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J. Gen. Appl. Microbiol. Doi 10.2323/Jgam.2019.04.005 ©2019 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

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J. Gen. Appl. Microbiol. Doi 10.2323/Jgam.2019.04.005 ©2019 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2019.04.005 ©2019 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 1 Genome Sequencing, Purification, and Biochemical Characterization of a 2 Strongly Fibrinolytic Enzyme from Bacillus amyloliquefaciens Jxnuwx-1 isolated 3 from Chinese Traditional Douchi 4 (Received November 29, 2018; Accepted April 22, 2019; J-STAGE Advance publication date: August 14, 2019) * 5 Huilin Yang, Lin Yang, Xiang Li, Hao Li, Zongcai Tu, Xiaolan Wang 6 Key Lab of Protection and Utilization of Subtropic Plant Resources of Jiangxi 7 Province, Jiangxi Normal University 99 Ziyang Road, Nanchang 330022, China * 8 Corresponding author: Xiaolan Wang, PhD, Key Lab of Protection and Utilization 9 of Subtropic Plant Resources of Jiangxi Province, Jiangxi Normal University 99 10 Ziyang Road, Nanchang 330022, China. Tel: 0086-791-88210391. 11 Email: [email protected]. 12 Short title: B. amyloliquefaciens fibrinolytic enzyme 13 14 * Key Lab of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province, Jiangxi Normal University 99 Ziyang Road, Nanchang 330022, China. Email：[email protected] (X.Wang) 1 15 Abbreviation 16 CVDs: Cardiovascular diseases; u-PA: urokinase-type plasminogen activator; t-PA: 17 tissue plasminogen activator; PMSF: phenylmethanesulfonyl fluoride; SBTI: soybean 18 trypsin inhibitor; EDTA: ethylenediaminetetraacetic acid; TLCK: N-Tosyl-L-Lysine 19 chloromethyl ketone; TPCK: N-α-Tosyl-L-phenylalanine chloromethyl ketone; pNA: 20 p-nitroaniline; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel 21 electrophoresis; GO: Gene Ontology 2 22 23 Summary 24 A strongly fibrinolytic enzyme was purified from Bacillus amyloliquefaciens 25 Jxnuwx-1, found in Chinese traditional fermented black soya bean (douchi). The 26 molecular mass of the enzyme, estimated by sodium dodecyl sulfate-polyacrylamide 27 gel electrophoresis (SDS-PAGE), was 29 kDa. The optimal pH and temperature for 28 the enzyme were 7.6 and 41°C, respectively. The enzyme was inhibited by 29 phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, ethylenediaminetetraacetic 3+ 2+ 30 acid, Fe , and Fe . The highest affinity exhibited by the enzyme was towards 31 N-Succinyl-Ala-Ala-Pro-Phe-pNA. These results indicated that it is a subtilisin-like 32 serine metalloprotease. The enzyme degraded both fibrinogen and fibrin, displaying 33 its highest degrading activity towards the Aα-chains followed by Bβ chains and Cγ 34 chains. The enzyme was also activated by plasminogen, indicating its ability to 35 degrade fibrinogen and fibrin in two ways: (a) by activating plasminogen conversion 36 into plasmin, or (b) by direct hydrolysis. It degraded thrombin, suggesting that it may 37 act as an anticoagulant to prevent thrombosis. Taken together, our results indicate the 38 potential of this enzyme in controlling cardiovascular disease. 39 40 Keywords: Bacillus amyloliquefaciens; Douchi; Fibrinolytic enzyme; Purification; 41 Subtilisin-like serine metalloprotease 42 3 43 Introduction 44 Cardiovascular diseases (CVDs) such as ischemic heart disease, high blood 45 pressure, and acute myocardial infarction are a leading cause of death, accounting for 46 approximately a third of all deaths worldwide (Mine et al. 2005). Thrombus formation 47 is one of the main causes of CVDs. Fibrin, the main component of thrombus, is 48 formed via catalysis of fibrinogen by thrombin (EC 3.4.21.5). Fibrin is degraded by 49 plasmin (EC 3.4.21.7), derived from plasminogen by the action of activators. Under 50 normal physiological conditions, the formation and degradation of fibrin remain in 51 equilibrium. However, any disturbance of such equilibrium, may lead to the 52 accumulation of fibrin, resulting in thrombus formation (Choi et al. 2011). 53 Fibrinolytic agents are currently categorized into plasmin-like proteins, which can 54 directly degrade the fibrin, and plasminogen activators, including urokinase-type 55 plasminogen activator (u-PA) (Duffy 2002), tissue plasminogen activator (t-PA) 56 (Collen and Lijnen 2004), and bacterial plasminogen activators, which indirectly 57 degrade fibrin by activating plasminogen conversion into plasmin. However, in 58 addition to being very expensive, clinical use of these agents result in undesirable side 59 effects, such as a short in vivo half-life, low specificity for fibrin, and excessive 60 bleeding (Wang et al. 2006; Lu et al. 2010; Choi et al. 2011; Liu et al. 2015). 61 Therefore, a search for alternatives, with fewer or no side effects, may prove useful. 