Abstract:Single- and double-copy expression vectors of lysozyme-containing glucoamylase signal peptide, controlled by promoter Pna II, were constructed following codon optimization of the GH25 lysozyme gene derived from Acremonium alcalophilum. The double-copy expression vector and CRISPR/cas9 techniques were applied. Lysozyme expression strains with high enzyme activity levels were obtained through PEG-mediated transformation of Aspergillus niger host HL-1. The lysozyme activity of the recombinant LyAa-C strain reached 2 291.37 U/mL, which is 2.63 times that of the LyAa-F strain transformed by double-copy expression vector only (869.74 U/mL) and 6.39 times that of the LyAa-T strain transformed by traditional single-copy expression vector only (358.41 U/mL). The recombinant lysozyme LyAa was purified with 6×His-tagging through nickel affinity chromatography, and its enzymatic properties were analyzed. The purified lysozyme LyAa had a size of 25.0 ku, an optimal pH value of 4.0, an optimal temperature of 30 ℃, and exhibited good pepsin stability. In conclusion, this study successfully optimized and improved the lysozyme expression in Aspergillus niger using double-copy vector and CRISPR systems.
