School of Marine Science and Technology, Huaihai Institute of Technology, Lianyungang, Jiangsu 222005, China.
Protein J. 2010 Nov;29(8):591-7. doi: 10.1007/s10930-010-9290-0.
A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7-8.5 at 30 °C for 1 h. ParAmy was activated by Mn(2+), K(+) and Na(+), and inhibited by Hg(2+), Cu(2+), and Fe(3+). N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K (m) and V (max) values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.
从北极假交替单胞菌 GS230 中克隆、测序并表达了一种冷适应的α-淀粉酶（ParAmy）基因，该基因作为 N 端 His 标签融合蛋白在大肠杆菌中表达。通过 DEAE-琼脂糖离子交换层析和 Ni 亲和层析对重组蛋白进行了生产和纯化。SDS-PAGE 测定 ParAmy 的分子量约为 55 kDa。ParAmy 的最适活性温度为 30°C，在 0°C 时具有 34.5%的最大活性，其活性在 40°C 以上急剧下降。ParAmy 在 30°C、pH 7-8.5 范围内稳定 1 h。ParAmy 被 Mn(2+)、K(+) 和 Na(+)激活，被 Hg(2+)、Cu(2+) 和 Fe(3+)抑制。N-溴代丁二酰亚胺对酶活性有显著的抑制作用。α-淀粉酶对可溶性淀粉的 K (m) 和 V (max) 值分别为 7.28 mg/mL 和 13.07 mg/mL·min。这项研究表明，ParAmy 具有成为洗涤剂工业中冷稳定和耐碱的淀粉酶的良好潜力。
