Purification and characterization of a keratinase from Stenotrophomonas maltophilia PU-LS07.

• Main Authors: Chia-ming Chang, 張家銘
• Other Authors: Yun-chin Chung
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/4er8xt

碩士 === 靜宜大學 === 食品營養研究所 === 97 === A feather-degrading bacterium was isolated from poultry manure. After sequencing the 16S rDNA and analyzing fatty acid profile, this strain was identified and named as Stenotrophomonas matophilia PU-LS07. The keratinase produced by PU-LS07 was induced by feather powder. Maximal enzyme production could be achieved by culturing PU-LS07 in a medium containing 0.1％ feather powder and 0.1％ yeast extract at 37℃ for 36 h. Maximal growth was achieved when PU-LS07 was cultured at 37℃ for 15 h. The crude keratinase produced by PU-LS07 in the culture broth was initially fractionated by Sephacryl S-200 HR gel filtration, which separated the keratinase into two isoforms, keratinase P1 and P2. The keratinase P1 isoform was further purified by PBE 94 chromatofocusing, which separated the keratinase P1 into a purified keratinase K1. By these steps, the purity of the keratinase K1 was increased by 17.7 fold with activity recovery of 12.9％. The optimal reaction pHs of the purified keratinase K1 were 8, 7 and 7~8 toward casein, soluble keratin and azokeratin, respectively; optimal temperatures were 40, 50~60 and 40℃; and the Km`s were 0.98, 2.89 and 14.5 mg/mL. The molecular mass of the purified enzyme was greater than 232 kDa, as estimated by gel filtration. The enzyme showed activity toward particular protein substrates as well as synthetic peptide substrates. Among the protein substrates, casein was the most sensitive substrate, followed by hemoglobin, fibrinogen, soluble keratin and so forth, respectively, in a decreasing order. Among the synthetic peptide substrates, N-succinyl-Ala-Ala-Pro-Phe-pNA was the most sensitive peptide substrate, followed by N-succinyl-Ala-Ala-Val- pNA. Among chemical modification agents, phenylmethanesulfonyl fluoride (PMSF), N-brmosuccinimide (NBS) and diethyl pyrocarbonate (DEPC), significantly inhibited enzyme activity, implying that serine, tryptophan and histidine were essential for enzyme activity. Incubaing the enzyme for 40 min at temperatures ranging from 30 to 80℃ revealed that the enzyme was thermally stable at the temperature range of 30 to 40℃. However, when the temperature was higher than 50℃, the enzyme activity was decreased significantly.