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Bioengineering Approach on Terpenoids Production
Published in Dijendra Nath Roy, Terpenoids Against Human Diseases, 2019
Two broad approaches have proved quite effective for improving the activity of enzymes, namely, directed evolution and rational design. Directed evolution is inspired by natural evolution whereby genetic diversity is created by mutagenesis and protein variants with desired functions are identified. Rational design method requires enough prior knowledge about the enzyme in terms of structure–function relationships to predict which mutations would result in the enhancement of enzyme activity. Lately, with the advent of powerful computing tools and the increasing amount of protein structures available, both these approaches have been combined and the combination is now referred to as ‘semi-rational design’. It involves site saturation mutagenesis or random mutagenesis over a specific part of the enzyme rather than over the entire enzyme (Porter et al. 2016).
Biocatalysis: An introduction
Published in Grunwald Peter, Biocatalysis and Nanotechnology, 2017
3DM analysis is an approach to create “smart” libraries, characterized by their significantly reduced size together with a comparatively high probability to identify hits; it is based on a software tool that can generate superfamily-specific databases, and has been developed by Jochens and Bornscheuer (2010). The applicability of this concept was demonstrated with the improvement of the enantioselectivity of an esterase from P. fluorescens (PFE) with the sterically demanding 3-phenyl butyric acid p-nitrophenyl ester (3-PB-pNP) as substrate. The enantioselectivity of PFE is influenced by the four positions W28, V121, F198, and V225 (Park et al., 2005). This amino acid distribution was determined by means of a structural alignment of 1751 sequences of α/β-hydrolase fold enzymes; frequently occurring residues/”mutations” that obviously have already proven during natural evolution to contribute to the integrity of the protein were defined as allowed (A) and rarely occurring ones at not allowed (NA). Based on this analysis an appropriate codon choice was possible. In this approach site-saturation mutagenesis was performed simultaneously at all four targeted positions in order to allow possible cooperative effects. With 3-PB-pNP as substrate the w-t PFE exhibits only very low activity and an enantioselectivity of E = 3.2, whereas in library Aa significant number of mutants with improved reaction rates towards either enantiomer of up to 240-fold and enantioselectivities up to E = 80 could be identified (as in similar cases the activity was determined by following the release of p-nitrophenol through measuring the absorbance at 410 nm).
A comprehensive review on enzymatic degradation of the organophosphate pesticide malathion in the environment
Published in Journal of Environmental Science and Health, Part C, 2019
Smita S. Kumar, Pooja Ghosh, Sandeep K. Malyan, Jyoti Sharma, Vivek Kumar
Organophosphorus hydrolase (OPH) obtained from bacteria is encoded by the opd gene (organophosphate degrading). It has been purified from Pseudomonas diminuta and Flavobacterium sp strain ATCC27551 and attacks various phosphoester bonds including P-O, P-CN, P-F, and P-S. However, the efficiency of cleavage of different bonds is different. For example, the rate of catalysis of the P-S bond is approximately 1000 times slower as compared to that of the P-O bond. So as to improve the specificity and efficiency of OPH, other variants have been created by intended and arbitrary approaches. Both of these approaches rely on mutagenesis of the opd gene followed by screening against the target of choice to optimize the mutant enzyme for a specific target. Very few studies have attempted to generate improved enzymes for P-S organophosphates. Schofield et al.108 carried out a study in order to generate an OPH variant with improved hydrolytic efficiency against the remediation of P-S organophosphates. Site-directed mutagenesis and saturation mutagenesis of active site residues followed by screening against demeton-S methyl and malathion were sequentially performed to identify variants with improved hydrolytic efficiency.
Development of capability for genome-scale CRISPR-Cas9 knockout screens in New Zealand
Published in Journal of the Royal Society of New Zealand, 2018
Francis W. Hunter, Peter Tsai, Purvi M. Kakadia, Stefan K. Bohlander, Cristin G. Print, William R. Wilson
There are, of course, caveats. CRISPR as a forward genetics tool is applicable only to phenotypes that are cell-autonomous, and thus identifiable in large populations of single, independent cells. The current sgRNA libraries do not necessarily mutate all isoforms from each gene. This is one of several reasons for considering the use of Cas9 libraries that inhibit gene expression, in parallel with knockout libraries (Rosenbluh et al. 2017). True saturation mutagenesis that includes non-coding RNA genes and intergenic regions is currently only possible for small regions of the genome. In addition, many technical challenges are being defined as the technology matures (Aguirre et al. 2016), including control of Cas nuclease-mediated toxicity and off-target mutagenesis (Morgens et al. 2017).