摘要
【目的】优化罗耳阿太菌源生淀粉糖化酶的发酵条件,并对其结构进行鉴定。【方法】以玉米淀粉和玉米黄浆作为发酵培养基的主要组分,对罗耳阿太菌进行发酵培养。固定接种量为5%(体积比),以罗耳阿太菌生淀粉糖化酶粗酶产量(U/mL)为评价指标,在单因素试验基础上,选择培养温度(℃)、培养时间(h)、摇床转速(r/min)为优化条件,采用响应面法对罗耳阿太菌源生淀粉糖化酶的发酵条件进行优化。利用扫描电子显微镜观察所得酶对玉米、小麦、木薯、红薯、豌豆生淀粉颗粒的水解效果,并用纳米级高效液相色谱与多级串联离子阱联用分析法(NanoLCESI-MS/MS),对所获得的蛋白(酶)进行结构鉴定。【结果】罗耳阿太菌源生淀粉糖化酶的最佳发酵条件为:培养温度38℃,培养时间81h,摇床转速215r/min,粗酶产量26.238 6U/mL。玉米、小麦、木薯、红薯、豌豆生淀粉经所得酶水解后结构被破坏。NanoLC-ESI-MS/MS分析结果与非冗余蛋白质数据库比对可知,所得酶为糖化酶,分子质量为61 966.69u。【结论】采用最优发酵条件获得的罗耳阿太菌生淀粉糖化酶可以水解玉米、小麦、木薯、红薯、豌豆生淀粉。
【Objective】This study was conducted to obtain the optimal fermentation conditions for raw starch-digesting glucoamylase fromAthelia rolfsii and identify its structure.【Method】The inoculation amount was fixed to 5%（volume ratio）using raw starch-digesting glucoamylase yield as indicator.The optimal fermentation conditions were determined using fermentation temperature（℃）,fermentation time（h）and shaking speed（r/min）as experimental factors based on single factor tests and response surface methodology.The hydrolysis effects of the enzyme on raw corn,wheat,cassava,sweet potato,and pea starch granules were observed using electron microscope,and the structure of protein（enzyme）was identified by Nanotechnology-High Performance Liquid Chromatography and multi-stage series ion trap MS（Nano LCESI-MS/MS）.【Result】The optimal fermentation conditions were temperature 38 ℃,fermentation time 81 h,and shaking speed 215 r/min.The primary enzyme production was 26.238 6 U/mL.The structures of corn,wheat,cassava,sweet potato,and pea starch granules hydrolyzed by enzyme were destroyed.The analysis results of Nano LC-ESI-MS/MS were compared with the non-redundant protein database（NR data-base,NCBI）to confirm that the protein（enzyme）fromAthelia rolfsii was glucoamylase with the molecular weight of 61 966.69 u.【Conclusion】The raw starch-digesting glucoamylase obtained from Athelia rolfsii under the optimal fermentation conditions could hydrolyze raw corn,wheat,cassava,sweet potato,and pea starch granules.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2017年第11期122-131,共10页
Journal of Northwest A&F University(Natural Science Edition)
基金
吉林省教育厅"十二五"科学技术研究项目(2015185)
关键词
罗耳阿太菌
生淀粉糖化酶
发酵条件优化
结构鉴定
Athelia rolfsii
raw starch-digesting glucoamylase
optimal fermentation conditions
structure identification
