米曲霉谷氨酰胺酶在黑曲霉中的重组表达与酶学特性

Recombinant Expression of Glutaminase from Aspergillus oryzae in Aspergillus niger and Its Enzymatic Properties

摘要:

该研究将米曲霉RIB40（Aspergillus oryzae RIB40）来源的谷氨酰胺酶（GahB）在黑曲霉（Aspergillus niger）宿主中分泌表达。基于该研究所建立表型鉴定板与孔板培养的通量鉴定筛选方法成功得到重组表达谷氨酰胺酶的转化子H-GahB，最高酶活力达到1.35 U/mL。为了增加目的基因在宿主中的拷贝数，采用CRISPR/Cas9基因组编辑技术重新构建高拷贝的谷氨酰胺酶表达菌株C-GahB，其酶活力提高到3.56 U/mL，约为H-GahB的2.64倍。进一步采用ARTP诱变技术对C-GahB进行传统诱变育种，获得了谷氨酰胺酶工程菌株A-GahB，其酶活力提升到4.16 U/mL，比出发菌株C-GahB的酶活力提高了0.17倍。纯化后的重组GahB的比酶活达到40.63 U/mg，最适温度为37 ℃，最适pH值为7.0，其在20~40 ℃及pH值5.5~8.0之间稳定性较好。K+对GahB的酶活力具有激活作用，Zn2+和Mn2+则对GahB的酶活力有较为强烈的抑制作用。当盐质量分数为18%时，GahB表现出35.38%的相对酶活力。该研究第一次在黑曲霉中实现了来源于米曲霉RIB40的谷氨酰胺酶GahB的重组分泌表达，为此后对米曲霉谷氨酰胺酶的研究提供了基础。

Abstract:

In this study, glutaminase (Gahb) from Aspergillus oryzae RIB40 was recombinantly expressed in Aspergillus niger. Based on the high-throughput screening method (phenotypic identification plate and pore plate culture) established in this study, the transformant of the recombinant glutaminase H-GahB was successfully obtained, and its highest enzyme activity was 1.35 U/mL. In order to increase the copy number of the target gene in the host, was reconstructed a high-copy glutaminase expressing strain C-GahB using the CRISPR/cas9 genome editing technology, and its enzyme activity increased to 3.56 U/mL (about 2.64 times that of H-GahB). Furthermore, C-GahB was subjected to traditional mutagenesis and breeding using ARTP mutagenesis technology, and an engineering strain of glutaminase, A-GahB, was obtained, with its enzyme activity upto 4.16 U/mL (0.17 times higher than that of the starting strain C-GahB). The specific enzyme activity of the purified recombinant GahB reached 40.63 U/mg, with the optimum temperature being 37 ℃ and the optimum pH being 7.0. The purified enzyme showed good stability from 20 ℃ to 40 ℃ and from pH 5.5 to 8.0. K+ could activate the enzyme activity of GahB, while Zn2+ and Mn2+ could strongly inhibit the enzyme activity of GahB. When the salt concentration was 18%, GahB showed a relative enzyme activity of 35.38%. In this study, glutaminase GahB from A.oryzae RIB40 was recombinantly expressed in A. niger for the first time, which provides a basis for subsequent investigations on the glutaminase from A. oryzae.