High-level Expression Of Keratinase And Its Application In Biosynthesis Of Silver Nanoparticles

Keratinase is a kind of special proteolytic enzyme that can degrade insoluble keratin.They have broad substrate spectrum and can be widely applied in the areas of medical,pharmaceutical,feed and textile industries.However,the current production level and enzymatic properties of keratinases cannot meet the requirements of commercial applications.Therefore,it is urgent to mine keratinases with favorable properties.Further genetic engineering pave the way for achieving the high-level expression of keratinase and optimization of its application properties.In this study,a series of work was carried out including mining of keratinase gene,heterologous expression,molecular modification of thermostability and its application in biosynthesis of silver nanoparticles.?1?A keratinase gene kerBv was successfully amplified from keratin-rich metagenomic DNA.The gene consisted of 1149 bp and encoded a protein of 382 amino acid residues.The keratinase gene was first expressed in Escherichia coli Rosetta?DE3?as intracellular enzyme,but it existed as the inclusion body.Bacillus subtilis had efficient secretory capacity.The recombinant keratinase could be released into the culture medium by its own signal peptide and the highest keratinase activity in the fermentation broth was 164.8 U/mL.The biochemical characterization of keratinase showed the optimum reaction temperature and pH were 60°C and10.0,respectively.Also,the keratinase displayed broad substrate spectrum.?2?In order to increase the production and application potential of recombinant keratinases,genetic engineering manipulation strategy was employed to realize the high-level expression of recombinant keratinase in B.subtilis WB600.Promoter is an important factor that affects the transcription of target gene.Selecting appropriate promoters can maximize the production of the recombinant protein.In this study,sixteen recombinant strains with promoter engineering were successfully constructed and seven of which showed an increase in the expression level.A maximum keratinase activity of 2605 U/m L was achieved in the fermentation broth by the recombinant strain with PaprE promoter.TB medium with addition of 15 g/L glycerol was used for the scale-up production of keratinase in a 5 L fermenter.The maximum keratinase activity in the fermentation broth was 7176 U/m L at 36 h,which was the highest production level reported in recombinant keratinase expression to date.This study may lay the foundation for the application of keratinase.?3?The recombinant keratinase had poor thermostability and its half-time was 14.7 min at60°C.A computer-aided site-directed mutagenesis strategy was carried out in this study to improve the keratinase thermostability.Based on the three-dimensional structure model of keratinase,eight sites in the highly flexible Loop region were selected for site-directed mutagenesis.The thermostability evaluation of the mutant keratinases showed that the thermostability of the N218S mutant was significantly improved and its half-life at 60°C was50.6 min,3.4 times higher than that of the control.?4?The keratinase displayed strong reducing power which was probably due to its disulfide bond-reducing activity towards keratin.On the base of previous strategies of genetic engineering manipulation,the keratinase was applied in the biosynthesis of silver nanoparticles.After optimization,silver nanoparticles were prepared with 200 U keratinase and 1.0 mM AgNO₃ after 48 h of reaction.The characterization results showed that the silver nanoparticles were uniform in size,which had a size range of 3-15 nm.The AgNPs had an absorption peaks at 420 nm and a Zeta potential of-22 mV.The antibacterial experiments showed that 100?g/mL of silver nanoparticles displayed good antibacterial activity.