摘要 :
The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H2O2-mediated substrate oxidation, has now been shown to exhibit a > 3.0-fold decrease in K-m,K-HOOH for 7-...
展开
The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H2O2-mediated substrate oxidation, has now been shown to exhibit a > 3.0-fold decrease in K-m,K-HOOH for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening > 3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained similar to 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T-m,T-app compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a > 2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.
收起
摘要 :
Enzyme is an efficient green catalyst, but its vulnerability and difficulties of separation from products limited the application in industry. Furthermore, a large number of oxidase enzymes rely on dihydronicotinamide adenine dinu...
展开
Enzyme is an efficient green catalyst, but its vulnerability and difficulties of separation from products limited the application in industry. Furthermore, a large number of oxidase enzymes rely on dihydronicotinamide adenine dinucleotide (NADH) or dihydronicotinamide adenine dinucleotide phosphate (NADPH) cofactors. These cofactors are too expensive to be used in industrial processes. Thus, cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD(P)+ in enzyme-catalyzed reactions. In this paper, Cu based metal nucleotides coordination polymers (MNCPs) with NAD(P)H oxidase activity were constructed by self-assembly of copper ions and nucleotides. Cu2+/AMP and Cu2+/GMP showed high catalytic activity and high reusability. Then, Cu2+/AMP with NADH oxidase activity was combined with NAD+ dependent alcohol dehydrogenase (ADH) to construct a bienzyme cascade system with circulating cofactor, which applied to the conversion of benzyl alcohol to benzaldehyde. In this system, ADH exhibited high activity due to the increase of substrate concentration and the inhibition of the diffusion of intermediate products. Besides, the bienzyme cascade system also showed high stability and reusability. After 8 cycles, the immobilized ADH remained more than 57% of the initial activity. The prepared bienzyme cascade system can extensively extended its application in industrial production.
收起
原文获取