粉红黏帚霉糖化酶基因的克隆、表达及酶学性质分析
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摘要
用黑曲霉(Aspergillusniger)G1为宿主菌表达粉红黏帚霉(Gliocladiumroseum)糖化酶。运用PCR技术从粉红粘帚霉中扩增得到一个疑似糖化酶基因序列(约1.8 kb),并将其连接到载体p Gm上组建成重组质粒p Gm-3440。将重组质粒转化到黑曲霉G1菌株中,经amd S筛选及PCR验证获得表达糖化酶的黑曲霉重组工程菌。重组菌的发酵结果显示,糖化酶基因在黑曲霉中得到了分泌表达,用国标法(QB/T 1803-1993)测得重组糖化酶活性达292 U/m L。进一步对其酶学性质进行分析发现,该重组酶最适温度和p H分别为50℃和5.0,该酶的耐热性较差,p H稳定性较好。
Glucoamylase from Gliocladium roseum was secretively expressed in Aspergillus niger G1 strain. A putative glucoamylase gene(about 1. 8 kb)was amplified by PCR using genomic DNA of G. roseum as template. The amplified products were ligated into the clone vector p Gm to construct recombinant plasmid p Gm-3440. The recombinant plasmid transformed into A. niger G1 strain, and amd S screening and PCR validation confirmed that an engineering Aspergillus strain of expressing glucoamylase was obtained. Fermentation of recombinant strain indicated that secretive expression of the glucoamylase gene was available in A. niger, and its enzyme activity was measured by method defined in national standard(QB/T 1803-1993), and it reached 292 U/m L. Further enzymatic analysis demonstrated that the optimum p H and temperature for the recombinant enzyme were 5. 0 and 50℃, respectively. Meanwhile, it had poor thermal resistance, but promising p H stability.
Glucoamylase from Gliocladium roseum was secretively expressed in Aspergillus niger G1 strain. A putative glucoamylase gene(about 1. 8 kb)was amplified by PCR using genomic DNA of G. roseum as template. The amplified products were ligated into the clone vector p Gm to construct recombinant plasmid p Gm-3440. The recombinant plasmid transformed into A. niger G1 strain, and amd S screening and PCR validation confirmed that an engineering Aspergillus strain of expressing glucoamylase was obtained. Fermentation of recombinant strain indicated that secretive expression of the glucoamylase gene was available in A. niger, and its enzyme activity was measured by method defined in national standard(QB/T 1803-1993), and it reached 292 U/m L. Further enzymatic analysis demonstrated that the optimum p H and temperature for the recombinant enzyme were 5. 0 and 50℃, respectively. Meanwhile, it had poor thermal resistance, but promising p H stability.
引文
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[2]Pandey A,Nigam P,Soccol CR,et al.Advances in microbial amylases[J].Biotechnol Appl Biochem,2000,31(2):135-152.
[3]Kaur P,Satyanarayana T.Production and starch saccharification by a thermostable,and neutral glucoamylase of a thermophilic mould Thermomucor indicae-seudaticae[J].World Journal of Microbiology&Biotechnology,2004,20(4):419-425.
[4]马丽娜,陈喜文,陈德富,等.曲霉属糖化酶基因的克隆及其在毕赤酵母中的表达[J].南开大学学报,2007,5(40):85-90.
[5]刘喜凤,王肇悦,张博润.糖化酶及糖化酶基因在酿酒酵母中表达的研究进展[J].酿酒,2007,34(4):73-76.
[6]梁新红,孙俊良,唐玉,等.黑曲霉糖化酶分离纯化与酶学性质研究[J].河南科技学院学报,2011,39(4):24-27.
[7]陈启和,何国庆.糖化酶及其基因研究进展[J].微生物学杂志,2000,20(4):46-50.
[8]Mertens JA,Skory CD.Isolation and characterization of two genes that encode active glucoamylase without a starch binding domain from Rhizopusoryzae[J].Curr Microbiol,2007,54(6):462-466.
[9]姚婷婷,王衍敏,顾建龙.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):567-571.
[10]康东亮.高产率糖化酶菌株的诱变选育[J].河南工业大学学报:自然科学版,2006,27(4):43-46.
[11]王家东,张茜,侯红萍.介孔分子筛MCM-41固定糖化酶的研究[J].中国酿造,2012,31(1):116-119.
[12]Durand H,Clanet M,Tiraby G.Genetic improvement of scale cellulase production[J].Enzyme and Microbial Technology,1988,10(6):341-346.
[13]Van den Hombergh JP,Van de Vondervoort PJ,Fraissinet-Tachet L,et al.Aspergillus as ahost for heterologous protein production:the problem of proteases[J].Trends Biotechnology,1997,15(7):256-263.
[14]Pavezzi FC,Carneiro AA,Martins DA,et al.Influence of different substatesonthe production of a mutant thermostableglucoamylase in submerged fermentation[J].Appl Bicohem Biotechnol,2011,163(1):14-24.
[15]何迎春,高必达.立枯丝核菌的生物防治[J].中国生物防治,2000,16(1):31-34.
[16]莫明和,董林茜,迟胜起,等.立枯丝核菌重寄生真菌的筛选[J].植物病理学报,2002,32(1):84-88.
[17]Vaili NG.Biological seed treatment of corn with mycopathogenic fungi[J].Journal of Phytopathology,1992,134(4):313-323.
[18]赵颖,巴再华,张春艳.双向选择标记amd S在黑曲霉中自发突变及amd S-检测[J].济宁医学院学报,2008,31(4):283-285.
[19]Duan YT,Wang Z,Wu W,et al.Polycistronic expression of human platelet factor 4 with heparin-neutralizing activity in Escherichia coli[J].Bioscience,Biotechnology,And Biochemistry,2012,76(10):1855-1860.
[20]李昆,李松,牛丹丹,等.黑曲霉糖化酶基因启动子功能鉴定[J].微生物学杂志,2008,28(6):5-9.
[21]贺莹,吕利华,张婵,等.黑曲霉IN7-31产糖化酶的液态发酵参数与技术优化研究[J].中国酿造,2011,1:48-51.
[22]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.