| Keratinases are a special kind of proteolytic enzymes that display the capability ofdegrading keratins. It can be used in animal feeding, textiles processing, detergent andmedicine. In this study, a Bacillus licheniformis BBE11-1with high-efficiency degradation ofwools was screened. The extracellular expression level of keratinase was enhanced byoptimizing the expression hosts, regulatory elements and culture conditions. Furthermore, therecombinant keratinase was characterized and modified to enhance the thermostability andα-keratin activity.(1) The strain of B. licheniformis BBE11-1that can efficiently degrade wools wasisolated. The ker gene was after expressed in E. coli, B. subtilis and P. pastoris, respectively.After optimized the concentration of IPTG and yeast concentration, the highest extracellularkeratinase activity obtained by recombinant E. coli was350U·mL-1. Besides, the highestkeratinase activity of recombinant B. subtilis WB600/pMA5-ker was386U·mL-1afteroptimized the carbon source and divalent mental ions. In another side, the keratinase activityof recombinant pPIC9K-ker/P. pastoris GS115was acheved at285U·mL-1when induced with10g·L-1methanol, the recombinant keratinase produced by P. pastoris was glycosylated. Theproduction of keratinase by recombinant B. subtilis WB600/pMA5-ker, B. subtilisWB600/pSTOP1622-ker and P. pastoris GS115/pPIC9K-ker were performed in a3-Lfermentor. The results showed that, the recombinants of B. subtilis WB600/pSTOP1622-kershowed better performance in production of keratinase, which obtained the highest keratinaseactivity of1840U·mL-1.(2) The corresponding recombinant keratinases were further purified and characterized,respectively. The optimal pH and pH stability of recombinant keratinases were similar,whereas the optimum temperature and thermostability of kerP were enhanced compared withKerE and KerB. The recombinant keratinases were serine proteases and exhibited same thepresence of metal ions, chelating agent and inhibitor. Moreover, KerE, KerB and KerPexhibited unusual stability in the presence of surfactants and H2O2. Kinetic parameters KmandVmaxof KerB were observed to be2.42mmol·L-1and35.84mmol·min-1, the kcatand kcat/Kmwere22.61s-1and9.34L·(s·mmol)-1, respectively. Coordinate with Savinase (1%OWFSavinase and60%OWF keratinase), the keratinase could efficient prevent shrinkage andeliminate fibres of wool at one hour. Furthermore, when treated with60%OWF keratinasealone can also prevent shrinkage well without tensile strengthen loss.(3) The computational design (B-FITTER, PoPMuSiC and NAMD) and consensusmutagenesis were applied to predict flexible sites of protein. Based on the results,12mutantswere constructed and4mutants (N122Y, N217S, A193P and N160C) have positive effect. Ofthese mutations, the N122Y substitution mutant best enhanced the half-life time at60°C, from9to23min. This substitution also led to an approximately5.6-fold increase in catalyticefficiency compared to that of the wild-type keratinase. The quadruple mutant displayedsynergistic or additive effects with an8.6-fold increase in the t1/2value at60°C. The reasonwas that the substitutions could result in the addition of six new putative hydrophobic interactions, one new putative cation-pi interactions, and11new putative hydrogen bonds.Among them, N122Y added three new putative hydrophobic interactions.(4) To improve the specificity of keratinase toward α-keratin substrate (wool scales),mutations were introduced at the bottom of S1and S4pocket in keratinase to change the sizeof pocket to investigate its effects on the substrate specificity of keratinase. The resultsshowed that, the specificity of keratinase towards the α-keratin could be decided by theconformation of substrate binding pocket. Increase the size of S4pocket, especially for theM134A mutant keratinase can efficiently improve the specificity of keratinase for α-keratin.Amino acids analysis, XPS experiment and kinetic analysis revealed that this preference ofM134A for α-keratin probably dues to it improves the affinity of S4pocket toward cystine,lysine and aspartic acid residues. The mutant M134A also provides satisfactoryshrink-property to wool fabrics compared with the wild-type keratinase.(5) Mutations were introduced at cleavage region of propeptide of keratinase toinvestigate it effect on the keratinase production. The results showed that, L(P1)A mutantcould facilite the secretion of mature keratinase. In addition, changed in primary structure atthe C-terminus of propeptide also affect the mature keratinase secretion. Deletion andreplacement of N-terminus of keratinase resulted in enzyme activity decreased prominently,indicated that the N-terminus of keratinase was important for enzyme activity. Althoughreplacement of N-terminus from thermitase decreased the activity of keratinase, thethermostability of mutant was enhanced compared with wild-type. The half-live of thermalinactivation (t1/2) was enhanced from9to20min at60°C. Moreover, the feeding rate ofglucose and induced cell density were investigated, separately. The highest yield of keratinasein recombinant B. subtilis WB600/pSTOP1622-ker was3017U·mL-1when constant feedingwith6g·(L·h)-1glucose and induced at OD600=15, which is the highest yield by recombinantstrains ever reported. |
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