Error prone polymerase chain

In 1989, a very practical mutagenesis method was described by Leung et al. 39), which was later improved by Cadwell and Joyce 40). Error-prone polymerase chain reaction (epPCR), as it is called, is based on the classical DNA amplification... 

The phbA, phbB, and phbC genes from Alcaligenes eutrophus (Ralstonia eutrophus) encoding the biosynthetic enzymes (3-ketothiolase, acetoacetyl-CoA reductase (NADPH-dependent), and PHB synthase, respectively, have been cloned into E. coli (Scheme 19.42).339-342 The use of in vitro evolution using error-prone polymerase chain reaction has led to enhanced accumulation of PHA in a resultant recombinant strain.343 Additional studies to enhance the biosynthesis of PHB through the use of metabolic engineering have been discussed.344... 

Evolution has been shown to be an efficient tool for the improvement of enzymes and pathways. Stochastic approaches to introduce point mutations in genes include error prone polymerase chain reaction (ePCR),11 the use of low-fidelity (mutator) strains,12 and chemical mutagens.13... 

In contrast to rational design methods, where changes in protein properties may be predicted from sequence changes, random mutation methods have been introduced to empirically improve protein properties in the absence of a priori knowledge of structure-function relationships. Error-prone polymerase chain reaction (PCR), for example, can be used to introduce approximately random mutations in amplified DNA sequences. The protein products of these mutations may then be screened to select successful or improved variants.2... 

The evolution of a transaminase from Arthrohacter citreus to a thermostable transaminase with increased specific activity and decreased inhibition by the amine product was accomplished using error prone polymerase chain reaction (PCR) [64] The reaction of substituted tetralone 75 and isopropylamine to produce substituted (S) aminotetralin 76 was carried out at greater than 50 °C to facilitate the removal of the acetone by product and drive reaction equilibrium (Figure 14.43). 

Microwave radiation increased the rate 1- to 14-fold and the ee 3- to 9-fold (up to 92% ee for one isomer and 96% ee for the other) in the lipase-catalyzed esterification of an alcohol with vinyl acetate.34 Ultrasound increased the rate 7-to 83-fold. Directed evolution can occasionally be used to improve the enantioselectivity of lipases. A lipase for the hydrolysis of racemic p-nitrophenyl 2-methyldecanoate that gave 2% ee was run through four generations of error-prone polymerase chain reactions to raise the selectivity to 81% ee.35... 

The method for random mutagenesis of genes using error-prone polymerase chain reaction (PCR) was adapted from previous reports (34, 35). An error rate of approximately 0.5% should be expected using this protocol. For a single yeast library of random mutants of approximately 105 clones, you should prepare enough reactions to yield 50—80 Llg error-prone amplified insert (between 3 and 8 reactions). 

A useful method for the generation of molecular diversity starting from a given gene is to use a method of random mutagenesis by error-prone polymerase chain reaction (error-prone PCR) (Figure 10.8). Using error-prone PCR, the mutation rate must be carefully tuned - beneficial mutations are rare but deleterious mutations are common. If the mutation... 

Directed evolution is a powerful tool used to improve lipase properties that does not depend on a comprehensive understanding of the relationship between enzyme structure and function. It rather depends on simple, yet powerfnl, random mutation and selection. The targeted genes are exposed to iterative cycles of random mutagenesis, expressed in an appropriate host and subsequently screened (Johannes and Zhao, 2006). Bacillus lipase was engineered by directed evolntion, where a lip gene was cloned and expressed in E. coli. The mutagenesis was executed by error-prone polymerase chain reaction (PCR). The mutation enhanced the specific activity of the lipase by twofold (Khurana et al., 2011). 

Martin, A., DiSanto, R., Plotnikov, I., Kamat, S., Shonnard, D., and Pannuri, S. (2007) Improved activity and thermostability of (S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine. Biochem. Eng. f, 37 (3), 246-255. 

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