Production, purification and characterization of keratinase from hot spring Bacillus licheniformis HS1

• Main Authors: Li, Jing-Shan, 李靜姍
• Other Authors: Tsai, Guo-Jane
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/x47zr2

碩士 === 國立臺灣海洋大學 === 食品科學系 === 105 === 　　Feather is protein-rich waste product of poultry processing industries and it contains over 90% crude protein in a form of keratin. It is resistant to proteolysis by common proteases due to cross-linked disulfide bridges, hydrogen bonds, and hydrophobic interactions; however, keratin can be degraded by keratinolytic bacteria. In previous experiment, a keratinolytic bacteria (Bacillus licheniformis HS1) was isolated from hot spring soil, and the optimal condition for keratinase production were established with the initial pH 7.3 of a feather suspension at 45oC for 72 hrs. Feather meal was used as the substrate medium in this study. The optimal feather concentration and shaking rate investigated in flask scale. The keratinase activity of 3.92 U/mL was obtained by HS1 in 1.6% feather meal incubated at 45oC, 160 rpm for 72 hrs. The enzyme activity could be further increased 7.76 fold with 30.42 U/mL after incubation of HS1 in 5 L fermentor at 45oC with 500 rpm, 0.2 vvm for 72 hrs. The feather meal degradation percentage was 78.16%. The keratinase was purified by DEAE Sepharose CL-6B and Sephacryl S-400 HR cheomatography, with 16.50 fold purification and 10.76% recovery percentage. The purified keratinase is an alkaline metalloprotease with a molecular weight of 37 kDa. Its optimal temperature and pH value are 60oC and pH 8.0, respectively. It is stable over a broad temperature of 20-60oC , and the pH range of 5.0-11.0. Tween 20, 2-Mercaptoethanol and Zn2+ had positive effect on the keratinase activity. This protease can degrade various proteins including bovine serum albumin, fibrinogen and casein with promising industrial applications.