Abstract: Considering that the industrial production of food-grade leucine aminopeptidase is low in China, in this study, we investigated the recombinant expression of the five leucine aminopeptidase encoding genes (lapA, lap1O, lap2, lap1S and lap1N) from Aspergillus oryzae, A. sojae and A. niger in aconidial A. niger strain HL-1 with a low-background of protein secretion. The encoding region was improved via signal peptide replacement, and the expression vector was constructed with the hybrid promoter PnaII and the auxotroph marker pyrG. The homologous recombination method combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was used to improve gene integration efficiency and protein expression. The LapA activity of the recombinant strain was 11 701.2 U/mL, about 4.7 times higher than that (only 2 476.0 U/mL) obtained without using CRISPR. The recombinant enzyme was purified using the 6 × His tag for enzymatic characterization. The molecular mass of the purified LapA was shown to be 35.0 kDa. The optimum reaction temperature and pH for the recombinant LapA were 65 ℃ and 8.5, respectively. Overall, the recombinant LapA was successfully overexpressed in A. niger.

Key words: leucine aminopeptidase; Aspergillus niger; high-level expression; enzymatic characteristics