Characterization,Heterologous Expression And Molecular Modification Of Brevibacillus Parabrevis Keratinase

Keratinase is a kind of protease,capable of hydrolyzing insoluble keratin substrate,which makes it potentially used widely in leather,detergent and medical industries.However,the native keratinase usually has some disadvantages,such as low catalytic efficiency and poor thermal stability,which hinder its industrial application.Therefore,a series of research on microbial keratinase was carried out from theory to application,including screening of keratinase-producing strains,purification and characterization,cloning and expression of keratinase gene,molecular engineering and dehairing application of keratinase.The main research results are listed as following:Samples were collected from keratin-enriched environment,such as leather factories and poultry ranches.Then,the keratinase-producing microorganisms were isolated by using keratin as the sole carbon and nitrogen source.Finally,a total of twenty-eight strains with high keratinase activity are derived.Collagenase activity was also determined,as the strains with low collagenase activity has potential in dehairing performance and they could minimize the damage on animal skin.Finally,the two bacterial strains,R-1 and R-7,were found to display high keratinase activity and low collagenase activity.Based on the morphological and biochemical characteristics as well as 16 S rRNA gene sequencing,the bacteria were identified as Acinetobacter sp.and Brevibacillus parabrevis,respectively.Meanwhile,Gibberella intermedia with high keratinase activity was screened from the culture collection of our lab.The dehairing performance of the three types of microbial keratinase were evaluated.The results showed that the keratinase could dehair wool from skin,and B.parabrevis keratinase displayed the highest dehairing efficiency,accomplishing the dehairing process of 7 h.In considering of the keratinase activity,the superior dehairing efficiency and the preferred collagenase activity,B.parabrevis was thus chosen for further study.B.parabrevis keratinase was purified to be explored its catalytic properties.The keratinase was purified to electrophoretic homogeneity by ammonium sulfate precipitation and DEAE fast flow from supernatant.The keratinase had an estimated molecular weight of 28 kDa,which was analyzed by SDS-PAGE and MALDI-TOF/TOF MS.Besides,the keratinase showed optimal activity at 60°C and pH 8.0.More than 80% of the keratinase activity was remained at pH 6.0-9.0 and 20-40°C after 1 h.The enzyme activity could be significantly increased in the presence of Na+ and Ca2+.But it suffered from inhibition by Zn2+,Mn2+,Fe2+,Cu2+ and Co2+.And it was inhibited by EDTA and PMSF,which indicated that the keratinase belongs to serine-metallo protease.The enzyme could remain stable in the presence of surfactants.Especially,5 mM Tween 40,Tween 80 and Triton-X 100 could improve the keratinase activity by 11%,8% and 30%,respectively.The substrate specificity results showed that B.parabrevis keratinase exhibited no activity toward type-I collagen.The kinetic parameters Km and Vmax were determined to be 15.67 mg?m L-1 and 1666.67 U?mg-1?min-1,respectively,with keratin as substrate.The keratinase gene from B.parabrevis was cloned and heterologously expressed.Using B.parabrevis genome DNA as template,PCR was carried out.We obtained the keratinase gene(kerbp)sequence of 1152 bp,encoding 383-aa protein chain,which was comprised of 31-aa signal peptide,77-aa propeptide and 275-aa mature peptide.The target gene was transformed into E.coli BL21(DE3)host strain.And the recombinant strain,E.coli BL21(DE3)/pET-22b(+)-kerbp,was constructed.After purification of recombinant keratinase to electrophoretic homogeneity by using Ni-NTA affinity column,it is found that the molecular weight was similar with that of the native keratinase.And the catalytic sites(Asp-32,His-64 and Ser-221)were confirmed by overlap extension PCR,which laid the foundation for the further molecular modification.The dehairing study showed that recombinant keratinase could depilate hide.And lipase and amylase were chosen to mix with keratinase in the system.The results showed the synergistic effect by lipase,amylase and keratinase was observed in depilation process.In order to further improve the applicable property,the computation software was adopted to design the modification strategy to improve the thermostability of keratinase.The 3-D model of the keratinase was constructed by SWISS-MODEL online software.Based on analysis of the normalized B-factor,molecular dynamic calculated by professional software and alignment of proteases,it was deduced that eight amino acids in the flexible loop structures were crucial for the thermostability of the keratinase.Of the 9 variants of those key amino acids by site-directed mutagenesis,three mutants(T218S,S236 C and N181D)had positive effect on thermal stability,with improvement of half-life at 60°C by 3.05-,1.18-and 1-fold,respectively;and with the increases of T50 by 5.4°C,4°C and 2°C,comparing to WT.We have constructed the recombinant mutant of the 3 amino acid sites.The results showed that it did not display further improvement effect.Only N181D-T218 S and N181D-S236 C showed better catalytic efficiency and thermal stability than WT.Comparing to WT,T50 of those 2 mutants were enhanced by 5.1 and 2.9°C.Additionally,half-life at 60°C prolonged with 4.09-and 1.54-fold,respectively.The reasons for thermostability improvement might be that the substitutions could result in the extra hydrogen bond formation.Especially,S236 C mutant resulted in a new salt bridge,beside of hydrogen bond.Comparison of local vacuum electrostatics potential of WT and mutants showed that the decrease of protein surface charge make the protein structure more compact.