Characterization of a novel alkali-stable and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda

Abstract

A novel gene (amyZ2) encoding an alkali-stable and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda (MCCC 1A01486) was cloned and expressed in Escherichia coli. The gene (1446bp) encodes a polypeptide with a predicted N-terminal signal peptide consisting of 24 amino acids and a mature α-amylase of 457 amino acids (AmyZ2). The estimated molecular mass of AmyZ2 was 50kDa by SDS-PAGE. AmyZ2 belongs to glycoside hydrolase family 13 and shares the highest identity (45%) with the characterized α-amylase from Pyrocoocus woesei (PDB id: 1MWO). The purified enzyme showed the maximum activity at 50°C, pH 7.0, and retained approximately 143 and 126% initial activity after 5 days of incubation (25°C) at pH 10.0 and 11.0, respectively, indicating its alkali stability. In addition, it had a salinity-tolerance range of 0–5M NaCl with a salt optimum at 2M (138% initial activity), retaining more than 130% initial activity at 0.5–2.5M and about 100% activity at 3.5M NaCl. AmyZ2 was relatively stable in 0–4M NaCl and its thermostability was significantly improved by the pre-incubation of enzyme solution with 2M NaCl at 50°C for 3h, which led to a 53.6-fold increase in the residual activity. The Km and kcat values of AmyZ2 were 11.71mgml−1 and 1449.43s−1, and the catalytic efficiency (kcat/Km) was 123.78mlmg−1s−1. AmyZ2 exhibited a specific activity of 1662.4U/mg toward soluble starch. The salt-tolerance and extreme alkali-stability of AmyZ2 suggests its potential applications in harsh industrial processes.