镰刀菌Q7-31T金属蛋白酶FQME14的分离纯化及鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2017-0896

生物技术通报 ›› 2018, Vol. 34 ›› Issue (4): 161-167.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0896

镰刀菌Q7-31T金属蛋白酶FQME14的分离纯化及鉴定

秦日甜¹, 卢玉丽¹, 梁高丽¹, 谢占玲^{1, 2}, 雷亚男³

1. 青海大学生态环境工程学院微生物实验室,西宁 810016;
2. 青海省高原作物种质资源创新与利用重点实验室,西宁 810016;
3. 西北农林科技大学,杨凌 712100

收稿日期:2017-10-24 出版日期:2018-04-20 发布日期:2018-05-04
作者简介:秦日甜,女,本科,研究方向：酶学;E-mail：qinritian97@126.com
基金资助:
国家自然科学基金项目（31260021）,青海省科技厅资助项目（2014-zj-903）

Isolation,Purification and Identification of Metalloprotease FQME14 from Fusarium sp. Q7-31T

QIN Ri-tian¹，LU Yu-li¹，LIANG Gao-li¹,XIE Zhan-ling^1,2,LEI Ya-nan³

1. College of Ecological and Environment Engineering,University of Qinghai,Xining 810016;
2. Key Laboratory Breeding Base for Innovation and Utilization of Plateau Crop Germplasm,Xining 810016;
3. Northwest Agriculture and Forestry University,Yangling 712100

Received:2017-10-24 Published:2018-04-20 Online:2018-05-04

摘要/Abstract

摘要： 从镰刀菌 Q7- 31T 燕麦秸秆诱导发酵的粗酶液中分离、纯化并鉴定金属蛋白酶。研究该蛋白酶的酶学特性,丰富和完善金属蛋白酶的基础资料;探究其在纤维素酶系降解纤维素过程中发挥的作用,旨在丰富和完善纤维素酶系降解机制。以燕麦秸秆为碳源诱导发酵镰刀菌Q7-31T,并在发酵第6天收酶。通过硫酸铵分级沉淀、Sephacryl S-100凝胶过滤层析和DEAE弱阴离子交换层析对粗酶液进行分离纯化得到金属蛋白酶,随后对其进行了酶学性质分析和串联质谱鉴定。结果显示,从镰刀菌Q7-31T菌株的粗酶液中分离得到一种蛋白酶FQME14,其相对分子质量为30 kD;串联质谱鉴定的结果表明FQME14属于M14家族;蛋白酶酶学性质研究表明：该蛋白酶的比酶活为2.85 U/mg,最适反应pH和温度分别为pH 7.0和40℃,在pH 5.0-pH 9.0的范围内具有很好的稳定性,蛋白酶在50℃以下稳定性很好,超过50℃酶活力降低。该蛋白酶对酪蛋白、卵清蛋白和牛血清白蛋白都有很高的降解能力。金属离子Mn²⁺和Co²⁺对酶活性有激活作用,Mg²⁺、Cu²⁺、Fe²⁺、K⁺、Ca²⁺和Na⁺对酶活性有不同程度的抑制作用。从镰刀菌Q7- 31T 粗酶液中分离纯化得到蛋白酶FQME14,并对其进行了酶学性质的研究和串联质谱鉴定,结果表明FQME14为M14家族蛋白酶,是一种新的金属蛋白酶。

关键词: 镰刀菌, 金属蛋白酶, M14家族

Abstract: The objectives of this work include：（1）to isolate the metalloproteinase from the fermented crude enzymatic solution of Fusarium sp. Q7-31T by oat straw,and then purify and identify them;（2）to study the enzymatic characteristics of protease for enriching and improving the basic data of metalloproteinases;and（3）to explore the role of cellulase in the degradation process of cellulose for understanding the mechanisms of cellulase degradation. Strain was cultured in liquid fermentation with oat straw as carbon source,and then crude enzyme solution was collected on the sixth day. The metalloprotease was purified using ammonium sulfate precipitation,Sephacry S-100 gel filtration chromatography and DEAE weak ion-exchange chromatography,its enzymatic properties were studied,and it was identified by MADIL-TOF-TOF. As results,the protease FQME14 isolated from the crude enzyme solution of Fusarium sp. Q7-31T was proved by SDS-PAGE to be a single band with a relative molecular mass of 30 kD. The MADIL-TOF-TOF results suggested that protease FQME14 belonged to M14 family. The characterization of purified protease showed that the specific activity of protease was 2.85 U/mg,its optimal pH and temperature for the enzyme activity were 7.0 and 40℃,respectively. The protease was very stable at pH 5.0-9.0 and temperature up to 50℃. The protease presented high degradation capacity to casein,ovalbumin and bovine serum albumin. The Co²⁺and Mn²⁺ were stimulators for protease,while Fe²⁺,Mg²⁺,K⁺,Ca²⁺,Zn²⁺and Na⁺ ions inhibited the activity of the protease at varied level. In conclusion,a protease from Fusarium sp. Q7-31T,named FQME14,was isolated,purified,identified and characterized. The enzymatic properties and MADIL-TOF-TOF results suggest that protease FQME14 belongs to M14 family,and is a new metalloprotease.

Key words: fusarium, metalloprotease, chromatography, identify, M14 family

秦日甜, 卢玉丽, 梁高丽, 谢占玲, 雷亚男. 镰刀菌Q7-31T金属蛋白酶FQME14的分离纯化及鉴定[J]. 生物技术通报, 2018, 34(4): 161-167.

QIN Ri-tian1LU Yu-li1LIANG Gao-li1XIE Zhan-ling1, 2LEI Ya-nan3. Isolation,Purification and Identification of Metalloprotease FQME14 from Fusarium sp. Q7-31T[J]. Biotechnology Bulletin, 2018, 34(4): 161-167.

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镰刀菌Q7-31T金属蛋白酶FQME14的分离纯化及鉴定

Isolation,Purification and Identification of Metalloprotease FQME14 from Fusarium sp. Q7-31T

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