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首页> 外文期刊>BMC Biotechnology >Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061
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Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061

机译：花青霉素生产者镰刀菌LF061的新型2-酮异戊酸还原酶的克隆和鉴定

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Background The ketoisovalerate reductase (EC 1.2.7.7 ) is required for the formation of beauvericin via the nonribosomal peptide synthetase biosynthetic pathway. It catalyzes the NADPH-specific reduction of ketoisovaleric acid to hydroxyisovalerate. However, little is known about the bioinformatics’ data about the 2-Kiv reductase in Fusarium . To date, heterologous production of the gene KivRFp from Fusarium has not been achieved. Results The KivRFp gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene KivRFp contained a 1,359?bp open reading frame (ORF) encoding a polypeptide of 452 amino acids with a molecular mass of 52?kDa. Sequence analysis indicated that it showed 61% and 52% amino acid identities to ketoisovalerate reductase from Beauveria bassiana ATCC 7159 (ACI30654) and Metarhizium acridum CQMa 102 (EFY89891), respectively; and several conserved regions were identified, including the putative nucleotide-binding signature site, GXGXXG, a catalytic triad (Glu405, Asn184, and Lys285). The KivRFp exhibited the highest activity at 35°C and pH 7.5 respectively, by reduction of ketoisovalerate. It also exhibited the high level of stability over wide temperature and pH spectra and in the presence of metal ions or detergents. Conclusions A new ketoisovalerate reductase KivRFp was identified and characterized from the depsipeptide-producing fungus F . proliferatum . KivRFp has been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering and directed evolution, towards the goal of developing efficient enzyme for downstream biotechnological applications.
机译：背景技术通过非核糖体肽合成酶的生物合成途径形成紫红霉素需要酮异戊酸还原酶（EC 1.2.7.7）。它催化NADPH特异性还原异戊酸酮成羟基异戊酸酯。然而，关于镰刀菌中2-Kiv还原酶的生物信息学数据知之甚少。迄今为止，尚未实现镰刀菌（Fusarium）的KivRFp基因的异源生产。结果使用pET表达系统将KivRFp基因亚克隆并在大肠杆菌BL21中表达。 KivRFp基因含有一个1,359？bp的开放阅读框（ORF），编码的452个氨基酸的多肽，分子量为52？kDa。序列分析表明，它与球孢白僵菌ATCC 7159（ACI30654）和a草甲基CQMa 102（EFY89891）的酮异戊酸还原酶分别具有61％和52％的氨基酸同一性。并鉴定了几个保守区域，包括推定的核苷酸结合特征位点，GXGXXG，催化三联体（Glu405，Asn184和Lys285）。通过还原异戊酸酮，KivRFp分别在35°C和pH 7.5时表现出最高的活性。在宽温度和pH光谱下以及存在金属离子或去污剂的情况下，它还表现出高水平的稳定性。结论从产二肽的真菌F中鉴定并鉴定了一种新的酮异戊酸酯还原酶KivRFp。增生。已经显示出KivRFp具有有用的特性，例如适度的热稳定性和广泛的pH最佳值，并且可以作为未来蛋白质工程和定向进化的起点，朝着为下游生物技术应用开发高效酶的目标迈进。

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