镰刀菌Q7-31T果胶酶PGL1的分离纯化、酶学性质鉴定及结构分析

doi:10.13560/j.cnki.biotech.bull.1985.2017-0864

摘要/Abstract

摘要： 从镰刀菌Q7-31T中分离纯化和鉴定果胶酶并进行生物信息学分析,旨在探究果胶酶降解植物细胞壁的作用,进一步完善镰刀菌属纤维素降解酶系信息。以0.8 % 蛋白胨为氮源,0.5% 燕麦秸秆为碳源诱导发酵培养镰刀菌Q7-31T;采用Sephacry S-100 凝胶柱层析和 DEAE 琼脂糖弱阴离子交换柱层析对粗酶液进行分离纯化得到果胶酶PGL1;对其进行了酶学性质分析和串联质谱鉴定;最后通过生物信息学分析结构及预测功能。PGL1分子量为36.21 kD,等电点为8.13;PGL1最适反应温度为40℃,最适反应 pH 为8;在20℃ 到50℃ 和pH 8.0到10.0能保持较高稳定性;Na⁺、K⁺、Mg²⁺、Zn²⁺、Ca²⁺ 和Fe²⁺对PGL1有抑制作用,串联质谱鉴定表明该蛋白为一种新型PL-1家族果胶酶。PGL1的二级结构构成元件中无规则卷曲和延伸链比例较高,分别占37.96%和31.17%;PGL1两端亲水性强而中间部分疏水性强;磷酸化位点共13个（Ser∶9;Thr∶3;Tyr∶1）;PGL1存在2个潜在的N-糖基化位点;跨膜结构域预测显示其为分泌酶;功能结构域预测出其降解植物细胞壁的作用。从镰刀菌Q7-31T中分离纯化得到一种新型PL-1家族果胶酶PGL1并对其进行了生物信息学分析。

关键词: 镰刀菌, 果胶酶, 分离纯化, 酶学特性, 生物信息学分析

Abstract: To explore the role of pectinase degrading cell wall and further improve the information of cellulose-degrading enzymes in Fusarium sp. Q7-31T,the pectinase from Fusarium sp. Q7-31T was isolated,purified,and identified,followed by bioinformatics analyses. Q7-31T was cultured by induction and fermentation with 0.8% peptone as nitrogen source and 0.5% oat straw as carbon source. Then the pectinase PGL1 was purified from crude enzyme liquid using Sephacry S-100 chromatography and DEAE-sepharose ion-exchange column chromatography. Further,its enzymatic properties were analyzed and MADIL-TOF-TOF identification was conducted. Finally,its structure was analyzed and the function was predicted by bioinformatics. The molecular weight and isoelectric point（pI）of PGL1 was 36.21 kD and 8.13,respectively. PGL1 had optimal activity at 50℃ and p H 8.0,was highly stable at 20℃ to 50℃ and pH 8.0 to 10.0,and was inhibited by Na⁺,K⁺,Mg²⁺,Zn²⁺,Ca²⁺,and Fe²⁺. MADIL-TOF-TOF identification showed that the PGL1 was a new PL-1 family pectinase. The secondary structure prediction suggested that random coil and extended strand were major secondary components（37.96% and 31.17% respectively）. PGL1 had two hydrophilic ends and a hydrophobic middle part. Its phosphorylation sites were 13（Ser：9;Thr：3;Tyr：1）. There were 2 potential N-glycosylation sites in PGL1. The prediction of transmembrane domain revealed that PGL1 was a secretory enzyme. The functional domain prediction of PGL1 showed the role of its degrading plant cell wall. Conclusively,a new PL-1 family pectinase PGL1 was isolated and purified from Fusarium sp. Q7-31T and analyzed by bioinformatics.

Key words: Fusarium sp., pectinase, isolation and purification, enzymatic properties, bioinformatics analysis

秦日甜, 谢占玲. 镰刀菌Q7-31T果胶酶PGL1的分离纯化、酶学性质鉴定及结构分析[J]. 生物技术通报, 2018, 34(4): 151-160.

QIN Ri-tian, XIE Zhan-ling. Isolation,Purification,Characterization and Structural Analysis of a Pectinase PGL1 Produced by Fusarium sp. Q7-31T[J]. Biotechnology Bulletin, 2018, 34(4): 151-160.

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