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Chitosan-alginate immobilized lipase based catalytic constructs: Development, characterization and potential applications

Rashid, Robina, Anwar, Zahid, Zafar, Muddassar, Rashid, Tayyba, Butt, Iqra
International journal of biological macromolecules 2018 v.119 pp. 992-1001
Aspergillus, alginates, ammonium sulfate, carboxylic ester hydrolases, chitosan, cross-linking reagents, dehairing, detergents, fractionation, gel chromatography, gels, glutaraldehyde, leather, metal ions, molecular weight, pH, polyacrylamide gel electrophoresis, tanneries, temperature
In this study, lipase (LIP) was isolated from Aspergillus crevinus, statistically optimized and purified via ammonium sulfate fractionation (ASF), and Sephadex G-100 gel permeation chromatography. LIP was 2.26-folds purified with a specific activity of 223.60 U/mg. The molecular mass was estimated to be 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5% stacking and a 12% resolving gel) (SDS-PAGE). The active LIP fraction was immobilized onto chitosan-alginate (CTS-ALG) beads developed in a uniform size, i.e., 2.0 ± 0.25 mm diameter using ultrasonically dispersed 2.0% (w/v) chitosan and alginate along with 0.5% (w/v) glutaraldehyde as a macromolecular crosslinking agent. Prior to exploit for detergent compatibility and dehairing purposes, various parameters including pH, thermal, Michaelis-Menten kinetic constants and influence of organic/inorganic and metal ions on PF-LIP and CST-ALG-LIPs fractions were investigated. The immobilized fractions were optimally active and stable over a broader pH (5–11) and temperature (75 °C) as compared to the free counterpart pH (7–8) and temperature (35 °C), respectively. However, the negligible difference between the KM and Vmax values of PF-LIP i.e., 0.133 ± 0.05 mg/mL and 255.0 ± 11.8 U/mL/min and CST-ALG-LIPs revealed that the conformational flexibility of LIP was retained as such. Comparative to PF-LIP, the CTS-ALG-LIPs were found much stable and retained most of their activity up to 80% in the presence of inhibitory molecules. After 75 min incubation, CTS-ALG-LIP3 retained >95% activity at pH 9.0 which was reduced to 80% at pH 10.0 and 44% at pH 11.0. Among all three samples, 100% dehairing was observed when the sheepskin was dipped for 30 min in CTS-ALG-LIP3. The dehairing of leather (sheepskin) was greatly affected by CTS-ALG-LIP3 rendering its potential candidatures for leather and tannery industry.