Oh Su-Won, Jang Myoung-Uoon, Jeong Chang-Ku, Kang Hye-Jeong, Park Jung-Mi, Kim Tae-Jip
Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Korea.
J Microbiol Biotechnol. 2008 Aug;18(8):1401-7.
The roles of conserved amino acid residues (Val329-Ala330- Asn331-Glu332), constituting an extra sugar-binding space (ESBS) of Thermus maltogenic amylase (ThMA), were investigated by combinatorial saturation mutagenesis. Various ThMA mutants were firstly screened on the basis of starch hydrolyzing activity and their enzymatic properties were characterized in detail. Most of the ThMA variants showed remarkable decreases in their hydrolyzing activity, but their specificity against various substrates could be altered by mutagenesis. Unexpectedly, mutant H-16 (Gly-Leu-Val-Tyr) showed almost identical hydrolyzing and transglycosylation activities to wild type, whereas K-33 (Ser-Gly-Asp-Glu) showed an extremely low transglycosylation activity. Interestingly, K-33 produced glucose, maltose, and acarviosine from acarbose, whereas ThMA hydrolyzed acarbose to only glucose and acarviosine-glucose, which proposes that the substrate specificity, or hydrolysis or transglycosylation activity of ThMA can be modulated by combinatorial mutations near the ESBS.
通过组合饱和诱变研究了构成嗜热麦芽糖淀粉酶（ThMA）额外糖结合空间（ESBS）的保守氨基酸残基（Val329 - Ala330 - Asn331 - Glu332）的作用。首先根据淀粉水解活性筛选了各种ThMA突变体，并详细表征了它们的酶学性质。大多数ThMA变体的水解活性显著降低，但通过诱变可以改变它们对各种底物的特异性。出乎意料的是，突变体H - 16（Gly - Leu - Val - Tyr）的水解和转糖基化活性与野生型几乎相同，而K - 33（Ser - Gly - Asp - Glu）的转糖基化活性极低。有趣的是，K - 33能从阿卡波糖产生葡萄糖、麦芽糖和阿糖氨基糖，而ThMA仅将阿卡波糖水解为葡萄糖和阿糖氨基糖 - 葡萄糖，这表明ThMA的底物特异性或水解或转糖基化活性可通过ESBS附近的组合突变进行调节。
