Maiangwa Jonathan, Hamdan Siti Hajar, Mohamad Ali Mohd Shukuri, Salleh Abu Bakar, Zaliha Raja Abd Rahman Raja Noor, Shariff Fairolniza Mohd, Leow Thean Chor
Department of Cell and Molecular Biology, Enzyme Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia Serdang, 43400, UPM Serdang, Selangor, Malaysia; Department of Microbiology Kaduna State University, Nigeria; Enzyme Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia Serdang, 43400, UPM Serdang, Selangor, Malaysia.
Department of Biochemistry, Enzyme Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia Serdang, 43400, UPM Serdang, Selangor, Malaysia.
J Mol Graph Model. 2021 Jun;105:107897. doi: 10.1016/j.jmgm.2021.107897. Epub 2021 Mar 10.
Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The k and k values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.
蛋白质在非水酶促过程中的应用，关键在于其与溶剂极性相关的结构动力学。从扎利哈芽孢杆菌T1脂肪酶衍生出的一组突变体，在有机溶剂（甲醇、乙醇、丙醇、丁醇和戊醇）中进行筛选，初步筛选出六个突变体（A83D/K251E、R21C、G35D/S195N、K84R/R103C/M121I/T272M和R106H/G327S）。定点诱变进一步产生了四重突变体A83D/M121I/K251E/G327S和A83D/M121I/S195N/T272M，这两个突变体在甲醇中孵育后活性均有所提高。这些突变体的k和k值在甲醇/底物混合物中与野生型酶略有不同。突变体的热诱导解折叠伴随着二级结构含量的一些损失。均方根偏差（RMSD）和B因子表明，结构组织的变化与蛋白质主链与有机溶剂的相互作用交织在一起。空间暴露的带电残基显示出甲醇溶剂的溶剂化动力学与残基疏水性之间的相关性。径向分布函数的短距离提供了形成氢键和疏水相互作用所需的距离。这些动态变化表明，基于溶剂流动性和突变残基，可以通过实验靶向并纳入新形成的结构相互作用。
