State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Extremophiles. 2014 Mar;18(2):271-81. doi: 10.1007/s00792-013-0614-9. Epub 2013 Dec 7.
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 (k cat/K m) 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.
从海洋细菌深红红螺菌（MCCC 1A01486）中克隆出一个编码冷活性和耐盐α-淀粉酶（AmyZ）的新基因，并在大肠杆菌中表达了该蛋白。该基因长 1785bp，编码一个 594 个氨基酸的α-淀粉酶，SDS-PAGE 估计其分子量为 66 kDa。该酶属于糖苷水解酶家族 13，与热解糖梭菌 R-47 中鉴定的 α-淀粉酶 TVA II 具有最高的同源性（25%）。重组α-淀粉酶在 35°C 和 pH 7.0 时表现出最大活性，在 0°C 时保留约 39%的活性。AmyZ 表现出极强的耐盐性，在 1.5 M NaCl 时活性最高，在 4 M NaCl 时活性仍高达 93%。在 35°C 和 pH 7.0 下，以可溶性淀粉为底物时，AmyZ 的催化效率（k cat/K m）从 115.51（无 NaCl）增加到 143.30 ml mg(-1) s(-1)（有 1.5 M NaCl）。此外，在 1.5 M NaCl 或 1 mM CaCl2 的存在下，酶的热稳定性显著提高。AmyZ 是少数既能耐受高盐度又能耐受低温的α-淀粉酶之一，使其成为基础和应用生物学研究的潜在候选者。
