Nitrobenzyl esterases

In addition to cutinases, various lipases, such as from C. antarctica, Candida sp. [13, 47], Thermomyces lanuginosus [2, 14, 15, 55, 56], Burkholderia (formerly Pseudomonas) cepacia [57] and esterases from Pseudomonas sp. (serine esterase) [58] and Bacillus sp. (nitrobenzyl esterases) [59, 60], have shown PET hydrolase... 

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. 

These investigations also showed that the conversion of ECB to ECB nucleus would proceed more rapidly if ECB were first solubilized in a suitable solvent such as methanol or acetone. However, if the concentration of solvent was too high, the enzyme activity was reduced. Ideally, the enzyme itself could be tailored to suit the industrially preferred conditions (e.g., to make it more resistant to solvent or active at a different pH). One method for achieving this is to use directed evolution [42], whereby genes encoding the enzyme are mutated, screened and then recombined in vitro. Although the contributions of individual amino acid mutations are small, the accumulation of multiple mutations by directed evolution allows significant improvement in the biocatalyst for reactions on substrates or under conditions not already optimized in nature. This approach was used by Arnold and Moore [43] to make a 150-fold improvement in the activity of a -nitrobenzyl esterase in the presence of 15% DMSO. 

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... 

Moore, J. C. and Arnold, F. H., Directed evolution of a/)ara-nitrobenzyl esterase for aqueous-organic solvents, Natl Biotechnol., 14, 458 67, 1996. 

Montenecourt BS, Eveleigh DE (1977) Preparation of mutants of Trichoderma reesei with enhanced ceUulase production. Appl Environ Microbiol 34(6) 777-782 Moore JC, Arnold EH (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat Biotechnol 14 458-467... 

Optically pure l-(3, 4 -methylenedioxyphenyl)ethanol is a key chiral intermediate for the synthesis of Steganacin and Salmeterol. Apara-nitrobenzyl esterase cloned from Bacillus amyloliquefaciens (BAE) was employed to hydrolyze... 

Figure 2.4 Stepwise and combinational strategies for modulating the enantioselectivity of a /tara-nitrobenzyl esterase cloned from Bacillus amylolique-faciens (BAE) in the hydrolysis of l-(3, 4 -methylenedioxyphenyl)ethyl acetate. ...

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]. 

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