J Peddu, C Chitturi, V Lakshmi
bacillus sp., feather degradation, keratinase
J Peddu, C Chitturi, V Lakshmi. Purification and Characterization of Keratinase from Feather Degrading Bacillus sp.. The Internet Journal of Microbiology. 2009 Volume 8 Number 2.
Microorganisms producing keratinases are gaining prominence in recycling of poultry waste feather. Keratinase producing
Feather is generated in bulk quantities as a by-product of poultry industry. It is estimated that 400 million chickens are processed every week. Typically as each bird has upto 125gms of feather, the weekly worldwide production of feather waste is about 3000 tons. Piling up of this waste material result in accumulation of dumps. Disposal of this bulk waste is a global environmental problem accounting to pollution of land and underground water resources. Thus, feather in spite of being made up of almost pure keratin protein is neither profitable nor environmentally friendly forming a produce of high volume with low profit margin (Mc Govern 2000)..
Various strategies are adopted to handle the volume of waste accumulating continuously. Feather waste generated by the member firms is disposed off to waste disposal sites, incinerated, sent to the feather meal industry or to gardeners/ farmers. These methods are inefficient or expensive adding to the cost for the producer. Traditionally, feather is processed by mechanical or chemical treatment and converted to feedstuff, fertilizers, glues, foils
Keratinases are emerging to play a vital role in the degradation of keratin and its conversion into digestible animal feed of higher nutritive value. Keratinases are produced by several microorganisms of which Bacillus
Viability of the enzyme production depends on the extent of downstream operations required for purification and extraction of enzyme, which is mainly determined by its application potential of the end product. Highly purified enzymes are required for applications in medicine and as pharmaceuticals. However, for most of the applications of keratinase including bioconversion of feather into animal feed, leather processing
Materials and Methods
The fermentation of two improved
Results and Discussion
Keratinase activity of the culture filtrate and the cell free extract was determined to find the localization of the enzyme. The activity of the culture filtrate was > 300 KU/ml for the isolates. On the other hand negligible enzyme activity was observed in the cell free extracts (only 0.04-0.07KU/ml) indicating that the keratinase was secreted into the medium and was thus primarily extracellular. The results of partial purification by ammonium sulfate fractionation showed that maximum purification was observed at 80% saturation with specific activity of the partially purified keratinase enzyme being 173-193KU/mg protein compared to that of culture filtrate which was about 6.9KU/mg to10.8KU/mg (Table1). Thus fractionation resulted in 16-27 fold purification. Specific activity of 85-86 units/mg protein was observed for keratinase enzyme purified from several
1-Marker, 2-MBF11, 3-MBF21
PPKs had optimum activity at 55C (398-635KU/ml) indicating thermo-tolerant nature of the obtained enzymes. However, good activity was observed between range 37C - 55C indicating versatility of application (Table 3). Further increase in the temperature resulted in decline in the enzyme activity. Optimum temperature of activity from majority of the mesophilic microorganisms producing keratinase has been observed to be between 28-45°C (Allpress
The reducing agents tested exhibited an enhancement in the keratinase activity
EDTA had a partial inhibitory effect on the activity of MBF enzymes whereas serine protease inhibitor PMSF strongly inhibited the activity (Table 5). There was significant inhibition of enzyme activity with benzimidine hydrochloride also. The results indicate that the keratinase produced by the MBF 11 and 21 isolates can be categorized as serine proteinase. Keratinase enzyme of serine protease group are also reported earlier to be strongly inhibited by inhibitor PMSF and benzimidine hydrochloride while EDTA exhibited only partial inhibition which were comparable with the results obtained in the presence study (Bockle
Solvents like DMSO, isopropanol and acetone, showed marginal inhibition of enzyme activity between 1-10% concentrations for MBF21. However, with PPK from MBF11, the enzyme activity was slightly enhanced with acetone at 5-10% concentration as compared to the controls (Table 5).Similar to our observation some of the keratinases characterized earlier are found to be quite stable in presence of organic solvents also (Mitsuiki
The response to SDS treatment varied with concentration. Similarly, slight enhancement in activity of the PPKs was observed on treatment with Triton X 100 at 0.5% concentration (Table 5). Proteins are known to be denatured by surfactants at concentrations higher than their critical micelle concentration (Henley and Sadana, 1985; Chaplin and Bucke, 1990; Rao and Deshpande, 1998).
The results of the present study show that serine protease group of keratinase obtained from MBF isolates are robust with good stability and can have versatile application potential.
The authors gratefully acknowledge Department of Biotechnology, New Delhi for the financial support.