粉红黏帚霉糖化酶基因的克隆、表达及酶学性质分析

Cloning,Expression and Characterization of Glucoamylase from Gliocladium roseum

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 pGm to construct recombinant plasmid pGm-3440. The recombinant plasmid transformed into A. niger G1 strain, and amdS 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/mL. Further enzymatic analysis demonstrated that the optimum pH and temperature for the recombinant enzyme were 5.0 and 50℃, respectively. Meanwhile, it had poor thermal resistance, but promising pH stability.