Screening And Fermentation Conditions Optimizing Of A Keratinase-producing Strain And Its Characterization

Keratinase has important value in the production of biological products and the recycle ofwaste biological resources. However, the existing keratinase producing strains have theproblems of low enzyme production levels, long fermentation period and high productioncosts, moreover, most keratinases can’t meet the requirements of industrial production.Therefore, we still need to screen keratinase producing strains with excellent enzymaticproperties and catalytic ability. Meanwhile, constant optimization of keratinase productionwas also necessary to reduce production costs and improve the production of keratinase.1. In this study, a keratinase producing strain was isolated with wool as the sole carbonand nitrogen source. Physiological and biochemical characteristics and molecular-biologicalidentification confirmed that it was Bacillus pumilus, and named K9. The effects offermentation conditions and medium composition on keratinase by B. pumilus K9wereinvestigated, and the results showed that, the addition of appropriate carbon (maltose) andnitrogen source (yeast extract) could increase the production keratinase when using woolkeratin as substrate. The optimal culture conditions were as follows, maltose15g/L, yeastextract10g/L, wool concentration25g/L, K2HPO40.4g/L, NaCl0.5g/L, initial pH8.5,liquid medium volume25mL/250mL, inoculum age12h, inoculum size6%, incubationtemperature at35oC, rotating rate220r/min and cultivation time48h, The keratinase activityreached up to1270U/mL, which was about2.54-fold compared with the unoptimizedconditions.2. The keratinase purification was carried out using ammonium sulfate precipitation,anion-exchange chromatography and size-exclusion chromatography. The keratinase waspurified to be a homogeneus protein band with a molecular mass of32kDa. The purificationsteps resulted in an overall39.80purification fold. The total activity yield of keratinase was8.98%, with a specific activity of18267.63U/mg.3. The purified enzyme exhibited optimum activity at60oC and pH9.0. And thiskeratinase was stable at pH9-12. The enzyme could be activated in the presence of Na+, Ba2+,Sr2+, and Rb+. It was inhibited by PMSF and EDTA, indicating that it is a metallo-serinekeratinase. This enzyme could remain stable in the presence of surfactants, including SDS,Triton and Tween. Especially, SDS and Tween40could substantially enhance the activity by7%and54%, respectively. The Kmand Vmaxof B. pumilus K9with keratin as substrate was7.33mg/mL and291.54μg/mL·min, respectively.4. The keratinase showed high activity when the substrates were casein, keratin, wool andfeather, but only little degradation on type I collagen. This might be an importantcharacteristic for its future application on collagen production.