Overexpression of a novel hydantoinase (hyuH) from P. aeruginosa (MCM B-887) in E. coli yielded optically pure carbamoyl amino acids. The use of optically pure carbamoyl amino acids as substrates facilitates the synthesis of non-proteinogenic amino acids. The enzyme hyuH shared a maximum of 92 % homology with proven hydantoinase protein sequences from the GenBank database, highlighting its novelty. Expression of hydantoinase gene was improved by >150 % by overexpressing it as a fusion protein in specialized E. coli CODON + host cells, providing adequate machinery for effective translation of the GC-rich gene. The presence of distinct residues in the substrate binding and active site of MCM B-887 hydantoinase enzyme explained its unique and broad substrate profile desirable for industrial applications. The purified enzyme, with a specific activity of 53U/mg of protein, was optimally active at 42 °C and pH 9.0 with a requirement of 2 mM Mn2+ ions. Supplementation of 500 mM of Na-glutamate enhanced the thermostability of the enzyme by more than 200 %.

中文翻译：

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一种新型、对​​映选择性、热稳定的重组乙内酰脲酶，有助于合成具有工业价值的非蛋白氨基酸

来自铜绿假单胞菌 (MCM B-887) 的新型乙内酰脲酶 (hyuH) 在大肠杆菌中的过度表达产生了光学纯的氨基甲酰基氨基酸。使用光学纯氨基甲酰基氨基酸作为底物促进非蛋白氨基酸的合成。酶 hyuH 与来自 GenBank 数据库的经证实的乙内酰脲酶蛋白序列具有最多 92% 的同源性，突出了其新颖性。通过在特化的大肠杆菌 CODON + 宿主细胞中将其作为融合蛋白过表达，乙内酰脲酶基因的表达提高了 >150%，为富含 GC 的基因的有效翻译提供了足够的机制。MCM B-887 乙内酰脲酶的底物结合和活性位点中存在不同的残基，解释了其独特而广泛的底物谱，适合工业应用。纯化的酶，具有 53U/mg 蛋白质的比活性，在 42 °C 和 pH 9.0 下具有最佳活性，需要 2 mM Mn2+ 离子。补充 500 mM 谷氨酸钠可将酶的热稳定性提高 200% 以上。