P-Nitrobenzyl esterase

Application of DNA Shuffling Recombination of p-Nitrobenzyl Esterase Genes... 

Eli Lilly (Indianapolis/IN, USA) needed a p-nitrobenzyl esterase to hydrolyze the p-nitrobenzyl ester of a )3-lactam compound in DMF. The purely organic route with zinc salts and large amounts of organic solvent was unattractive, so an enzyme was sought which could hydrolyze the ester. One with rather low activity was found which served as a starting point for directed evolution. As nature never had to hydrolyze an antibiotic in DMF, this approach seemed very promising. 

In this directed evolution experiment, increased thermostability was accompanied by an increase in catalytic activity at all temperatures (Fig. 6), as measured on the substrate used during screening, jfr-nitrophenyl acetate. Variant 8G8 is 3.7 times more active than wild type at the wild-type Topt the improvement is 4.5-fold at 8G8 s new Topt of 60°C. Activity was allowed to decrease in the first generation (variant 1A5D1 was an intermediate product of a separate evolution experiment to increase p-nitrobenzyl esterase activity in aqueous organic solvent and differs from wild type at five positions), but was recovered in later generations. 

A common aim of directed evolution is to increase the stability of an enzyme to conditions of practical use that may be very different from those the enzyme naturally functions in. Factors such as heat, altered pH, and the presence of oxidants or organic solvents can lead to denaturation or loss of enzyme function. Many researchers have successfully increased the stability of an enzyme to thermal denaturation (41, 42). Work with p-nitrobenzyl esterase uicreased the melting temperature 14°C after six rounds of EP-PCR and recombination without forfeiting enzyme activity (41). As another example, phosphite dehydrogenase catalyzes the formation of phosphate from phosphite, by reducing NAD+ to NADH. However, the usefulness... 

A carboxylesterase (EC 3.1.1.1) from T. fusca [8, 86] and a steryl esterase (EC 3.1.1.13) from Melanocarpus albomyces [59] have also shown activity with PET oligomers and fabrics. The enzyme from M. albomyces with high specificity for fatty acid esters of sterols increased the hydrophilicity of PET fabrics. The highly hydrophobic serine hydrolase from T. fusca with a catalytic triad composed of serine, glutamic acid, and histidine hydrolyzed CTR and PET nanoparticles. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. In addition, p-nitrobenzyl esterases from Bacillus subtilis and B. licheniformis that hydrolyzed short chain dialkylphthalates and PET nanoparticles have been reported [74, 87]. 

Fig. 2. Comparison of activities on target (p-nitrobenzyl) and screening (p-nitrophenyl) substrate of selected active pNB esterases isolated after fourth generation of random mutagenesis and screening, relative to parent enzyme from the 3rd generation. The five most active variants were pooled for random recombination...

Fig. 4. Reaction rates for evolved pNB esterases from the first four generations of directed evolution. Activity towards p-nitrobenzyl loracarbef nucleus in 1% (a) and 15% (b)DMF in 0.1 M PIPES, pH 7.0, 30°C. Four times as much substrate can be dissolved in 15% DMF as in 1% DMF...

Fig. 7. Molecular model of the pNB esterase showing positions of antibiotic p-nitrobenzyl ester substrate (white CPK structure), catalytic residues (S189, E310, and H399), and beneficial mutations accumulated during directed evolution. Mutations at positions 322 and 370 are believed to improve expression, while the remaining six substitutions improve specific activity [2]. Arrows indicate the position of new mutations found after DNA shuffling...

Hep),14-9, 31, 32, 98-1001 the 2-bromoethyl (EtBr) I4-6, 31, 32, 1011 and the p-nitrobenzyl (PNB) esters11021 as carboxy protecting groups for peptide synthesis which can be enzymatically removed by means of lipases or esterases, respectively (Fig. 18-11). 

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