Department of Biochemical Engineering, University College London, Bernard Katz, London WC1E 6BT, UK.
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK.
Enzyme Microb Technol. 2020 Sep;139:109592. doi: 10.1016/j.enzmictec.2020.109592. Epub 2020 May 12.
Epoxide hydrolases (EHs) catalyse the conversion of epoxides into vicinal diols. These enzymes have extensive value in biocatalysis as they can generate enantiopure epoxides and diols which are important and versatile synthetic intermediates for the fine chemical and pharmaceutical industries. Despite these benefits, they have seen limited use in the bioindustry and novel EHs continue to be reported in the literature. We identified twenty-nine putative EHs within the genomes of soil bacteria. Eight of these EHs were explored in terms of their activity. Two limonene epoxide hydrolases (LEHs) and one ⍺/β EH were active on a model compound styrene oxide and its ring-substituted derivatives, with low to good percentage conversions of 18-86%. Further exploration of the substrate scope with enantiopure (R)-styrene oxide and (S)-styrene oxide, showed different epoxide ring opening regioselectivities. Two enzymes, expressed from plasmids pQR1984 and pQR1990 de-symmetrised the meso-epoxide cyclohexene oxide, forming the (R,R)-diol with high enantioselectivity. Two LEHs, from plasmids pQR1980 and pQR1982 catalysed the hydrolysis of (+) and (-) limonene oxide, with diastereomeric preference for the (1S,2S,4R)- and (1R,2R,4S)-diol products, respectively. The enzyme from plasmid pQR1982 had a good substrate scope for a LEH, being active towards styrene oxide, its analogues, cyclohexene oxide and 1,2-epoxyhexane in addition to (±)-limonene oxide. The enzymes from plasmids pQR1982 and pQR1984 had good substrate scopes and their enzymatic properties were characterised with respect to styrene oxide. They had comparable temperature optima and pQR1984 had 70% activity in the presence of 40% of the green solvent MeOH, a useful property for bio-industrial applications. Overall, this study has provided novel EHs with potential value in industrial biocatalysis.
环氧化物水解酶（EHs）催化环氧化物转化为顺式二醇。这些酶在生物催化中具有广泛的应用价值，因为它们可以生成手性纯的环氧化物和二醇，这些都是精细化工和制药行业重要且用途广泛的合成中间体。尽管具有这些优势，但它们在生物工业中的应用仍然有限，并且新的 EHs 仍在文献中不断报道。我们在土壤细菌的基因组中鉴定了 29 种推定的 EHs。其中 8 种 EHs 的活性得到了研究。两种柠檬烯环氧化物水解酶（LEHs）和一种 α/β EH 对模型化合物苯乙烯氧化物及其环取代衍生物具有活性，转化率为 18-86%。进一步用对映体纯的（R）-苯乙烯氧化物和（S）-苯乙烯氧化物探索了它们的底物范围，显示出不同的环氧化物开环区域选择性。两种酶，分别来自质粒 pQR1984 和 pQR1990，不对称地拆分了内消旋环氧环己烯氧化物，形成了具有高对映选择性的（R,R）-二醇。两种来自质粒 pQR1980 和 pQR1982 的 LEHs 催化了（+）和（-）柠檬烯氧化物的水解，对（1S,2S,4R）-和（1R,2R,4S）-二醇产物具有非对映选择性。来自质粒 pQR1982 的酶对 LEH 具有良好的底物范围，对苯乙烯氧化物、其类似物、环氧环己烯氧化物和 1,2-环氧己烷以及（±）-柠檬烯氧化物均具有活性。来自质粒 pQR1982 和 pQR1984 的酶具有良好的底物范围，其酶学性质已针对苯乙烯氧化物进行了表征。它们具有相似的最适温度，并且在 40%的绿色溶剂甲醇存在下，质粒 pQR1984 的活性为 70%，这是生物工业应用的有用特性。总的来说，这项研究提供了具有工业生物催化潜在价值的新型 EHs。
