Functional Characterization And Thermostability Modification Of Deinococcus Keratinases

Keratin is a recalcitrant fibrous protein.Since keratinous substrates are rich with S-S bonds and hydrophobic structure,it can resist the common proteases such as pepsin and trypsin.Keratins can be found as major components in agricultural wastes.Due to the increasing production of feathers from poultry industries,the untreated feathers could become pollutants.Keratinase could specifically attack disulfide bond in the keratin to convert them from complex to simplified forms.The keratinase thermal stability has drawn attention to various biotechnological industries.It is significant to identify keratinases and improve their thermostability from microorganism in extreme environment,especially in hot environment.Therefore,in this study,the keratinases DgoKer and DgeKer were identified in Deinococcus gobiensis I-0 form the gobi and Deinococcus geothermalis from hot spring,respectively.Furthermore,the thermostability of keratinase was improved by molecular modification.The main results are described as follows:1.The analysis of sequence showed that DgoKer and DgeKer displayed similarity to MtaKer from M.taiwanensis WR-220 with 58.68% and 58.96% identity,respectively.The structure analysis found that DgoKer and DgeKer are composed of signal peptide,N-terminal propeptide,mature domain with a catalytic triad.The catalytic triad of DgoKer was formed by Asp171-His203-Ser358.And the catalytic triad of DgeKer was formed by Asp171-His203-Ser354.DgeKer also contained a C-terminal extension.Moreover,DgoKer shared 66.91%identity with DgeKer.2.In this study,DgoKer and DgeKer were expressed and purified by E.coli BL21.DgoKer showed maximum activity at 60°C and p H 7 with specific activity of 51147 mg/m L on soluble keratin.And DgoKer had a half-life(t1/2)of 46.52 min at 60?.DgeKer showed maximum activity at 70°C and p H 9 with specific activity of 40356 mg/m L on soluble keratin.And DgoKer had a t1/2 of 103.45 min at 60?.DgoKer and DgeKer could degrade feathers at 60°C.TheKer A from Bacillus licheniformis PWD-1 had a t1/2 of 26.46 min at 60°C.It can be seen that the t1/2 of DgoKer at 60? was higher thanKer A,but lower than DgeKer.3.In order to study the effect of C-terminal extension on the DgeKer thermal stability,the Cterminal extension-truncated enzyme(DgeKer-C)was constructed.DgeKer-C showed maximum activity at 70°C and p H 10 with specific activity of 41814 mg/m L on soluble keratin.DgeKer-C had a t1/2 of 169.10 min at 60?,which was 65.65 min higher than DgeKer.In short,the thermal stability of the mutation DgeKer-C was improved.4.In order to improve the temperature stability of DgoKer,the mutant protein DgoKer-3 was constructed based on schema-guided recombination.The result showed that the optimum temperature of DgoKer-3 was 60?,and the relative enzyme activity at 70? was 98%.DgoKer-3 displayed a 3.2-fold longer half-life(110.02 min)at 60°C than wild-type enzyme DgoKer.DgoKer-3 could rapidly degrade feathers within 60 minutes,while wild-type DgoKer only degrade part of feathers at 70°C.In short,the stability of mutation DgoKer-3 at 60°C and the enzyme activity at 70°C were improved.In summary,DgoKer and DgeKer keratinases were identified from Deinococcus microorganisms.The mutant keratinases DgoKer-3 and DgeKer-C with better thermal stability had been obtained by rational design,which have great potentials in industrial applications.