title: Purification and characterization of a novel fibrinolytic α chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji.
abstract: 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 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°C, respectively. Enzymatic activity was inhibited by Cu(2+) and Co(2+). 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 μg purified enzyme have two fold more activity than 1 unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the Aα-chain followed by the Bβ- and γ-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L. shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease.
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Sung-Min
authors: SM
authors: Moon
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Jae-Sung
authors: JS
authors: Kim
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Heung-Joong
authors: HJ
authors: Kim
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea; Department of Dental Hygiene, Chodang University, Muan-ro, Muan-eup, Muan 534-701, Republic of Korea.
authors: Mi Suk
authors: MS
authors: Choi
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Bo Ram
authors: BR
authors: Park
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Su-Gwan
authors: SG
authors: Kim
authors: Department of Dental Hygiene, Chodang University, Muan-ro, Muan-eup, Muan 534-701, Republic of Korea.
authors: Hoon
authors: H
authors: Ahn
authors: Department of Biotechnology, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Hong Sung
authors: HS
authors: Chun
authors: Foundation of Industry-Academy Cooperation, Semyung University, 65 Semyung-ro, Jecheon, Chungcheongbuk-Do 390-711, Republic of Korea.
authors: Yong Kook
authors: YK
authors: Shin
authors: Chunbuk Technopark Biocenter, Ochang, Chungcheonbuk-Do 363-883, Republic of Korea.
authors: Jong-Jin
authors: JJ
authors: Kim
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Do Kyung
authors: DK
authors: Kim
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
authors: Sook-Young
authors: SY
authors: Lee
authors: Korean Basic Science Institute, Gwangju Center Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea.
authors: Young-Woo
authors: YW
authors: Seo
authors: Korea Institute of Planning & Evaluation for Technology in Food, Agriculture Forestry & Fisheries, Burim-ro, Dongan-gu, Anyang-si, Gyeonggi-do 431-810, Republic of Korea.
authors: Yong Hwan
authors: YH
authors: Kim
authors: Oral Biology Research Institute, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea. Electronic address: cskim2@chosun.ac.kr.
authors: Chun Sung
authors: CS
authors: Kim
mesh_terms: D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D012694:Serine / Q000378:metabolism; D013696:Temperature; D013927:Thrombosis
chemical_list: D012694:Serine; D005337:Fibrin; D005340:Fibrinogen; D045726:Metalloproteases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: Edible mushroom; Fibrinogenolysis; Fibrinolysis; Lyophyllum shimeji; Thrombosis