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