Biocatalysts: application and engineering for industrial purposes.

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

Expression of A. niger US368 xylanase in E. coli: purification, characterization and copper activation.

The XAn11 cDNA was cloned in pET-28a(+) and the recombinant plasmid was transformed in Escherichia coli. The His-tagged r-XAn11 was purified using Ni-NTA affinity and anion exchange chromatography. The enzyme showed a specific activity of 415.1 U mg(-1) and a molecular mass of 25 kDa. It had an optimal activity at pH 5 and 50°C. It was stable in a wide range of pH and in the presence of some detergents and organic solvents. In the presence of 3mM Cu2+, the relative activity of the His-tagged r-XAn11 was enhanced by 54%. This is the first work reporting that copper is a strong activator for xylanase activity making this enzyme very attractive for future industrial applications. Molecular modeling suggests that the contact region between the catalytic site and the N-terminal His-tag fusion peptide could be responsible for the different behavior of the native and recombinant enzyme toward copper.

Changes in the catalytic properties and substrate specificity of Bacillus sp. US149 maltogenic amylase by mutagenesis of residue 46.

Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the K(m) values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward ß-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium.

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/mL) obtained for CGTase activity was very close to the predicted value (9.27 U/mL).