A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning, heterologous expression and biochemical characterization
A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (kcat/Km) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg−1 s−1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl2. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.
Keywordsα-Amylase Cold active Salt tolerant Thermostability Zunongwangia profunda
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