关键词: 抗菌肽, moricin, 毕赤酵母, 高效表达, 抑菌活性, 生化特性

Abstract: The excessive use of antibiotics has brought about many adverse effects to natural ecology. Finding suitable alternatives to antibiotics to reduce the use of antibiotics is becoming a research hotspot. The aim of this study was to highly express the antimicrobial peptide moricin derived from the diamondback moth Plutella xylostella in Pichia pastoris, and study its antibacterial properties and biochemical properties. The mature peptide gene mor-3 of moricin from Plutella xylostella (L.) was amplified by PCR, and then cloned into the pGAPZα A vector, and an expression plasmid of pGAPZα A-mor3 was constructed. The linearized plasmid pGAPZα A-mor3 was transformed into P. pastoris SMD1168 by electroporation, and the high copy recombinant transformants were screened out by using high concentration of Zeocin. The molecular mass of the recombinant protein expressed in P. pastoris SMD1168 was approximately 5 kD, corresponding to the theoretical molecular mass of this target peptide. Up to 248.98 mg/L recombinant protein in the supernatant after 60 h fermentation was obtained. The recombinant protein in the supernatant showed obvious antibacterial activity against several microorganisms detected by an improved agar diffusion method, including Escherichia coli and Staphylococcus aureus. The minimum inhibitory concentration (MIC) against E. coli CMCC44103 was 6.25 μg/mL, while that against S. aureus ATCC25923 was only 124.49 μg/mL. It showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria, especially Gram-negative bacteria. In addition, the recombinant protein also had good thermal stability, and strong resistance to acid and alkali. In particular, the antimicrobial activity was maintained in an acidic environment, and so it had good adaptability to the animal intestinal environment. Due to these unique antibacterial properties and biochemical characteristics it has a broad development and application prospect with good social and environmental benefits.

Key words: antimicrobial peptides, moricin, Pichia pastoris, efficient expression, antimicrobial activity, biochemical characteristics