Purification and characterization of a novel fibrinolytic 伪 chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji

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• 作者：Sung-Min Moon¹ ; ^&Dagger ; ; Jae-Sung Kim¹ ; ^&Dagger ; ; Heung-Joong Kim¹ ; Mi Suk Choi¹ ; ² ; Bo Ram Park¹ ; Su-Gwan Kim¹ ; Hoon Ahn² ; Hong Sung Chun³ ; Yong Kook Shin⁴ ; Jong-Jin Kim⁵ ; Do Kyung Kim¹ ; Sook-Young Lee¹ ; Young-Woo Seo⁶ ; Yong Hwan Kim⁷ ; Chun Sung Kim¹ ; ^{cskim2@chosun.ac.kr" class="auth_mail}
• 关键词：Lyophyllum shimeji ; Edible mushroom ; Fibrinolysis ; Fibrinogenolysis ; Thrombosis
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：May, 2014
• 年：2014
• 卷：117
• 期：5
• 页码：544-550
• 全文大小：1089 K

文摘

A novel fibrinolytic enzyme was purified from Lyophyllum shimeji, a popular edible mushroom in Asia. The enzyme was purified using combination of anion exchange chromatography on a Mono Q 5/5 column and size exclusion gel filtration chromatography on Superdex 200 100/300 column. This purification protocol resulted 80.9-fold purification of the enzyme and a final yield of 5.7%. The molecular weight of the purified enzyme was estimated to be 21聽kDa by SDS-PAGE and size exclusion gel filtration. The N-terminal amino acid sequence was found to be ITFQSASP, which is dissimilar from that of known fibrinolytic enzymes. The purified enzyme was a neutral protease with an optimal reaction pH and temperature of 8.0 and 37掳C, respectively. Enzymatic activity was inhibited by Cu²⁺ and Co²⁺. It was also significantly inhibited by PMSF and TPCK. Furthermore, it was found to exhibit a higher specificity for S-7388, a well-known chymotrypsin chromogenic substrate, indicating chymotrypsin like serine metalloprotease. The relative fibrinolytic activity of 5聽渭g purified enzyme have two fold more activity than 1聽unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the A伪-chain followed by the B尾- and 纬-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L.聽shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease.