Liu Yi-Han, Lu Fu-Ping, Li Yu, Yin Xiang-Bin, Wang Yi, Gao Chen
Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, 300457, Tianjin, People's Republic of China.
Appl Microbiol Biotechnol. 2008 Feb;78(1):85-94. doi: 10.1007/s00253-007-1287-z. Epub 2007 Dec 21.
Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134-->Arg and Ser320-->Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.
基于地衣芽孢杆菌的原始耐热α-淀粉酶基因，通过聚合酶链反应对两个氨基酸进行定点诱变以获得一个新基因。该基因具有Leu134→Arg和Ser320→Ala的突变，以前具有耐酸性。为便于产物的纯化，以蛋白酶缺陷型枯草芽孢杆菌WB600作为宿主，实现了成熟、真实且稳定的重组诱变α-淀粉酶的高水平表达和分泌。然后通过硫酸铵分级沉淀、阴离子交换和凝胶过滤依次纯化具有4700 U/mL活性的重组诱变α-淀粉酶。通过多步纯化，重组蛋白的比活性高达916.7 U/mg，纯化倍数为187.1倍。发现诱变后的蛋白比天然蛋白更耐酸。诱变蛋白的最适pH和pH稳定范围分别为4.5以及4.0至6.5，而天然蛋白的最适pH和pH稳定范围分别为6.5以及5.5至7.0。
