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

作　　者：秦日甜 卢玉丽 梁高丽 谢占玲 雷亚男 QIN Ri-tian;LU Yu-li;LIANG Gao-li;XIE Zhan-ling;LEI Ya-nan(College of Ecological and Environment Engineering, University of Qinghai, Xining 810016;Key Laboratory Breeding Base for Innovation and Utilization of Plateau Crop Germplasm, Xining 810016;Northwest Agriculture and Forestry University, Yangling 712100)

机构地区：[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

出　　处：《生物技术通报》2018年第4期161-167,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31260021);青海省科技厅资助项目(2014-zj-903)

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 k D;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p H和温度分别为p H 7.0和40℃,在p H 5.0-p H 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Mn^(2+)和Co^(2+)对酶活性有激活作用,Mg^(2+)、Cu^(2+)、Fe^(2+)、K^+、Ca^(2+)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。The objectives of this work include ：（1）to isolate the metalloproteinase from the fermented crude enzymatic solution ofFusarium sp. Q7-31 T by oat straw,and then purify and identify them;（2）to study the enzymatic characteristics of protease for enrichingand improving the basic data of metalloproteinases;and（3）to explore the role of cellulase in the degradation process of cellulose forunderstanding the mechanisms of cellulase degradation. Strain was cultured in liquid fermentation with oat straw as carbon source,and thencrude 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 byMADIL-TOF-TOF. As results,the protease FQME14 isolated from the crude enzyme solution of Fusarium sp. Q7-31 T was proved by SDSPAGE to be a single band with a relative molecular mass of 30 k D. The MADIL-TOF-TOF results suggested that protease FQME14 belongedto M14 family. The characterization of purified protease showed that the specific activity of protease was 2.85 U/mg,its optimal p H andtemperature for the enzyme activity were 7.0 and 40℃,respectively. The protease was very stable at p H 5.0-9.0 and temperature up to 50℃.The protease presented high degradation capacity to casein,ovalbumin and bovine serum albumin. The Co-（2＋）and Mn-（2＋） were stimulators forprotease,while Fe-（2＋）,Mg-（2＋）,K-＋,Ca-（2＋）,Zn-（2＋）and Na＋ ions inhibited the activity of the protease at varied level. In conclusion,a protease fromFusarium sp. Q7-31 T,named FQME14,was isolated,purified,identified and characterized. The enzymatic properties and MADIL-TOFTOF results suggest that protease FQME14 belongs to M14 family,and is a new metalloprotease.

关 键 词：镰刀菌 金属蛋白酶 M14家族 

分 类 号：Q936[生物学—微生物学]