烟草源高产淀粉酶菌株的分离鉴定、发酵条件优化及固体淀粉酶制剂的制备
Isolation,identification and fermentation conditions optimization of high-yield amylase strains from tobacco and preparation of solid amylase preparations
孙志康 1李选文 1季嫱 1吴娜 1郑超 1吴晗 1叶长文 2黄阔 2郝捷1
作者信息
- 1. 内蒙古昆明卷烟有限责任公司,内蒙古呼和浩特 010020
- 2. 中国烟草总公司郑州烟草研究院,河南郑州 450001
- 折叠
摘要
该研究从分离自植烟土壤的具有产淀粉酶能力的20株细菌和17株真菌中筛选高产淀粉酶的菌株,结合形态学观察、生理生化试验和分子生物学技术对筛选菌株进行鉴定,通过单因素试验对其产酶条件优化,并通过澄清、分离纯化制备固体淀粉酶制剂,研究其稳定性.结果表明,筛选到1株高产淀粉酶的菌株MK10163,其被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其最佳产酶发酵条件为发酵时间44 h,发酵温度35℃,装液量50 mL/500 mL,摇床转速200 r/min,初始pH 6.0.在此条件下,淀粉酶活力为(224.63±7.34)U/mL,较优化前提高34.21％.通过1％氯化钙和1％磷酸氢二钠澄清和2倍体积的体积分数为95％乙醇醇沉,将沉淀冷冻干燥后得到固体淀粉酶制剂,其在储存60 d时仍具有91.12％的相对酶活,表明其具有较强的稳定性.
Abstract
The high-yield amylase strains were screened from 20 strains of bacteria and 17 strains of fungi with amylase production ability isolated from the tobacco-growing soil.The screened strains were identified by morphological observation,physiological and biochemical tests and molecular biology techniques.The enzyme production conditions were optimized by single factor experiments,and solid amylase preparations were prepared through clarification,separation,and purification,and their stability was studied.The results showed that a high-yield amylase strain MK10163 was screened and identified as Bacillus amyloliquefaciens.The optimal fermentation conditions for amylase production were fermentation time 44 h,temperature 35 ℃,liquid loading volume 50 ml/500 ml,shaking speed 200 r/min,initial pH 6.0.Under these conditions,the amylase activity was(224.63±7.34)U/ml,which was 34.21％higher than that before optimization.The solid amylase preparation was obtained by clarifying with 1％calcium chloride and 1％disodium hydrogen phosphate and alcohol precipitation with 2 times volume of 95％ethanol.After freeze-drying,solid amylase preparation was ob-tained.The relative enzyme activity of solid amylase preparation was still 91.12％after storage for 60 d,indicating that it had strong stability.
关键词
淀粉酶/筛选/鉴定/产酶条件优化/固体淀粉酶制剂/稳定性Key words
amylase/screen/identification/enzyme production conditions optimization/solid amylase preparation/stability引用本文复制引用
基金项目
中国烟草总公司重点研发项目(110202102019)
出版年
2024
NETL
NSTL
万方数据