Esterase mutant

Fig. 7. Distribution of activity and stability in a library of random pNB esterase mutants.

Scheme 14.3. Principle of the assay system used to identify active variants of esterase from Pseudomonas fluorescens (PFE) acting on the sterically hindered 3-hydroxy ester 1. Both substrates 1a,b yield the free acid leading to a color change 1b also releases the carbon source glycerol leading to enhanced growth of esterase mutant producing E. coli clones.

Detoxification Cholinesterases enzymes hydrolyzing OP monoclonal antibodies against OP Butyrylcholin esterase. Mutants Acetylcholinesterase Triesterase Paraoxonase Very persjjective PROTEXIA ... 

In another study a hyperthermophilic esterase from Aeropyrum pemix K1 (APE1547) was used as a catalyst in the hydrolytic kinetic resolution of rac-3-octanol acetate [53]. Following a single round of epPCR, a mutant displaying a 2.6-fold increase in enantioselectivity was identified having five amino acid substitutions, which were shown to be spatially distal to the catalytic center. 

Modulation of the quantity and/or quality of poly(3HAMCL) synthesized in peroxisomes was also achieved by modifying the endogenous fatty acid biosynthetic pathway [58]. For example, expression of the peroxisomal PHA synthase in an A. thaliana mutant deficient in the synthesis of triunsaturated fatty acids [59] resulted in the synthesis of a PHA having an almost complete absence of triunsaturated 3-hydroxyacid monomers [58]. In a different strategy, expression of a fatty acyl-ACP thio esterase in the plastid was combined with the expression of a peroxisomal PHA synthase [58]. Fatty acyl-ACP thioesterases are... 

Sanders They release acetylcholine to the same extent. We measured the output of acetylcholine from the nerves, and this was the same in wild-type and mutant animals. It appears that the close association between the nerve terminals and the interstitial cells is very important. As acetylcholine is released, it is broken down by the esterase. If the esterase is inhibited, a response in the smooth muscle can be seen, but this is not physiological. 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Directed evolution involves multiple rounds of random mutation and selection combined with gene shuffling to evolve enzymes towards desired properties (reviewed in Arnold and Moore, 1997 Kuchner and Arnold, 1997). The group of Arnold has succeeded in evolving a dimethylformamide (DMF)-sensitive esterase for the cleavage of the loracarbef-/>-nitrobenzyl ester into an esterase that remains active in 15% DW (Moore et al, 1997). Most of the mutations that had been found in the solvent-resistant mutants could not have been predicted using current computational methods. 

Plettner E., DeSantis G., Stabile M. and Jones J. B. (1999) Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants. J. Am. Chem. Soc. 121, 4977-4981. 

LiCata and Ackers report that mutations that are not directly in contact can be non-additive (1995). They find that most mutations exhibit some degree of non-additivity that cannot be explained by short-range disruptions. A structural study of mutants of pNB esterase generated by directed evolution supports this observation (Spiller et al., 1999 see chapter by Orencia, Hanson, and Stevens in this volume). In this case, the influence of a mutation was realized through small backbone shifts, spatially distant from the mutated residue. Non-additivity can result when these perturbed regions overlap (Skinner and Terwilliger, 1996). 

Fig. 6. Dependence of activity on temperature for wild-type jfrNB esterase and thermo-stabilized mutants from the first, third, fifth, sixth, and eighth generations.

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

Kinetic resolution of a chiral ester catalyzed by a mutant esterase... 

Pseudomonas fluorescens esterase MS growth/ pH-indicator active double mutant acting on sterically hindered substrate 22, 24, 26... 

Although this method was useful to turn an inactive wild-type enzyme into an active esterase, the best mutant showed only modest enantioselectivity. Further work showed that the enantioselectivity of PFE could be increased from E = 3.5 to E = 5.2 - 6.6 in a single round of mutation [26]. Libraries were created by epPCR as well as by using the... 

Henke, E., Bomscheuer, U.T. (1999), Directed evolution of an esterase from Pseudomonas fluorescens. Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity... 

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