构建转天冬酰胺酶基因菌株及其固体发酵产酶培养基优化

Construction of Trichoderma reesei Transasparaginase Strain and Optimization of Solid Fermentation Medium

构建一株转L-天冬酰胺酶基因的里氏木霉重组菌株,并采用固体发酵的方法研究其发酵产酶。首先以贝莱斯芽孢杆菌质粒为骨架重组天冬酰胺酶基因,验证其天冬酰胺酶的表达;随后探讨不同培养基配方(配方1和配方2)发酵产酶效果,通过单因素和正交试验优化其优势产酶培养基。结果表明:构建的里氏木霉可以正确表达天冬酰胺酶;在固体培养基配方1和配方2中,重组的里氏木霉菌株发酵产天冬酰胺酶分别为4.52 U/g和2.21 U/g,配方1产酶量远高于配方2。因此,以配方1作为优选配方,通过单因素及正交试验对其进一步优化。配方1中,当麦麸与玉米芯的比例为3∶1、初始含水量为1∶4.4、初始pH值为5.5时,该里氏木霉转L-天冬酰胺酶基因重组菌株发酵产酶活力为5.965 U/g,与原配方相比,优化后的固体培养基发酵产酶活力提高了24.22%。

A recombinant strain of Trichoderma reesei with L-asparaginase gene was constructed and its fermentation for enzyme production was studied by solid fermentation method.Firstly,the expression of asparaginase gene was verified by recombinant plasmid of Bacillus Velez.Then,the effect of enzyme production through fermentation by different medium formulations(formula 1 and formula 2) was investigated.The dominant enzyme producing medium was optimized through single factor and orthogonal test.The results showed that the constructed Trichoderma Reesei expressed asparaginase correctly.In solid medium formula 1and formula 2,the asparaginase production of the recombinant Trichoderma Reesei strains was 4.52 U/g and 2.21 U/g,respectively,indicating that the enzyme production by formula 1 was much higher than that by formula 2.Therefore,formula 1 was selected as the candidate formula,which was further optimized by single factor and orthogonal test.For formula 1,when the ratio of wheat bran to corn cob was 3∶1,the initial water content was 1∶4.4,and the initial pH was 5.5,the enzyme production activity of the L-asparaginase recombinant strain was 5.965 U/g,which was increased by 24.22% compared with the original formula.