Purification and fibrinolytic/anticoagulant activity of a novel fibrinolyticenzyme from the fruiting bodies of Agrocybe aegerita
LI Guanlong1,3, LIU Xiaolan1,3, ZHENG Xiqun1,2*
1(College of Food and Biological Engineering, Qiqihar University, Qiqihar 161006, China) 2(College of Food, Bayi Agricultural University, Daqing 163319, China) 3(Key Laboratory of Corn Deep Proessing Theory and Techology, Qiqihar 161006, China)
Abstract: In recent years, the increasing incidence of thrombotic diseases has seriously threatened human life and health. Some functional compounds in foods can increase the activity of human fibrinolytic system, thus gradually restore human's normal physiological state and reduce the tendency of thrombophilia. A novel fibrinolytic enzyme was purified from the fruiting body of Agrocybe aegerita by ammonium sulfate precipitation, Sephadex G-25 gel filtration, CM-Sepharose FF ion exchange, Source 15PHE hydrophobic chromatography and Mono S ion exchange. The fibrinolytic enzyme had a specific activity of 1 731.05 U/mg, with the purification fold of 246.94. The enzyme was named ACase and comprised two subunits. The molecular weights of the two subunits were estimated to be approximately 31.4 kDa and 21.2 kDa, respectively, by SDS-PAGE. Only one band appeared on Native-PAGE and it was found to show fibrinolytic activity. The analysis by Edman degradation method showed that the N-terminal sequences of the two subunits were AIVTQTNAPWGL and SNADGNGHGTHV, respectively. The enzyme was identified as a newfibrinolyticenzymebysequencecomparisoninGenBank. ACase could degrade the three chains (α, β and γ) of fibrinogen in order and exhibited the functionality of anticoagulant in vitro. Agrocybe aegerita has the potential application as functional food ingredient in food industry.
李冠龙,刘晓兰,郑喜群. 茶树菇子实体新纤溶酶的纯化及其溶栓抗凝性质研究[J]. 食品与发酵工业, 2020, 46(11): 105-111.
LI Guanlong,LIU Xiaolan,ZHENG Xiqun. Purification and fibrinolytic/anticoagulant activity of a novel fibrinolyticenzyme from the fruiting bodies of Agrocybe aegerita[J]. Food and Fermentation Industries, 2020, 46(11): 105-111.
 《中国血栓性疾病防治指南》专家委员会.中国血栓性疾病防治指南[J].中华医学杂志,2018,98(36):2 861-2 888.  WHO.全球非传染性疾病现状报告[J/OL].2017. https://www.who.int/zh/news-room/fact-sheets/detail/cardiovascular-diseases.  胡盛寿,高润霖,刘力生,等.《中国心血管病报告2018》概要[J].中国循环杂志,2019,34(3):209-220.  王鸿利,王学锋.血栓病临床新技术[M].北京:人民军医出版社,2003.  PENG Y, YANG X, ZHANG Y. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo[J]. Applied Microbiology and Biotechnology, 2005, 69(2):126-132.  李冠龙,郑喜群,刘晓兰,等.蛹虫草玉米肽功能性饮料的研制及其抗氧化活性研究[J].食品与发酵工业,2019,45(14):163-169.  MAN L L, XIANG D J, ZHANG C L. Strain screening from traditional fermented soybean foods and induction of nattokinase production in Bacillus subtilis MX-6[J]. Probiotics and Antimicrobial Proteins, 2018, 11(1):283-294.  赵睿秋,马高兴,杨文建,等.6种食用菌子实体水提物对肠道菌群的影响[J].食品科学,2017,38(5):116-121.  XU JT. Chinese Medicinal Mycology[M]. Beijing: Combined Press of Beijing Medical University and Peking Union Medical College, 1997.  陈子涵,刘金娟.六种食用菌体外抗氧化及抗细胞增殖活性研究[J].生物技术通报,2019,35(11):104-108.  CHOI D B, CHA W S, PARK N, et al. Purification and characterization of a novel fibrinolytic enzyme from fruiting bodies of Korean Cordyceps militaris[J]. Bioresour Technology, 2011, 102(3):3 279-3 285.  LIU XL, KOPPARAPU N, SHI X, et al. Purification and biochemical characterization of a novel fibrinolytic enzyme from culture supernatant of Cordyceps militaris[J]. Journal of Agricultural & Food Chemistry, 2015, 63(8):2 215-2 224.  LIU XL, KOPPARAPU N, LI Y, et al. Biochemical characterization of a novel fibrinolytic enzyme from Cordyceps militaris[J]. International Journal of Biological Macromolecules, 2016, 94(Pt B):793-801.  CHOI J H, KIM K J, KIM S. Purification and antithrombotic potential of a fibrinolytic enzyme from shiitake culinary-medicinal mushroom, Lentinus edodes GNA01 (Agaricomycetes)[J]. International Journal of Medicinal Mushrooms, 2018, 20(1):47-59.  臧琬婷,梅余霞,梁运祥.冬虫夏草固态培养菌丝中纤溶酶的纯化和酶学性质[J].微生物学通报, 2020,47(2):562-570.  CHOI J H, KIM D W, KiIM S, et al. Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae[J]. Prep Biochem Biotechnol, 2016, 47(6):539-546.  LIU XL, ZHENG XQ, QIAN PZ, et al. Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus[J]. Journal of Microbiology & Biotechnology, 2014, 24(2):245-253.  ZHAO H, LI J, ZHANG J, et al. Purification, in vitro, antioxidant and, in vivo, anti-aging activities of exopolysaccharides by Agrocybe cylindracea[J]. International Journal of Biological Macromolecules, 2017, 102:351-357.  MUZGONJA N, MILOVANOVIC I, VUKOJEVIC J, et al. Antioxidant and antifungal potential of Pleurotus ostreatus and Agrocybe cylindracea basidiocarps and mycelia[J]. Current Pharmaceutical Biotechnology, 2015, 16(2):179-186.  LOWRY O H, ROSEBROUGH N J, FARR A L, et al. Protein measurement with the flin phenol reagent[J]. Journal of Biological Chemistry, 1951, 193(1):265-275.  ASTRUP T, STEN M. The fibrin plate method for estimating fibrinolytic activity[J]. Archives of Biochemistry and Biophysics, 1952, 40(2):346-351.  张雯舒,刘晓兰,郑喜群,等. 蛹虫草无性型深层培养液中纤溶酶的分离纯化和酶学性质研究[J].微生物学通报,2012,39(12):1 787-1 795.  DENG Y, LIU X, KATROLIA P, et al. A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila[J]. International Journal of Biological Macromolecules, 2018, 109:1 338-1 343.  CHOI J H, KIM D W, PARK S E. Purification and partial characterization of TFase, a fibrinolytic enzyme from the fruiting bodies of the medicinal and edible mushroom, Tremella fuciformis[J]. Applied Biochemistry and Microbiology, 2015, 51(6):712-719.  CHA W S, PARK S S, KIM S J, et al. Biochemical and enzymatic properties of a fibrinolytic enzyme from Pleurotus eryngii cultivated under solid-state conditions using corn cob[J]. Bioresource Technology, 2010, 101(16):6 475-6 481.  CHOI B S, SAPKOTA K, JUN-HUI C. Herinase: a novel bi-functional fibrinolytic protease from the monkey head mushroom, Hericium erinaceum[J]. Applied Biochemistry and Biotechnology, 2013, 170(3):609-622.  KIM J S, SAPKOTA K, PARK S E, et al. A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris[J]. The Journal of Microbiology, 2007, 44(6):622-631.