耐酸性黑曲霉β-甘露聚糖酶的克隆及其在毕赤酵母中的表达分析

Cloning of an Acid-Stable β-Mannanase from Aspergillus niger AN070902 and its Expression Analysis in Pichia pastoris

目的:克隆黑曲霉β-甘露聚糖酶基因,研究该基因在毕赤酵母中的表达情况。方法:运用RT-PCR从黑曲霉AN070902中克隆β-甘露聚糖酶cDNA片段,与载体pPIC9K相连,构建重组载体VMAN-pPIC9K,电转化毕赤酵母GS115,筛选产酶最高菌株进行5 L液体发酵,对该菌株所产重组酶进行酶学性质分析。结果:克隆获得1152 bpcDNA,编码由383个氨基酸残基组成的蛋白质,该蛋白质属于GH5家族,理论pI和相对分子质量分别为4.48和41.6×103;筛选获得的重组菌株VMAN-pPIC9K-GS115在5 L液体发酵中上清酶活达11 785 U/mL;表达的重组酶是一种酸性β-甘露聚糖酶,最适反应pH值为3.0,经pH2.0～9.0处理2 h后剩余酶活保持90%以上;该重组酶最适反应温度为65℃,70℃处理1 h后剩余酶活保持75%以上;该重组酶活性被1 mmol/L的Fe3+和Mn2+显著抑制,被1mmol/L的Co2+显著激活。结论:重组耐酸性β-甘露聚糖酶的特性,决定了其在工业生产中,特别是动物饲料和食品加工中具有应用价值。

Objective：To obtain β-mannanase gene from Aspergillus niger,then to study the expression of the gene in Pichia pastoris.Methods：The cDNA sequence of β-mannanase from A.niger AN070902 was obtained by RT-PCR.The cDNA fragment was cloned into the expression vector pPIC9K and the linearized recombinant vector VMAN-pPIC9K was transformed to P.pastoris GS115 by electroporation.After screening,the recombinant strain that expressed the β-mannanase at the highest level was cultivated in 5 L fermenter.The recombinant mannanase characteristic was analyzed subsequently.Results：A 1152 bp cDNA was obtained.The deduced amino acid sequence has a theoretical pI 4.48 and Mr 41.6 kD,belongs to glycoside hydrolase family 5.The recombinant strain VMAN-pPIC9K-GS115 was screeninged and reached a maximum mannanase activity of 11 785 U/mL in 5 L fermenter.The recombinant β-mannanase was acidophilic and acid stable,exhibiting maximal activity at pH3 and retaining above 90% of the initial activity over the pH range 2.0～9.0.The recombinant mannanase had an optimal temperature of 65℃ and maintained over 70% of original activity after incubation at 75℃ for 5 min.The enzymatic activity was not significantly affected by 1 mmol/L K＋,Mg2＋,Ca2＋,Zn2＋,Fe2＋,Cu2＋,EDTA but inhibited by Fe3＋,Mn2＋ and enhanced by Co2＋.Conclusion：All these favorable properties make it useful in many industrial applications,especially for animal feed and food processing.