Site-specific mutagenesis at positions 272 and 273 of the Bacillus sp SAM1606 α-glucosidase to screen mutants with altered specificity for oligosaccharide production by transglucosylation
Site-specific mutagenesis at positions 272 and 273 of the Bacillus sp SAM1606 α-glucosidase to screen mutants with altered specificity for oligosaccharide production by transglucosylation
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DOI：
10.1016/s1381-1177(01)00071-6
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发表时间：
2002-02-18
影响因子：
--
通讯作者：
Ueda, T
中科院分区：
文献类型：
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作者：
Okada, M;Nakayama, T;Ueda, T
SAM1606 α -葡萄糖苷酶催化蔗糖的转糖基化，与其他二糖、三糖和四糖一起产生雄糖（6- o -g -葡萄糖-蔗糖）作为主要的转移产物。为了获得α -葡萄糖苷酶变体更丰富地产生雄性糖，我们进行了位点特异性诱变研究，其中α -葡萄糖苷酶的推定催化位点（Glu271）附近的氨基酸残基（Gly273或Thr272）被所有其他天然存在的氨基酸取代。每个突变体的浓度为2.6 U/ml（蔗糖水解单位），在60℃和pH 6.0条件下与1.75 M蔗糖反应，通过HPLC监测低聚糖生产过程，系统分析氨基酸取代对转糖基化特异性的影响。分析清楚地表明，催化位点附近的取代对低聚糖生产特异性的差异影响取决于位点和残基。例如，273位被芳香氨基酸或His取代的突变体实际上失去了通过转糖基化产生低聚糖的能力。在272位被比野生型Thr大的氨基酸取代的突变体显示四糖的产量增加；而在273位被赖氨酸和精氨酸取代的突变体只产生双糖转移产物。T272I突变体的雄糖生成特异性最高（即反应产物中雄糖的含量最高），其雄糖的产量（每蔗糖水解单位）是野生型酶的1.74倍。(C) 2002 Elsevier Science B.V.版权所有
The Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce theanderose (6-O-G-glucosylsucrose) as the major transfer product along with the other di-, tri-, and tetrasaccharides. To obtain an alpha-glucosidase variant(s) producing theanderose more abundantly, we carried out site-specific mutagenesis studies, in which an amino acid residue (Gly273 or Thr272) near the putative catalytic site (Glu271) of this alpha-glucosidase was replaced by all other naturally-occurring amino acids. Each mutant, whose concentration was set at 2.6 U/ml (sucrose-hydrolyzing units), was reacted at 60 C and pH 6.0 with 1.75 M sucrose, and the course of the oligosaccharide production was monitored by HPLC to systematically analyze the effects of amino acid substitutions on the specificity of transglucosylation. The analysis clearly showed site- and residue-dependent differential effects of substitution near the catalytic site on the specificity of oligosaccharide production. For example, mutants with substitution at position 273 by aromatic amino acids or His virtually lost the ability to produce oligosaccharides by transglucosylation. Mutants with substitution at position 272 by amino acids that were bulkier than the wild-type Thr showed enhanced production of tetrasaccharides; whereas, mutants with substitution at position 273 by Lys and Arg exclusively produced disaccharidal transfer products. The highest specificity for theanderose formation (i.e. the highest content of theanderose in the reaction product) was obtained with the T272I mutant, which showed 1.74 times higher productivity (per sucrose-hydrolyzing unit) of theanderose than that of the wild-type enzyme. (C) 2002 Elsevier Science B.V. All rights reserved.