| Microorganism Keratinases have wide substrate specificities,which do not only degrade soluble protein substrats,but also degrade insoluble protein substrates.Because of its superiority,keratinases are considered as good replacements of traditional proteases in detergent additives,leather,textile,feed additives and other industries.For its strongly hydrolytic abilities toward keratin substrates such as feather,wool,hoof,hair and so on,which are hardly degraded by common proteases,keratinases are widely used in leather,textile and keratin wastes degradation industries.Till now,the aim to isolate keratiase-producing microorganisms and to study their keratinase industrial and commercial applications,are considered as important,but not solved problems.There are many microorganisms with high keratinase-producing abilities stored in our lab,and we chose three typical strains from them for further study.1.Bacillus subtilis Y3-4 has strong ability to degrade wool(a-keratin)and feather(?-keratin).In order to study its keratinase,we partly purified its extracellular fermentant and studies the basic characterization of the crude keratinase such as optimal catalytic temperature,and the effects of reducing agents and detergents on the keratinase activities.We also studied applications of keratinase in dealing with keratin wastes.2.As the main extracellular production of Bacillus subtilis FJ-3-16,keratinase KerFJ has high specific activity,good performance in resistance,and has many applications in degrading keratin wastes,as detergent additives and used for enzymatic dehairing.Here we optimized the keratiase-producing conditions of the strain using one-time-variation,Plackett-Burman design and Response surface method,and built a keratinase fermentation method relying on 50 Liter fermenter.We tested the dehairing effects of the fermentant on the hardly-dehaired pig fur and hoof.Beside these,We also studied the keratinase gene kerFJ heterogenous expression in Pichia pastoris.3.The strain Bacillus cereus Y-15 which was isolated from the silt of a sewage pipe which was submerged in feather wastes for long times,has a strong feather degradation ability.We observed the structure disruption of feathers during the degradation process by visual oberservation and Scanning Electron Microscope(SEM).We also optimized its keratiase-producing conditions using RSM method.These work applied good platform for further applications of the strain.The results are as follows:1.The partly purified keratinase of B.subtilis Y3-4 has optimal catalytic activity at 55? and pH8.0 for keratin hydrolysis,which indicated it belonged to a mesophilic,alkaline keratinase.The keratinase was stable at pH 6.0-10.0 range,and lost its activities very fast when the temperature exceed 50?.Its activity was almost inhibited by PMSF(serine protease inhibitor)totally and not inhibited by 1,10-Phenanthrolinemonohydrate(specific chelates zinc ion),which suggested that it is a kind of serine keratinase.The metal ions chelating agents EDTA and EGTA inhibited 80%-90%of the keratinase activity.The reducing agents DTT and ?-ME were defined as effective reagents to improve its keratinase activity,but DTNB had no effect on it.Metal ions Cu2+,K2+ and Ca2+ could improve the keratinase activities,but Ni2+ and Co2+ effects were conversely.The keratinase resistance toward oxidate agents such as H2O2,NaCIO and sodium cholate was not good,but its activities were improved or almost not changed in reducing agents exisiting conditions.These suggested that the keratinase may have a specific resistance to unfavorable environments.The activites of the keratinase were stable in some laundry detergents,and it showed good performance in decontamination as detergent additives.Beside these,the keratinse also showed a good performance in dehairing.2.The optimized ferment medium was described as follows:corn meal 1.41%,soybean flour 3.62%,K2HPO4 0.3%,casein 2%,CaCl2 0.16%,KH2PO4 0.1%(pH 8.0)and the cultivation condition is at 37? for 48 h,with a rotate shaking of 250 r/min.The total activities of the extracellular keratinase were increased from 73.82 U/mL to 166.08 U/mL,increased by 1.25 times.The total activities of the extracellular keratinase were further increased to 207.6 U/mL in the amplified 50 L fermenter,and the fermentative time was greatly shortened from 48 h to 18 h at the same time.The dehairing effects of the ferment toward of the skins of the Bama pig's hoof and head were excellent.When dehairing small pieces,it took 6 h.When dehairing the whole hoof,it took 10 h.Using 20 Liter fermentation could deal with 40 kg hoof/head/fur.The products showed intact surface after dehairing.This study applied a platform for the application of strain B.subtilis FJ-3-16 in hardly-dehaired pig's dehairing industry.In addition,we sequenced the whole genome sequence of the B.subtilis FJ-3-16 strain and obtained its keratinase gene kerFJ.The gene was amplified and and heterogenous expressed in Pichia pastoris,finally eight transformants were obtained.3.B.cereus Y-15 strain was grown rapidly in the medium with feather as a sole carbon and nitrogen source,the feather-degradation rate was 47.5%at 48 h,and the feather was almost completely degraded after cultivation lasted for 5 d(120 h),these data illustrated that strain Y-15 has a strong ability in feather degradation.The structural desruption of feather after enzymatic hydrolysis was observed by visual observation and SEM.The results illustrated that the whole structure of the feather,including the pinnule and pinna rachis,was destroyed seriously during the hydrolytic process.The optimized medium was:corn meal 2.23%,bran 2%,bean pulp 0.88%,K2HPO4 0.1%,CaCl2 0.22%,KH2PO4 0.1%,the keratinase activity was greatly improved by 20 times after optimization.Studies on keratinase thermal stability experiment showed that the enzyme has good thermal stability and the half-life is 20 minutes when incubating in 70?.Meanwhile,the keratinase has good pH stability,and has good resistance toward detergents.All these characterization indicated its prospect applications in keratin wastes degradation. |
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