Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
N Biotechnol. 2010 Feb 28;27(1):78-84. doi: 10.1016/j.nbt.2009.11.002. Epub 2009 Nov 10.
The industrial importance of Trigonopsis variabilisd-amino acid oxidase (TvDAAO) is represented by its biocatalytic oxidative deamination of cephalosporin C (CPC) to yield glutaryl-7-aminocephalosporanic acid (GL-7-ACA). The process has been incorporated into a two-step bioconversion to produce 7-aminocephalosporanic acid, the crucial synthetic nucleus for several semi-synthetic cephalosporin antibiotics. A homology model of TvDAAO indicated that residue F54 is in a close proximity to the in silico docked CPC. Substitution of this F54 to Tyr (F54Y) resulted in 6-fold improvement in k(cat,app) and approximately 2.5-fold increase in K(i) of GL-7-ACA. Heat treatment (55 degrees C, 60 min) did not decrease the activity of F54Y. It is suggested that the Tyr substitution might initiate hydrogen bond formation with the amino group of CPC and facilitate deamination. Faster substrate turnover, reduced GL-7-ACA inhibition and improved thermostability of the F54Y substitution render it a useful candidate in industrial production of semi-synthetic cephems.
三角酵母天冬氨酸氨基氧化酶（TvDAAO）在工业上的重要性体现在其能够生物催化地氧化头孢菌素 C（CPC），从而生成 7-氨基头孢烷酸（GL-7-ACA）。该过程已被纳入两步生物转化，以生产 7-氨基头孢烷酸，这是几种半合成头孢菌素抗生素的关键合成核。TvDAAO 的同源模型表明，残基 F54 与计算机对接的 CPC 非常接近。将此 F54 突变为 Tyr（F54Y），导致 k(cat,app)提高了 6 倍，而 GL-7-ACA 的 K(i)增加了约 2.5 倍。热处理（55°C，60 分钟）并没有降低 F54Y 的活性。这表明 Tyr 取代可能会与 CPC 的氨基形成氢键，并促进脱氨。F54Y 取代物更快的底物转化、减少 GL-7-ACA 的抑制以及提高的热稳定性，使其成为半合成头孢菌素工业生产的有用候选物。
