Heterologous expression of Bacillus licheniformis E7 aminopeptidase and its application for efficient proteolysis

Abstract:

[Objective] Aminopeptidase gene pepN from Bacillus licheniformis E7 was cloned into Escherichia coli BL21 for heterologous expression of recombinant aminopeptidase EcPepN. The enzymatic properties of the recombinant EcPepN were studied. The combination of EcPepN with alkaline protease efficiently improved the hydrolysis of soybean protein and casein and released multitudinous peptides and amino acids. [Methods] With the genomic DNA of E7 as template, the aminopeptidase gene pepN was cloned into the expression vector pET28a to construct recombinant expression plasmid pET28-pepN, which was transformed into the competent cells of E. coli BL21. Through DNA sequencing verification, the recombinant E. coli BL21/pET28-pepN was obtained. The recombinant enzyme was purified by nickel-affinity chromatography. The pH and temperature stability, half-life, and NaCl tolerance of the pure enzyme were tested. With the commercial aminopeptidase combined with alkaline protease as a control, the combination of EcPepN and alkaline protease was used to hydrolyze soybean protein and casein, and the constituents of active small peptides and free amino acids in the hydrolysate were determined. [Results] The recombinant EcPepN was expressed in E. coli BL21. SDS-PAGE analysis showed that the purified target protein presented a single band at about 52 kDa. Among the seven substrates tested, the preferred substrate of EcPepN was Ala-pNA. Under optimal conditions (pH 9.0 and 50. After 22 days of incubation in 3.0 mol/L NaCl, the residual activity was more than 55%. When the recombinant aminopeptidase EcPepN worked synergistically with alkaline protease to hydrolyze soybean protein and casein, its hydrolysis efficiency was similar to that of the combination of commercial aminopeptidase and alkaline protease. More than 70% of the constituents of hydrolysate were peptides with molecular weight of less than 500 Da and it also contained more than 2 000 mg/L of free amino acids. [Conclusion] The recombinant strain containing aminopeptidase EcPepN was developed. The recombinant EcPepN shows pH and temperature stability and strong tolerance to NaCl of high concentration. It can efficiently catalyze the hydrolysis of soybean protein and casein and release small molecular active peptides and abundant free amino acids. This study provides important methods for enhancing deep protein hydrolysis and improving the values of protein-rich bioresources.