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首页> 外文期刊>Biochemistry >Engineering of the pH Optimum of Bacillus cereus beta-Amylase:Conversion of the pH Optimum from a Bacterial Type to a Higher-Plant Type
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Engineering of the pH Optimum of Bacillus cereus beta-Amylase:Conversion of the pH Optimum from a Bacterial Type to a Higher-Plant Type

机译：芽孢杆菌β-淀粉酶的pH值的工程：从细菌类型到高等植物类型的pH值转化

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摘要

The optimum pH of Bacillus cereus beta-amylase (BCB,pH 6.7) differs from that of soybean beta-amylase (SBA,pH 5.4) due to the substitution of a few amino acid residues near the catalytic base residue (Glu 380 in SBA and Glu 367 in BCB).To explore the mechanism for controlling the optimum pH of beta-amylase,five mutants of BCB (Y164E,Y164F,Y164H,Y164Q,and Y164Q/T47M/Y164E/ T328N) were constructed and characterized with respect to enzymatic properties and X-ray structural crystal analysis.The optimum pH of the four single mutants shifted to 4.2-4.8,approx2 pH units and approx1 pH unit lower than those of BCB and SBA,respectively,and their kappa_(cat) values decreased to 41-3% of that of the wild-type enzyme.The X-ray crystal analysis of the enzyme-maltose complexes showed that Glu 367 of the wild type is surrounded by two water molecules (Wl and W2) that are not found in SBA.Wl is hydrogen-bonded to both side chains of Glu 367 and Tyr 164.The mutation of Tyr 164 to Glu and Phe resulted in the disruption of the hydrogen bond between Tyr 164 Oeta and Wl and the introduction of two additional water molecules near position 164.In contrast,the triple mutant of BCB with a slightly decreased pH optimum at pH 6.0 has no water molecules (Wl and W2) around Glu 367.These results suggested that a water-mediated hydrogen bond network (Glu 367···Tyr 164···Thr 328) is the primary requisite for the increased pH optimum of wild-type BCB.This strategy is completely different from that of SBA,in which a hydrogen bond network (Glu 380···Thr 340···Glu 178) reduces the optimum pH in a hydrophobic environment.
机译：芽孢杆菌β-淀粉酶（BCB，pH6.7）的最佳pH与大豆β-淀粉酶（SBA，pH 5.4）的含量不同，由于催化基质残留件附近的几个氨基酸残基（SBA和SBA中的Glu 380以及BCB中的Glu 367）。探讨控制β-淀粉酶的最佳pH的机制，构建并表征酶的BCB（Y164E，Y164F，Y164H，Y164M / Y164E / T328N）的五个突变体特性和X射线结构晶体分析。四个单突变体的最佳pH变化为4.2-4.8，约2 pH单位和大约1个pH单位，分别低于BCB和SBA，其Kappa_（CAT）值降至41野生型酶的-3％。酶 - 麦芽糖复合物的X射线晶体分析表明，野生型的Glu 367被在SBA中未发现的两个水分子（WL和W2）包围。 WL是氢键合到Glu 367和Tyr 164的两个侧链。Tyr 164至Glu和Phe的突变导致I. n Tyr 164 oeta和WL之间的氢键破坏以及引入两个额外的水分子接近位置164.在对比度下，BCB的三突变体在pH6.0下略微降低的pH值，没有水分子（WL和W2 ）在Glu 367周围。这些结果表明，水介导的氢键网络（Glu 367··Tyr 164···Thr 328）是野生型BCB的pH值增加的主要必要条件。该策略完全不同从SBA的那个中，其中氢键网络（Glu 380···Thr 340···11148）降低了疏水环境中的最佳pH值。

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  • 来源
    《Biochemistry》 |2004年第39期|共9页
  • 作者

    Akira Hirata; Motoyasu Adachi; Shigeru Utsumi; Bunzo Mikami;

  • 作者单位

    Laboratory of Food Quality Design and Development Graduate School of Agriculture Kyoto University Gokasho Uji Kyoto 611-0011 Japan;

    Laboratory of Food Quality Design and Development Graduate School of Agriculture Kyoto University Gokasho Uji Kyoto 611-0011 Japan;

    Laboratory of Food Quality Design and Development Graduate School of Agriculture Kyoto University Gokasho Uji Kyoto 611-0011 Japan;

    Laboratory of Food Quality Design and Development Graduate School of Agriculture Kyoto University Gokasho Uji Kyoto 611-0011 Japan;

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  • 正文语种 eng
  • 中图分类 生物化学;
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