Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi 10600, Viet Nam.
J Microbiol Biotechnol. 2012 May;22(5):607-13. doi: 10.4014/jmb.1107.07033.
Improvement of endoglucanase activity was accomplished by utilizing error-prone rolling circle amplification, supplemented with 1.7 mM MnCl2. This procedure generated random mutations in the Bacillus amyloliquefaciens endoglucanase gene with a frequency of 10 mutations per kilobase. Six mutated endoglucanase genes, recovered from six colonies, possessed endoglucanase activity between 2.50- and 3.12-folds higher than wild type. We sequenced these mutants, and the different mutated sites of nucleotides were identified. The mutated endoglucanase sequences had five mutated amino acids: A15T, P24A, P26Q, G27A, and E289V. Among these five substitutions, E289V was determined to be responsible for the improved enzyme activity. This observation was confirmed with site-directed mutagenesis; the introduction of only one mutation (E289V) in the wild-type endoglucanase gene resulted in a 7.93-fold (5.55 U/mg protein) increase in its enzymatic activity compared with that (0.7 U/mg protein) of wild type.
通过易错滚环扩增，辅以 1.7mM MnCl2，提高了内切葡聚糖酶的活性。该方法使枯草芽孢杆菌内切葡聚糖酶基因随机突变的频率达到每千碱基 10 个突变。从 6 个菌落中回收的 6 个突变内切葡聚糖酶基因的内切葡聚糖酶活性比野生型高 2.50-3.12 倍。我们对这些突变体进行了测序，并确定了核苷酸的不同突变位点。突变的内切葡聚糖酶序列有 5 个突变的氨基酸：A15T、P24A、P26Q、G27A 和 E289V。在这 5 个取代中，E289V 被确定为导致酶活性提高的原因。通过定点突变证实了这一观察结果；在野生型内切葡聚糖酶基因中仅引入一个突变（E289V），其酶活性（5.55U/mg 蛋白）比野生型（0.7U/mg 蛋白）增加了 7.93 倍。