62 Over the past decade, fibrinolytic enzymes in fermented foods have been 63 investigated (Fujita et al. 1993; Yong et al. 2003; Rajendran et al. 2016). Examples of 64 this include Japanese natto (Sumi et al. 1987), Asian fermented shrimp paste (Hua et 4 65 al. 2008), Chinese douchi (Yong et al. 2003; Wang et al. 2008b), red beans (Chang et 66 al. 2012), and Korean Chungkook-Jang (Kim et al. 1996). These may be considered 67 useful sources for exploring mechanisms underlying fibrinolysis, as well as for 68 identifying potential drugs for clinical use. 69 Douchi is a fermented food used in food flavoring, which has been produced in 70 China and several other countries for thousands of years (Chen et al. 2011b). Douchi 71 is also used in Chinese traditional medicine to treat dyspepsia, restlessness, and 72 asthma and to stimulate sweating (Chen et al. 2007; Chen et al. 2011a). In 1994, 73 Barnes’s team found that douchi may inhibit prostate and breast cancer (Messina et 74 al. 1994). It also displays anti-diabetic activity (Wang et al. 2008a) and provides 75 protection against osteoporosis and cardiovascular diseases (Ishida et al. 1998). 76 Several studies have antecedently indicated that fibrinolytic enzymes have been 77 purified from douchi and characterized (Yong et al. 2003; Wang et al. 2006; Wang et 78 al. 2008b). However, studies conducted on fibrinolytic enzymes in douchi are scarce. 79 The objective of the present study was to isolate, purify, and characterize a strongly 80 fibrinolytic enzyme from douchi using submerged fermentation culture. 81 Materials and Methods 82 Materials 83 DEAE-Sepharose fast flow and Superdex 75, PD-10 columns were purchased from 84 GE Healthcare Co. (USA). Bovine fibrinogen, bovine thrombin, human fibrinogen, 85 human thrombin, phenylmethanesulfonyl fluoride (PMSF), soybean trypsin inhibitor 5 86 (SBTI), pepstatin A, aprotinin, ethylenediaminetetraacetic acid (EDTA), 87 N-Tosyl-L-Lysine chloromethyl ketone (TLCK), N-α-Tosyl-L-phenylalanine 88 chloromethyl ketone (TPCK), N-Succinyl-Ala-Ala-Pro-Phe-pNA, 89 N-Benzoyl-Phe-Val-Arg-pNA, N-(p-Tosyl)-Gly-Pro-Lys-pNA, 90 N-Succinyl-Ala-Ala-Ala-pNA, and p-nitroaniline (pNA) were purchased from 91 Sigma-Aldrich Co. (USA). Protein standard markers (ranging from 14.3 to 97.2) were 92 purchased from Takara Biotechnology (Dal Lan) Co. Ltd. All other chemicals used 93 were of analytical grade. 94 Bacterial and culture condition 95 We prepared a suspension by soaking and shaking the douchi with sterile saline. 96 Douchi suspension was cultured in a nutrient broth medium (peptone 10 g/L, beef 97 dipping powder 5.0 g/L, NaCl 5.0 g/L) for 48 h at 37°C. Subsequently, the obtained 98 strains were inoculated to a fibrinolytic enzyme-producing strain screening medium 99 (Na2HPO4 0.2%, NaCl 0.5%, skim milk powder 1%, agar 2%), and cultured at 37°C 100 for 48 h to observe whether the colony produced a transparent hydrolyzed circle. 101 Twenty-four bacterial colonies with different activities were isolated from the douchi 102 fermentation process. The strain, Jxnuwx-1, showed the highest fibrinolytic enzyme 103 activity and was identified as B. amyloliquefaciens following alignment with NCBI 104 database sequences. B. amyloliquefaciens Jxnuwx-1 was deposited with the China 105 Center for Type Culture Collection (CCTCC No: M2014638). Fermentation was 106 carried out with the fermentation medium, comprising 30 g/L corn starch, 20 g/L beef 107 powder, 1 g/L NaCl, 1 g/L K2HPO4·3H2O, 0.5 g/L MgSO4·7H2O, 1 g/L CaCl2, for 72 6 108 h at 37°C and 170 rpm. The supernatant from centrifuged (10000 rpm, 15 min, 4°C) 109 fermentation broth was considered as the crude enzyme extract. 110 Genome Sequence of B. amyloliquefaciens Jxnuwx-1 111 Genome sequencing was performed using Illumina Solexa Hiseq4000 at Novogene 112 Bioinformatics Technology Co., Ltd, Beijing, China. A library containing 350-bp 113 inserts was constructed. Sequencing was performed with the pair-end strategy of (150, 114 150)-bp reads to produce 2.0 Gb of filtered sequences, representing a 500-fold 115 coverage of the genome. 116 Genome annotation was performed with the NCBI Prokaryotic Genome Annotation 117 Pipeline 2.0. Open reading frames were identified by Glimmer 3.02 (Delcher et al. 118 2007) and GeneMark (Besemer et al. 2001). The resulting translations were used in a 119 BLASTP (Altschul et al. 1990) search against the GenBank NR database, as well as 120 against KEGG (Kanehisa et al. 2008) and COG (Tatusov et al. 2000) databases. tRNA 121 and rRNA genes were identified via tRNAscan-SE (Lowe and Eddy 1997) and 122 RNAmmer (Lagesen et al. 2007), respectively. 123 Enzyme assay and protein determination 124 The fibrinolytic enzyme content of each solution was determined using the method 125 of Astrup and Müllertz (1952), with slight modifications. The fibrin plates consisting 126 of a fibrinogen solution (2 mg of fibrinogen in 8 mL of 40 mM sodium barbital buffer 127 (pH 7.8), 20 IU of thrombin solution, and 8 mL of 12 g/L agarose) in petri dishes (10 128 cm in diameter), were left to stand for 1 h at room temperature to facilitate clotting, 7 129 following which 10 µL of the enzyme solution was carefully added onto each fibrin 130 plate and subsequently incubated at 37°C for 18 h. Activity of the fibrinolytic enzyme 131 was estimated by measuring the diameter of the lytic cycle according to the 132 calibration curve, using urokinase as a standard.
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