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柳梅梅,谢敏,杨燕芳,刘滔滔,杜丽琴,梁智群,韦宇拓.绿色糖单孢菌麦芽糖α-淀粉酶基因在枯草芽孢杆菌中的高效分泌表达[J].广西科学,2016,23(1):12-18. [点击复制]
- LIU Meimei,XIE Min,YANG Yanfang,LIU Taotao,DU Liqin,LIANG Zhiqun,WEI Yutuo.High Level Secretion Expression of Maltogenic α-amylase from Saccharomonospora viridis in Bacillus subtilis[J].Guangxi Sciences,2016,23(1):12-18. [点击复制]
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| 绿色糖单孢菌麦芽糖α-淀粉酶基因在枯草芽孢杆菌中的高效分泌表达 |
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柳梅梅1,2, 谢敏1,2, 杨燕芳1,2, 刘滔滔1,2, 杜丽琴1,2, 梁智群1,2, 韦宇拓1,2
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| (1.广西大学生命科学与技术学院, 广西南宁 530005;2.亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530005) |
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| 摘要: |
| [目的]构建高产麦芽糖α-淀粉酶的工程菌株并实现高效分泌表达。[方法]PCR扩增麦芽糖α-淀粉酶基因sva,再与大肠杆菌-枯草芽孢杆菌麦芽糖诱导型穿梭载体pHCMCO4-Pglv连接,构建重组质粒pHCMCO4-Pglv-sva并转入枯草芽孢杆菌进行表达,对重组酶进行SDS-PAGE分析,然后对重组菌株的生长及发酵条件进行优化。[结果]成功构建重组质粒pHCMCO4-Pglv-sva并在枯草芽孢杆菌中实现分泌表达,SDS-PAGE分析发现,在55kDa处得到特异性蛋白条带。单因素试验结果显示,重组菌的最适诱导温度为35℃,最适接种量为4%(V/V),最适装液量为30mL。正交实验结果显示,重组菌的最佳发酵条件组合是诱导温度37℃、接种量5%(V/V)、装液量25mL,在此条件下发酵液粗酶活力达到257.3698U/mL。装液量对重组菌的产酶量影响显著。[结论]成功构建能高效分泌表达麦芽糖α-淀粉酶的枯草芽孢杆菌工程菌株,经发酵条件优化后,重组酶产量显著提高10倍左右。 |
| 关键词: 麦芽糖α-淀粉酶 枯草芽孢杆菌 高效表达 优化 |
| DOI：10.13656/j.cnki.gxkx.20160315.008 |
| 投稿时间：2015-11-16修订日期：2016-02-28 |
| 基金项目:国家自然科学基金项目(31160311)和广西自然科学基金项目(2012GXNSFAA053051)资助。 |
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| High Level Secretion Expression of Maltogenic α-amylase from Saccharomonospora viridis in Bacillus subtilis |
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LIU Meimei1,2, XIE Min1,2, YANG Yanfang1,2, LIU Taotao1,2, DU Liqin1,2, LIANG Zhiqun1,2, WEI Yutuo1,2
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| (1.College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;2.State Key Laboratory for Conservation and Utilizaiton of Subtropical Agro-bioresources, Nanning, Guangxi, 530005, China) |
| Abstract: |
| [Objective] Engineering strains for high yield of maltose alpha amylase were constructed in order to implement efficient secrection expression.[Methods] The gene that had a length of 1 440 bp was amplified by PCR.The recombinant plasmid pHCMCO4-Pglv-sva was constructed and transformed into Bacillus subtilis.The recombinant protein was analyzed by SDSPAGE, and the growing conditions of recombinant strains were further optimized.[Results] Therecombinant strain pHCMCO4-Pglv-sva was successfully constructed and expressed in Bacillus subtilis.The molecular weight of the recombinant protein was approximately 55 kDa by SDS-PAGE analyis.Single factor optimization of fermentation conditions revealed that the recombinant strain had the optimal induction temperature at 35℃, the optimal inoculated quantity of 4% (V/V)and the optimal loading fluid amount of 30 mL.Orthogonal experiment resultsshowed that the optimum fermentation conditions were induction temperature at 37℃, quantity of 5% (V/V)and fluid volume 25 mL, under which the enzyme activity of recombinant maltose alpha amylase reached to 257.3698U/mL.Among them, liquid loading quantity had significant effects on enzyme activity of maltose alpha amylase.[Conclusion] The recombinant plasmid pHCMCO4-Pglv-sva was successfully built and maltose alpha amylase was highly increased to ten times after optimizing fermentation condition. |
| Key words: maltose α-amylase Bacillus subtilis high efficient expression optimization |
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