Enzymes optimization

Fox, R.J. and. Huisman, G.W., Enzyme optimization moving from blind evolution to statistical exploration of sequence-function space. Trends Biotechnol, 2008, 26, 132-138. 

Lactate produced during glycolysis lowers pH and assists in pathogen killing by lysosomal enzymes optimal at acid pH. 

In addition to the usual a-D-mannosidase having a pH optimum between 4 and 5, a similar enzyme (optimal activity at pH 6.5) has been observed in rat liver by Marsh and Gourlay,76 and in pig kidney,... 

Stereoselective enzymatic degradation of nerve agents is also a current issue in developing both novel noncorrosive decontamination systems and new therapeutics making use of recombinant mutated enzymes optimized for fast and exhaustive hydrolysis of most toxic isomers (Blum and Richardt, 2008 Furlong, 2007 Ghanem and Raushel, 2005 Li et al, 2001 Tsugawa et al, 2000). 

Mass screening of our compound collection, utilizing the Amersham SPA technology, afforded pyrone and coumarin non-peptide templates as initial lead structures. X-ray cocrystallization and structure-based design were utilized to assist in the design of more potent inhibitors. These efforts resulted in the design of the 5,6-dihydropyrones, which afforded a more flexible template from which to fill the internal pockets of the enzyme. Optimization of the dihydropyrone series afforded a potent antiviral agent,... 

Figure 4-1. Evolutionary enzyme optimization. One or several parent genes are chosen and subjected to mutagenesis and/or recombination. The mutant gene library (genotype) is then expressed in vivo (or in vitro) where it is linked to the enzyme produced (phenotype). These enzymes are tested for the targeted property by screening or selection. DNA from the most fit clone(s) is isolated and subjected to a new cycle of mutagenesis and screening or selection. This procedure is repeated until the desired goal is reached or until no further improvements are observed.

Investigations into avoid this danger have taken place. Extended anionic surfactants have been employed to extract corn oil instead of hexane [17]. These extractions were shown to extract 83% of the oil available and also maintained the composition of the corn oil when compared to extractions carried out using hexane. A mixture of enzymes to extract oil from Irvingia gabonensis kernels was employed controlled, ordered addition of the enzymes optimized the extraction of the oil up to 90% efficiency [18]. Similar protocols were applied to the extraction of canola oil [19] and showed that the enzymatically assisted extractions were more efficient than by using water alone and produced better oil quality than both aqueous and solvent extractions. The efficiency of the process in this instance was significantly increased from those previously stated (circa 25%). 

Scheme 1. Optimization of oligosaccharide linking by conventional one at a time linkage plus enzyme optimization or by convergent strategy of optimizing one enzyme reaction and then coupling linkers.

We had two patients with allergic patch-test reactions to cellulase and xylanese (Tarvainen et al. 1991b, Table 3). Cellulase and xylanase were tested in a dilution series (3.3%> i%> 0.33%) in petrolatum and aqua. As a control, 20 non-exposed subjects (10 atopies) were patch tested with a concentration of 3.3% w/v in petrolatum, with negative results. Currently, scanty data are available on how to patch test with industrial enzymes. Optimal patch test concentrations need to be established for each enzyme, and controls are necessary. 

Reaction engineering has been used to improve the performance of the synthesis of HPP. Whole cells or cell-free extracts were studied and shown to be more efficient than the purified enzyme.Optimal pH (5 to 8) and temperature (20°C to 40°C) were determined and found to be broad. Finally, immobilization techniques, notably on silica carrier, were also investigated to improve the performance and stability of the enzyme. 

Resch V, Schrittwieser JH, Wallner S, Macheroux P, Rroutil W. Biocatalytic oxidative C—C bond formation catalysed by the berberine bridge enzyme optimal reaction conditions. Adv. Synth. Catal 2011 353 2377-2383. 

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