Purification and characterization of a novel extracellular neutral metalloprotease from <i>Cerrena albocinnamomea</i>
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- Hamada Shigeki
- Faculty of Agriculture and Life Science, Hirosaki University
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- Kubota Keisuke
- Faculty of Agriculture and Life Science, Hirosaki University
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- Sagisaka Masanobu
- Faculty of Science and Technology, Hirosaki University
抄録
<p>We selected a fungus secreting a neutral protease from soil and identified it as the basidiomycete fungus Cerrena albocinnamomea according to its ITS-5.8S rDNA and 28S rDNA-D1/D2 sequences. A major extracellular protease isolated from C. albocinnamomea was purified approximately 44-fold through two purification steps. SDS-PAGE analyses of the purified protease revealed a single band, and its molecular mass of 39,756 Da was determined using MALDI-TOF-MS. The enzyme was optimally active at approximately pH 7.0 and 45°C. The Km and Vmax values for the hydrolysis of azocasein were 2.46 mg/mL and 989 units/min/mg protein, respectively. The enzyme was stable at pH 3.6–8.6 for 16 h and at temperatures ≤35°C for 1 h. Enzymatic activity was completely inhibited by Cu2+ and Zn2+ and markedly by EDTA and phosphoramidon. The N-terminal amino acid sequence ASYRVLPIT is highly similar to those of the members of the metalloprotease family M36, such as keratinase and elastinase. However, the protease did not detectably hydrolyze keratin or elastin. In contrast, the protease hydrolyzed fibrinogen, although there were no significant sequence similarities to the N-terminal amino acid sequences of other fibrinolytic enzymes. These results suggest that the purified protease represents a new neutral metalloprotease with fibrinogenolytic activity.</p>
収録刊行物
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- The Journal of General and Applied Microbiology
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The Journal of General and Applied Microbiology 63 (1), 51-57, 2017
公益財団法人 応用微生物学・分子細胞生物学研究奨励会