A strategic approach of enzyme engineering by attribute ranking and enzyme immobilization on zinc oxide nanoparticles to attain thermostability in mesophilic Bacillus subtilis lipase for detergent formulation

Abstract

The present study envisaged rationalized protein engineering approach to attain thermostability in a mesophilic Bacillus subtilis lipase. Contributing amino acids for thermostability were analyzed from homologous thermophilic-mesophilic protein dataset through relative abundance and generated ranking model. Analyses divulged priority of charged amino acids for thermostability. Ranking model was used to predict thermostabilizing mutations. Three lipase mutants, bsl_the1 (V149K, Q150E), bsl_the2 (F41K, W42E, V149K, Q150E) and bsl_the3 (F41K, W42E, P119E, Q121K, V149K, Q150E) were generated and validated through in silico and in vitro approaches for improved activity and thermostability. ZnO nanoparticles were synthesized by precipitation method and functionalized using polyethylenimine, APTES and glutaraldehyde for lipase immobilization. The immobilization was confirmed through various analytical techniques. Analysis revealed bsl_wt showed optimum activity at 35 °C and pH 8 which was increased to 60 °C and pH 10 in case of ZnO-bsl_the3. The ZnO-bsl_the3 showed 80% of their initial activity after 60 days of storage stability and retained 78% of activity after 20 cycles of reuse. Lipases were applied for oil and grease stain removal from fabric. ZnO-bsl_the3 removed 90% and 82% of oil and grease stains, respectively. Conclusively, it revealed a promising perspective of low-cost nanobiocatalysts in detergent formulation.