Engineering Cyclohexanone Monooxygenase for the Production of Methyl Propanoate

详细信息    查看全文

• 作者：Hugo L. van Beek ; Elvira Romero ; Marco W. Fraaije
• 刊名：ACS Chemical Biology
• 出版年：2017
• 出版时间：January 20, 2017
• 年：2017
• 卷：12
• 期：1
• 页码：291-299
• 全文大小：455K
• ISSN：1554-8937

文摘

A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer–Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP⁺ binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography–mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively). Another interesting mutant is the T56S AcCHMO mutant, which exhibits a higher conversion yield (92%) and k_cat (0.5 s^–1) than wild type AcCHMO (52% and 0.3 s^–1, respectively). Interestingly, the uncoupling rate for the T56S AcCHMO mutant is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.