Investigation On Screening Microbial Strains Of Producing Keratinase And Its Fermentation Conditions

A large number of poultry feathers and wool wastes were produced in China each year.The main components of poultry feathers and wool wastes are keratin.Keratin is resistant to degradation by common proteases such as trypsin,pepsin and papain due to its high degree of disulfide bridges,hydrogen bonds and hydrophobic interactions.So most of them have not been utilized,even some has caused environmental pollution.Many kinds of microorganism are able to produce keratinase.Feathers and wool waste keratin could be degradated into soluble protein,peptide or amino acid by keratinase,turning waste keratins into treasure and reducing pollution.Keratinase also could be used to modify wool surface.In this study,screening of keratinase produced strains and fermentation conditions were investigated using wool keratin as substrate.In this work,50 microorganisms strains,which were able to grow on wool as the sole carbon and nitrogen source,were screened from 7 soil samples of different sources.After the initial identification of morphology of colony and mycelidium, 32 of 50 strains were actinomycete and 18 of 50 strains were bacteria.A bacteria strain FYM601 which had the maximal keratinase activity was gotten from first screened 50 strains by shakeflask rescreening.its ability of degrading wool was higher,and its keratinase activity was 10.1U/mL.Since keratinase activity of FYM601 was low,which did not meet requirements of industry.ultraviolet mutation and microwave mutation were used to improve keratinase activity of FYM601.Comparing with UV irradiation mutation,improvement with microwave mutation was poorer.The keratinase activity was 1.5 times as many as the parent strain.A strain UV09 was screened by UV irradiation mutation,whose keratinase activity was 26.0U/mL and soluble protein was 0.217mg/mL,was 2.6 and 2.4 times as many as the parent strain respectivly.Culture conditions and medium composition had significant effects on cells growth and producting keratinase.In the study,the effects of culture conditions and medium composition on keratinase production of UV09 were investigated.The optimal culture conditions were as follow:wool concentration 1%,temperature 37℃,pH 9.0,inoculum10%,rotating rate 180 r/min and fermentation time 72 hours.The keratinase activity and soluble protein were 42.3U/mL and 0.616 mg/mL respectivly.In the study,it found that adding carbon,nitrogen and metal ions had significant effects on keratinase prduction of UV09.keratinase activity was improved by adding glucose(2%),soluble starch (2%) and sucrose (2%). but lactose had inhibition on keratinase activity . The maximal keratinase activity (49U/mL) was gotten with glucose(2%).Four kinds of inorganic nitrogen sources were tested. Inhibition of (NH4)2CO3 added on keratinase produced n was significant,whose keratinase activity was only 40% of the control.Among four organic nitrogen sources,0.1% casein could intensively stimulate the keratinase activity,achieving 77U/mL and increasing by about 70%. But keratinase produced was inhibited largely by 0.1% beef extract.In the study,it also found that keratinase produced was stimulated by Mn2+,while Mg2+,Ca2+ inhibited significantly and reduced keratinase produced activity by about 25%.