黑曲霉糖化酶基因克隆及在酿酒酵母中的表达
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摘要
为了以酿酒酵母S78为宿主菌异源高效表达糖化酶基因,进一步扩大糖化酶基因在工业生产上的应用。运用RT-PCR法从黑曲霉中克隆得到糖化酶基因(glaA)cDNA,去除其信号肽编码区后的序列重组到酵母表达载体pVT102U/αADH1强启动子下游,并与α因子分泌肽信号序列融合。用PEG/LiAc法将构建的重组表达载体转入酿酒酵母S78菌株,筛选出的转化菌点种到可溶性淀粉平板上培养,用碘染法鉴定重组基因的表达情况。鉴定出了典型水解圈的酵母转化子,转化子接种到YPD培养基中摇瓶培养后,取发酵上清液经SDS-PAGE检测到分子量大小约为80 kDa的目标蛋白带,上清液用DNS法检测其淀粉酶最高酶活为28.86 IU/m L。表明糖化酶基因已在酿酒酵母S78中成功表达并能有效分泌到细胞外。
In order to express the glucoamylase gene efficiently with Saccharomyces cerevisiae S78 as a host strain and to further expand the application of gla A gene in industrial production.The glaA gene was cloned from Aspergillus niger by RT-PCR,and the sequence removed the natural signal peptide coding region was recombined into the downstream of the yeast expression vector pVT102 U/α ADH1 strong promoter and fused with the sequence ofα factor secretion peptide signal.The recombinant expression vector was transformed into Saccharomyces cerevisiae S78 strain by PEG/LiAc method.The selected transformants were spread on soluble starch plates and the expression of recombinant genes was identified by iodine staining.The yeast transformants of the typical hydrolytic circles were identified and the transformants were inoculated into YPD medium.The molecular weight of the target protein about80 k Da was detected by SDS-PAGE.The supernatant was detected by DNS method and the highest activity of enzyme was 28.86 IU/m L in normal bottle fermentation.It indicated that the glucoamylase gene had been successfully expressed in Saccharomyces cerevisiae S78 and the enzyme could be effectively secreted.
In order to express the glucoamylase gene efficiently with Saccharomyces cerevisiae S78 as a host strain and to further expand the application of gla A gene in industrial production.The glaA gene was cloned from Aspergillus niger by RT-PCR,and the sequence removed the natural signal peptide coding region was recombined into the downstream of the yeast expression vector pVT102 U/α ADH1 strong promoter and fused with the sequence ofα factor secretion peptide signal.The recombinant expression vector was transformed into Saccharomyces cerevisiae S78 strain by PEG/LiAc method.The selected transformants were spread on soluble starch plates and the expression of recombinant genes was identified by iodine staining.The yeast transformants of the typical hydrolytic circles were identified and the transformants were inoculated into YPD medium.The molecular weight of the target protein about80 k Da was detected by SDS-PAGE.The supernatant was detected by DNS method and the highest activity of enzyme was 28.86 IU/m L in normal bottle fermentation.It indicated that the glucoamylase gene had been successfully expressed in Saccharomyces cerevisiae S78 and the enzyme could be effectively secreted.
引文
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[14]张贺迎,武金霞.耐热糖化酶的分离纯化及部分性质[J].食品研究与开发,2007,28(9):10.
[15]林艳,马莹莹,张宿义,等.米曲糖化酶活力测定方法的比较研究[J].酿酒科技,2015,161(6):23-27.
[16]杨丽娟,余少文.黑曲霉高产糖化酶的分子水平研究方法概论[J].食品研究与开发,2016,37(8):204-208.
[17]王海燕,秦浚川,王敖全,等.黑曲霉酸性α曲淀粉酶基因和糖化酶基因对工业酒精酵母的整合及其共表达[J].微生物学报,2004,44(4):483-486.
[18]施慧琳,孙靖淳,张荣凯,等.电子嗅在线反馈控制毕赤酵母糖化酶发酵过程中甲醇浓度新方法的应用[J].中国生物工程杂志,2016,36(3):68-76.
