同源重组整合vgb基因提升枯草芽孢杆菌亚麻脱胶能力  

Improvement of Flax Degumming by Chromosomal Integration of Vitreoscilla Hemoglobin Gene(vgb)in Bacillus subtilis

作　　者：张懿翔 丁若垚[1,2,3] 陈婷[1] 郁崇文[1,2,3] 张兴群[1] 

机构地区：[1]东华大学生态纺织教育部重点实验室,上海201620 [2]东华大学纺织面料技术教育部重点实验室,上海201620 [3]东华大学纺织学院,上海201620

出　　处：《中国麻业科学》2015年第2期87-94,共8页Plant Fiber Sciences in China

基　　金：中国博士后科学基金面上资助(2014M561386);国家麻类产业技术体系建设专项资金资助项目(CARS-19);中央高校基本科研业务专项资金(14D110524);大学生"小平科技创新团队"(麻类纤维生物脱胶技术产业化集成研发团队)

摘　　要：【目的】将透明颤菌血红蛋白(VHb)基因(vgb)在枯草芽孢杆菌中进行整合表达,以改善枯草芽孢杆菌的呼吸强度,通过高密度培养提高果胶酶的表达量,最终明显改善脱胶效果。【方法】构建含有vgb基因、amy E基因以及启动子P43的p SK-vgb(+)质粒,通过同源重组的方法,将vgb基因整合到枯草芽孢杆菌A5的染色体上,得到稳定表达透明颤菌血红蛋白的枯草芽孢杆菌A5-vgb(+)。分别采用PCR和CO差光谱分析法检测vgb基因的整合状态与血红蛋白的稳定表达,并通过发酵摇瓶试验研究VHb对菌株果胶酶产量的影响。将构建好的A5-vgb(+)菌株进行脱胶试验,通过XRD结晶度与电镜扫描分析鉴定脱胶效果。【结果】PCR与CO差光谱检测表明,vgb基因成功整合在A5菌株中,且正确表达的VHb具有生物学活性,在150 rpm,37℃,p H=8.0,16小时发酵的条件下,vgb基因的表达促使A5-vgb(+)菌株的果胶酶产量较不含vgb基因的菌株提升了29.5%。XRD与电镜扫描结果表明,构建的A5-vgb(+)菌株的脱胶效果相较于不含vgb的菌株,在结晶度上提高了0.55%,表面更光滑。【结论】将vgb基因整合至枯草芽孢杆菌A5菌株中,可提高A5菌株的脱胶效果。The gene Vitroscilla Hemoglobin was integrated and expressed in Bacillus subtilis to en-hance the effect of degumming by improving pectinase production.Promoter p43 and gene amyE,pSK -vgb （＋）were integrated into expression vector.Through the method of homologous recombination, gene vgb was integrated into Bacillus subtilis A5,and then got a vgb integrated expression strain named Bacillus subtilis A5 -vgb （＋）.The result was identified by PCR analysis and CO -spectral analysis.A5 -vgb （＋）strain was applied in flax degumming,shake flask experiment was used to study the func-tion of protein VHb in production of Pectinase.X -ray diffraction （XRD）and canning electron micro-scope （SEM）were uesd to analysis the effect of degumming.PCR analysis and CO -spectral analysis showed that the gene vgb was succeed in being integrated into Bacillus subtilis A5 and protein VHb had bioactivity.The results of shake flask experiment showed that the expression of gene vgb could improve Pectinase production,and the activity of Pectinase was 29.5% higher than that of the control under 150 rpm,37℃,pH =8.0,16 hours .The data of XRD and SEM showed that being compared with the strain without gene vgb,the degumming effect of A5 -vgb （＋）strain improved 0.55% in crystallinity, and had a smoother surface.To some extent,the VHb could enhance the degumming effect of Bacillus subtilis A5.

关 键 词：生物脱胶 果胶酶 枯草芽孢杆菌 VGB基因 同源重组 

分 类 号：Q789[生物学—分子生物学]