镰刀菌Q7-31T内切葡聚糖酶Egn21的分离纯化及酶学性质

Purification and characterization of endoglucanase Egn21 from Fusarium sp. Q7-31T

摘要:

摘要：【目的】为进一步研究镰刀菌Q7-31T产生的植物细胞壁降解酶的酶系信息。【方法】以1% (W/V) 蛋白胨为氮源，0.5% (W/V)燕麦秸秆为碳源，20 °C、120 r/min振荡培养3 d，诱导发酵培养菌株，获得的粗酶液经过Sephacry S-100凝胶柱层析和DEAE琼脂糖弱阴离子交换柱层析，最终得到纯化的内切葡聚糖酶，并对其进行酶学性质分析及串联质谱鉴定。【结果】研究表明：Egn21的分子质量为44.25 kDa，等电点为4.91；酶学特性研究显示：Egn21降解羧甲基纤维素的最适反应温度为40 °C，在45 °C以下比较稳定。该酶最适pH为6.0，在pH为5.0–8.0条件之间比较稳定。Co2+、Zn2+和Mg2+对其没有明显作用，而Fe2+、Ca2+、K+、Na+和Mn2+对酶活性有抑制作用，Hg2+会使酶失去活性。【结论】从Q7-31T中分离纯化得到的内切葡聚糖酶Egn21，经过酶学特性与串联质谱鉴定结果显示其属于GH5家族。

关键词:关键词:镰刀菌，内切葡聚糖酶，分离纯化，鉴定，酶学特性

Abstract:

Abstract: [Objective] The objective of this research was to study plant cell wall degradation enzymes from Fusarium sp. Q7-31T. [Methods] Strain was cultured in liquid medium with 1% (W/V) peptone as nitrogen source, 0.5% (W/V) oat straw as carbon source, 120 r/min shaking at 20 °C for 3 days. The endoglucanase Egn21 was purified by using Sephacry S-100 chromatography and DEAE-sepharose ion-exchange column chromatography. Then the enzymatic properties and MADIL-TOF-TOF identification were analyzed. [Results] The molecular weight and isoelectric point (pI) of Egn21 was 44.25 kDa and 4.91, respectively. Egn21 had optimal activity with carboxymethyl cellulose at 40 °C and pH 6.0, stable at 45 °C and pH between 5.0 and 8.0, inhibited by Fe2+, Ca2+, K+, Na+, Mn2+ and inactivated by Hg2+, whereas Co2+, Zn2+ and Mg2+ had no effect. [Conclusion] The enzymatic properties and MADIL-TOF-TOF results suggested that Egn21 belongs to GH5 family.

Key words:Keywords: Fusarium sp., endoglucanase, protein purification, identification, enzymatic properties