[19]汤斌,钱鹏.黑曲霉糖化酶基因在毕赤酵母中的克隆和表达[J].食品与发酵工业,2012,38(6):53-56.
[20]唐国敏,钟丽婵,杨开宇,等.具有糖化酶活性的工业啤酒酵母菌的构建及其发酵特性[J].生物工程学报,1996,12(4):489-491.
[2]Acourene S,Ammouche A.Optimization of ethanol,citric acid,and alpha-amylase production from date wastes by strains of Saccharomyces cerevisiae,Aspergillus niger,and Candida guilliermondii[J].Journal of Industrial Microbiology&Biotechnology,2012,39(5):759-766.
[3]郭彦言,王红蕾,左小明,等.糖化酵母糖化酶基因在酿酒酵母中的克隆与表达[J].安徽农业科学,2015,38(22):8-10.
[4]Favaro L,Jooste T,Basaglia M,et al.Codon-optimized glucoamylases GAI of Aspergillus awamori improves starch utilization in an industrial yeast[J].Applied Microbiology and Biotechnology,2012,95(4):957-968.
[5]徐荣燕,张亚波,谢晨,等.疏绵状嗜热丝孢菌糖化酶基因(gla)的克隆及在毕赤酵母中的表达[J].农业生物技术学报,2010,18(2):362-367.
[6]姚婷婷,王衍敏,顾建龙,等.携多拷贝gla A的重组黑曲霉过量合成糖化酶的研究[J].生物工程学报,2006,22(4):9.
[7]曹慕琛,徐健勇,罗立超,等.黑曲霉糖化酶基因的克隆及其在毕赤酵母X33中的表达[J].安徽农业科学,2011,39(14):8226-8230.
[8]王强,徐义兵,郭春和,等.黑曲霉糖化酶基因在毕赤酵母X33中的高效表达[J].中国畜牧兽医,2012,39(4):21-24.
[9]Sidra,Pervez,Nadir,et al.Phenotypic and molecular characterization of Aspergillus species for the production of starch-saccharifying amyloglucosidase[J].Annals of Microbiology,2015,65(4):2287-2291.
[10]张水龙,陈东,曹树威,等.黑曲霉木聚糖酶基因xyn B的克隆及在酿酒酵母中的表达[J].广西科学,2013,20(2):148-151.
[11]林志楷,叶冰莹,潘海芳,等.p VT102U/α-bgln重组真核表达质粒的构建和鉴定[J].福建师范大学学报:自然科学版,2007,23(2):17.
[12]杜济良,赵洪亮,薛冲,等.不同信号肽对胞外β-(1,3)-葡聚糖酶在巴斯德毕赤酵母中表达的影响[J].生物技术通讯,2010,21(6):12.
[13]崔铁忠,卢康荣,李银霞,等.酿酒酵母遗传缺陷型菌株化学转化方法研究[J].现代生物医学进展,2016,16(8):1420-1423.
[14]张贺迎,武金霞.耐热糖化酶的分离纯化及部分性质[J].食品研究与开发,2007,28(9):10.
[15]林艳,马莹莹,张宿义,等.米曲糖化酶活力测定方法的比较研究[J].酿酒科技,2015,161(6):23-27.
[16]杨丽娟,余少文.黑曲霉高产糖化酶的分子水平研究方法概论[J].食品研究与开发,2016,37(8):204-208.
[17]王海燕,秦浚川,王敖全,等.黑曲霉酸性α曲淀粉酶基因和糖化酶基因对工业酒精酵母的整合及其共表达[J].微生物学报,2004,44(4):483-486.
[18]施慧琳,孙靖淳,张荣凯,等.电子嗅在线反馈控制毕赤酵母糖化酶发酵过程中甲醇浓度新方法的应用[J].中国生物工程杂志,2016,36(3):68-76.
[19]汤斌,钱鹏.黑曲霉糖化酶基因在毕赤酵母中的克隆和表达[J].食品与发酵工业,2012,38(6):53-56.
[20]唐国敏,钟丽婵,杨开宇,等.具有糖化酶活性的工业啤酒酵母菌的构建及其发酵特性[J].生物工程学报,1996,12(4):489-491.