维氏气单胞菌噬菌体裂解酶在毕赤酵母中的表达及抑菌活性分析OA

Aeromonas veronii is a common Gram-negative pathogen in aquaculture that is difficult to kill by a variety of antibiotics and antimicrobial agents due to the shielding effect of the self-produced extracellular membrane.In this study,an amino acid sequence with the N-terminal catalytic structural domain and C-terminal amphiphilic α-helical structural motif unique to the structure of Gram-negative bacterial phage endolysin was screened from the PhageScope database by bioinformatic analysis.To determine whether this amino acid sequence has antimicrobial activity,the encoding gene was optimized and synthesized according to the yeast codon and then transformed into Pichia pastoris GS115 for constitutive secretion expression,yielding recombinant A.veronii phage endolysin(LYS2)with a secretion expression level of 140 mg·L-1.The supernatant of the fermentation broth containing this endolysin exhibited significant bacteriostatic activity against A.veronii Hm091.The minimum in vitro inhibitory concentration of LYS2 against A.veronii Hm091 was 3 µg·mL-1;the optimal in vitro inhibitory conditions were 42℃and pH 4～10;the activity was stable at 4～50℃and pH 4～7;and the endolysin showed good tolerance to common metal ions and proteases.An Aeromonas veronii phage-derived endolysin with significant in vitro antimicrobial activity was experimentally verified,which provided a new antimicrobial protein with potential application for the prevention and treatment of A.veronii experimentally verified in aquaculture,and also laid a theoretical foundation for the production and application of this antimicrobial protein.