葡萄几丁质酶基因在毕赤酵母中的表达及其生物活性研究

Expression and Functional Analysis of Grape Chitinase in Pichia pastoris GS115

采用一步法逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR),以葡萄幼叶为材料克隆几丁质酶基因的cDNA序列,序列分析表明,其包含基因完整的编码序列,编码由314个氨基酸组成的几丁质酶,通过抑菌试验证明,所纯化的几丁质酶对尖孢镰刀菌菌丝生长有抑制作用,同时用水解CM-chitin-RBV的方法测定纯化的几丁质酶活性,在37℃,pH 5时分解几丁质的比活力为0.78△A 550/(μg.h),而该酶的粗提物的比活力为0.21△A 550/(μg.h)。

The gene encoding the chitinase was cloned from cDNA of grape（Vitis vinifera L. ） leaves by reverse transcriptase polymerase chain reaction （RT - PCR） amplification. The open reading frame （ORF） had 940 bp that encodes a protein of 314 amino acids. To explore the properties of the chitinase, we had expressed and purified recombinant chitinase. Using the assay of hydrolysis of CM -chitin -RBV,the activity of the purified MpChi -1 was 0.78△A 550/（μg·h） at 37 ℃ and pH5 ,comparing to 0. 21△A 550/（μg · h） of the crude extracted enzyme. The recombinant protein inhibited the growth of the Fusarium occysporum F. sp. vasinfecturn in vitro.