Efficient production and biochemical characterization of a thermostable carboxypeptidase from Bacillus megaterium and its application on flavor improvement of soy isolate protein hydrolysates.

A carboxypeptidase M32 (CPM32) gene (cpm32) from Bacillus megaterium was cloned. CPM32 exhibited the highest sequence similarity of 79.3% with the same from Priestia veravalensis. CPM32 was fused with the signal peptides AmyQ (SPamyQ), AprE (SPaprE), NprB (SPnprB) in Bacillus subtilis WB600, and the highest CPM32 activity of 5320 U/mL was achieved when it was fused with SPamyQ. The purified enzyme (CPM32), 58.6 kDa, was most active at pH 8.0 and 60 °C, respectively, and it was stable over a broad pH range of 5.0–9.0 and an extensive temperature of 30–60 °C. Co2+ and Zn2+ could activate the enzyme activity by 600% and 334%, respectively. CPM32 could cleave most amino acid residues except Pro from chain's C-terminus. N-benzyloxycarbonyl-l-phenylalanyl-l-tyrosine (Z-Phe-Tyr) was the optimal substrate, for which CPM32 exhibited Km 0.6 mmol/L and kcat/Km 82.2 L/mmol s. Debittering soybean isolates using CPM32 (supplemented with 4% alkaline protease, w/w) resulted in a reduction in bitterness value of 47.6% to a commercially acceptable level. Applied to the hydrolysis of soy isolate protein, the bitterness of the hydrolyzates is significantly reduced and the flavor of the soy protein isolate hydrolysates is effectively improved. [ABSTRACT FROM AUTHOR]