Research On Keratin-degrading Bacteria With High Disulfide Bond-Reducing Capacity

Keratin is stable and hard to be degraded duing to high degree of cross-linking of disulfide bonds,hydrophobic interactions, and hydrogen bonds. The key steps of degrading keratin are breakingdisulfide bonds and destroying its spatial structure. Comparing with the physical and chemical methods,biodegradation keratin is more moderate, pollution-free and lower amino acid damaging. In this paper,bacteria with high keratinolytic and disulfide bond-reducing capacity would be screened, and theirkeratinolytic mechanisms, fermentation conditions and keratinase properties would be explored toprovide guides for keratin degrading efficiently.Screening bacteria with high keratinolytic and disulfide bond-reducing capacity. In this part,27keratin-degrading bacteria were screened from decomposed feathers and sheep fur. According toclearance zone diameter and complete feather degradation,4strains with high keratinolytic activitywere gotten and named CP-7、CP-12、CP-16、YMD-5. Especially, the strain CP-7and CP-16, whichwere both identified as Bacillus licheniformis by colony morphological characteristic observing and16SrDNA sequeneing, could completely degrade native feathers in132h and96h, respectively. Furtherly,the fermentation process of4strains screened and strain10（Stenotrophomonas maltophilia SHMP-2）previous reserved was monitored, results showed that sulfhydryl group increased significantly infermentation broth of strain CP-16,CP-7and strain10, indicated that disulfide bond was restored largelyand they contained high disulfide bond-reducing capacity.Keratin degradation mechanism. Intracellular disulfide reductase activity of the strain CP-7,CP-16and strain10was detected, but extracellular broth was opposite. Intracellular broth was no keratinolyticactivity, but intracellular broth could significantly enhanced extracellular keratinolytic（feather power,wool power） activity （P <0.01）, while the caseinolytic activity was negative. In this part, sulfiteproduction was positive. In addition, whole feather degradation only occurred with live bacterial cells,while these enzymes alone alone failed to degrade the feather. These results suggested that intracellulardisulfide reductase may be one of breaking disulfide bonds reasons and keratin degradation mechanismsfor the three bacteria, sulfitolysis and bacteria may also play a important role in feather degradation bythe bacterium.The characteristies of Bacillus Licheniformis CP-16and strain10crude enzymes. The optimumtemperature and pH of Bacillus Licheniformis CP-16keratinase activity with better temperature andalkaline tolerance were50℃and pH9.0. Keratinase activity was inhibited strongly by PMSF（1mM）,had a5-fold and1-fold enhancement in presence of DTT and β-ME, respectively. This enzyme belongsto a thiol-activated and thermostable alkaline serine protease. The keratinase activity was significantlyinhibited by SO2--3（10mM）, S22O3（10mM）and Zn²⁺. In addition, it has high keratinolytic andelastinolytic activity without collagenolytic activity. The optimum temperature and pH of strain10keratinase activity were45℃and pH9.0, keratinase activity was completely inhibited by EDTA at10mM, belongs to metalloproteinases. The reducing agent, DTT and β-ME, extremely increased the keratinase activity, while S2O²⁺₃could significantly inhibit keratinase activity（residual31.79%）, butSO²⁺₃（10mM） was positive （enhanced more than71.83%）, and then keratinase activity wassignificantly inhibited by Zn²⁺and Cu2+（P<0.01）.Optimizing the fermentation conditions of Bacillus Licheniformis CP-16. The results showed thatthe optimal fermentation conditions were temperature40℃and initial pH6.5. Adding carbon andnitrogen sources， the results showed that1%soluble starch and0.1%ammonium sulfate couldsignificantly stimulate keratinase-producing（P<0.05）, while sucrose, glucose and maltose stronglyinhibited keratinase-producing, peptone and ammonium chloride were no effects.