Purification, characterization and applications of keratinase and protease from a newly isolated Bacillus cereus H10 from hog hair
Date Issued
2008
Date
2008
Author(s)
Huang, Ju-Chieh
Abstract
Keratin, which is increasingly accumulating in the environment mainly in the form of feathers and hair, becomes a part of solid waste management. However, keratin is difficult to degrade due to its highly rigid structure rendered by extensive disulfide bonds and cross-linkages. There is a demand for developing alternatives for recycling of such wastes. Thus, the purpose of this study was to purify and characterize a keratinase secreted by a high keratinolytic bacterium isolated from hog hair. In addition, the potential for utilization of the hydrolyzates of hog hair and hog hair meal as ingredients in animal feed was also evaluated.solation results indicated that a bacterium designated H10 from hog hair showed high keratinolytic and proteolytic activities. Further identified by PCR-DGGE and 16S rRNA sequencing, H10 was placed in a cluster with Bacillus cereus. The unique of this strain is comprised two types of extra-cellular proteins: proteolytic and keratinolytic. After purification of these enzymes secreted by B. cereus H10 using ion exchange chromatography, the specific activity of the finally purified enzyme for proteolysis and keratinolysis was 29080 and 6391.25 U/mg, respectively. The molecular mass of keratinase and protease were 45.4 kDa and 102.3 kDa, respectively. The enzyme sample possessed two proteases which displayed a very different active temperature and pH range for proteolysis and keratinolysis. Response surface methodology (RSM) combining with sequential quadratic programming (SQP) helps in evaluating the effect factors and in building models. Thus, RSM with SQP was used in the following work to develop a prediction model for the optimal pH and temperature for the enzyme sample from B. cereus H10. Optimization results indicated that the optimal pH and temperature were at pH 7.57 and 59 °C. The results on the biochemical properties showed that both proteases belonged to the metalloproteases and were stable toward solvents, detergents and reduced agents. Among the metal ions, calcium and zinc ion showed an inhibition for enzyme activities, but the keratinase was activated by ferric ion. On the keratinolytic activity, the enzyme displayed stability at 59, 70 and 80 °C, with more than 95% of its initial activity up to 2hr, whereas, the proteolytic activity only remained 30~40% activity.valuating the possible applications of this enzyme, immobilized enzymes could stand higher temperature than free enzymes on both activities. After 6 times usages, the immobilized enzymes displayed stability with more than 80% of its initial activity, whereas, the proteolytic activity also remained 40~60% activity. Testing the protease activity on various protein substrates indicated this enzyme sample could easily hydrolyze hog hair meal which showed no significant difference with hydrolyzing soy bean meal. However, it displayed a relatively low digestibility for feather meal and hog hair. Further comparison with proteinase K, the hydrolyzation of hog hair by B. cereus H10 enzyme was 2-fold higher than that of proteinase K. Nutrition improvement test proved that hydrolyzates of hog hair and hog hair meal might have potential for utilization as ingredients in animal feed. n conclusion, this enzyme sample is very effective in hog hair degradation and shows the high thermal stability, presenting potential use for biotechnological processes involving keratin hydrolysis. To the best of our knowledge, this is the first report on isolation and purification of keratinase and protease from hog hair and cooperation of these two enzymes resulting in the effective degradation of keratin.
Subjects
Bacillus cereus
hog hair
keratinase
protease
purification
SDGs
Type
thesis
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