Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea.
Biotechnol Lett. 2011 Feb;33(2):353-8. doi: 10.1007/s10529-010-0445-z. Epub 2010 Oct 24.
For the removal of galactose inhibition, the predicted galactose binding residues, which were determined by sequence alignment, were replaced separately with Ala. The activities of the Ala-substituted mutant enzymes were assessed with the addition of galactose. As a consequence, amino acid at position 349 was correlated with the reduction in galactose inhibition. The F349S mutant exhibited the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The K (i) of the F349S mutant (160 mM), which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β-galactosidase. The wild-type enzyme hydrolyzed 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzed more than 99%.
为了消除半乳糖的抑制作用，通过序列比对确定的预测半乳糖结合残基分别用丙氨酸取代。用半乳糖评估 Ala 取代突变酶的活性。结果表明，第 349 位氨基酸与半乳糖抑制作用的降低有关。与 349 位测试突变酶在无半乳糖条件下的活性相比，F349S 突变体在有半乳糖存在时表现出最高的活性。F349S 突变体的 K（i）（160mM）是野生型酶的 13 倍，是已报道的β-半乳糖苷酶中最高的。在添加 30g 半乳糖/l 的情况下，野生型酶水解 100g 乳糖/l 的 62%，而 F349S 突变体水解超过 99%。
