Characterization and heterologous expression of a novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from Lysinibacillus sphaericus

Abstract

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co²⁺-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 μM Co²⁺, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 10⁴ vs. 3.37 × 10⁴ U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. KEY POINTS: • A novel Co²⁺-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. • The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. • B. subtilis- and C. glutamicum-based expression systems are built to promote secretory (soluble) Amp0279 expression.

Keywords: Bacillus subtilis; Corynebacterium glutamicum; Leucyl aminopeptidase; Lysinibacillus sphaericus; Secretory expression.