葡萄穗霉中β-甘露聚糖酶基因在黑曲霉中的表达及酶学性质

Expression in Aspergillus niger and characterization of β-mannanases from Stachybotrys chartarum

摘要:

[目的] 以黑曲霉（Aspergillus niger）为宿主菌来表达葡萄穗霉（Stachybotrys chartarum）中的β-甘露聚糖酶（β-mannanase）基因。[方法] 通过对葡萄穗霉全基因组进行比对分析，获得编码β-甘露聚糖酶基因s16942和s331的序列信息，通过设计引物，PCR扩增得到基因s16942和s331，连接到载体pGm上，并转化到黑曲霉中，获得的转化子经过amdS二筛平板复筛，测序验证，得到高效表达此基因的工程菌株G1-pGm-s16942和G1-pGm-s331。[结果] SDS-PAGE检测结果显示，G1-pGm-s16942和G1-pGm-s331表达的蛋白分子量大小分别约为48 kDa和60 kDa，且阴性对照中没有此条带。粗酶液的酶学性质表明，G1-pGm-s16942表达的β-甘露聚糖酶的最适反应温度为60℃，最适反应pH为7，粗酶液的酶活最高达521 U/mL；G1-pGm-s331表达的β-甘露聚糖酶的最适反应温度为50℃，最适反应pH为7，粗酶液的酶活最高达84 U/mL。[结论] 本研究首次将葡萄穗霉的β-甘露聚糖酶基因转化到黑曲霉中并成功表达，并且具有较高的活性。

Abstract:

[Objective] Using Aspergillus niger as host to express β-mannanases from Stachybotrys chartarum.[Methods] Through sequence analysis of Stachybotrys chartarum genome, two β-mannanase genes (s16942 and s331) were identified. The primers were designed based on the DNA sequence and the β-mannanase genes (s16942 and s331) were obtained, and then inserted to the vector pGm. The expression plasmids were transferred into Aspergillus niger. β-mannanase producing strains (G1-pGm-s16942 and G1-pGm-s331) were isolated after screening several transformants using amdS selection plates and confirmed by PCR fragment sequencing.[Results] The molecular weight of the enzymes from G1-pGm-s16942 and G1-pGm-s331 were about 48 kDa and 60 kDa respectively by SDS-PAGE gel analysis, and the recombinant proteins did not present in the negative control. Assays of enzymatic property using the crude enzyme preparations indicated that the enzyme from G1-pGm-s16942 exhibited maximum activity (521 U/mL) under the optimum conditions (pH 7 and 60℃); the enzyme from G1-pGm-s331 exhibited maximum activity (84 U/mL) under the optimum conditions (pH 7 and 50℃).[Conclusion] This was the first study of the heterologous expression of the β-mannanase genes from Stachybotrys chartarum in Aspergillus niger host and the β-mannanase genes could be expressed successfully with high activities and protein titers.