Cloning,expression and activity identification of aminopeptidase peptidase A from Klebsiella pneu-moniae
Objective To amplify and prokaryotically express a probable peptidase A(PepA)from Klebsiella pneumoniae(KP),and identify its enzymatic activity characteristics.Methods According to the KP PepA sequence published by the(US)National Center for Biotechnology Information(NCBI),the PepA open reading frame(ORF)was amplified by polymerase chain reaction(PCR),then cloned into the pET-30a vector,and transformed into Escherichia coli BL21(DE3),induced by isopropyl β-D-thiogalacto-side(IPTG),and the recombinant protein rPepA was obtained after Ni+chromatography gel purification.Using Leucine-p-Nitroaniline(Leu-pNA)as the substrate,the hydrolysis ability of KP PepA was deter-mined,and the effects of different reaction temperatures and pH conditions on the hydrolysis reaction were explored.Different divalent metal ions were added to the reaction system to observe the effect of each ion on the activity of KP PepA aminopeptidase.The t test was used for comparison between groups.Results The KP PepA gene with a length of 1 527 bp was amplified,and the soluble recombinant KP PepA protein was obtained with a size of 56.1 × 103,consistent with the predicted sizes.The pNA concentration(reaction product)in the group with KP PepA addition was significantly higher than that in the group without KP PepA[(205.00±5.00)vs.(8.00±2.00)μmol/L,t=63.36,P＜0.01],indicating that KP PepA can hydrolyze Leu-pNA.The optimal temperature for hydrolyzing Leu-pNA by KP PepA was 37℃,and the op-timal pH value was 8.0.Both Co2+and Mn2+can increase the activity of KP PepA enzyme,among which Co2+had the strongest activation effect.The relative enzyme activity of the group adding Co2+was signifi-cantly higher than that of the group without metal ions(181.70±7.64 vs.95.00±0.00,t=16.44,P＜0.01);Zn2+and Fe2+can inhibit KP PepA enzyme activity,among which Zn2+had the strongest inhibito-ry effect.The relative enzyme activity of the group adding Zn2+was significantly lower than that of the group without metal ions(44.67±8.97 vs.95.00±0.00,t=8.49,P＜0.01).Conclusion PepA an-notated in Klebsiella pneumoniae possesses aminopeptidase activity in vitro.
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