶表达。利用镍柱亲和层析纯化得到高纯度的重组几丁质酶。【结果】成功构建产低温几丁质酶的重组乳酸克鲁维酵母并纯化获得高纯度的重组几丁质酶。经SDS-PAGE分析在110 k Da与90 k Da附近出现符合预期大小的蛋白条带。铁氰化钾法测得Chi A和Chi C的酶活分别为51.45 U/mg与108.56 U/mg。最适反应温度分别为20°C和30°C,最适p H分别为8.0和9.0。在低于40°C,p H 8.0–12.0时,Chi A和Chi C重组酶较稳定。Chi A和Chi C对胶体几丁质以及粉状底物α-几丁质与β-几丁质具有明显的降解活性,且具有一定协同降解能力。【结论】首次实现假交替单胞菌来源的低温几丁质酶在乳酸克鲁维酵母中的重组表达、纯化、酶学性质及其降解产物分析,为其他低温几丁质酶的研究提供借鉴意义。

[Objective]We aimed to construct recombinant Kluyveromyces lactis(K.lactis)strains to produce cold-adapted chitinases(chitinase A,chi A;chitinase C,chi C)of Pseudoalteromonas sp.DL-6.We purified and characterized the recombinant proteins.[Methods]We synthesized optimized-codons chitinase genes.The recombinant K.lactis expression plasmids(p KLAC1-chi A,p KLAC1-chi C)were constructed successfully and transformed into K.lactis by electric field pulses to achieve soluble expression of cold-adapted chitinases.The recombinant proteins were further purified using Ni-NTA chromatography.[Results]The recombinant K.lactis producing cold-adapted chitinases was successfully constructed and obtained high purity.The purified recombinant proteins,Chi A and Chi C,appeared as a single band of approximately 110 k Da and 90 k Da by SDS-PAGE,respectively.The activities of Chi A and Chi C were 51.45 U/mg and 108.56 U/mg measured by Schales’method.The optimum temperature was 20°C and 30°C,and the optimum p H was 8.0 and 9.0,respectively.Chi A and Chi C were stable at p H 8.0–12.0 and below 40°C.Chi A and Chi C have obvious degradation activity to colloidal chitin and powdered chitin includingα-chitin andβ-chitin.Besides,they acted synergistically to efficiently degrade chitin substrates.[Conclusion]The expression,purification,enzymatic properties and degradation products of cold-adapted chitinases in K.lactis were achieved for the first time.These research results provide reference for the study of other cold-adapted chitinases.