In 1987, a novel fibrinolytic enzyme (nattokinase) was discovered from a typical and popular fermented Japanese soybean food called natto . Since then, several fibrinolytic enzymes have been discovered in traditional foods, such as douche, brewing rice wine, tempeh, chungkook-jang, salty fermented fish, and fermented shrimp paste [2,3]. Due to the prevalence of nattokinase as a health food especially in Asia, numerous biochemical studies have been carried out to characterize this enzyme. It was found that nattokinase is a highly conserved serine protease which is a very effective extracellular enzyme composed of 275 amino acids . The nattokinase shows higher enzymatic activity than plasmin with direct fibrinolytic properties both in vitro and in vivo . Most of the proteases derived from the Bacillus subtilis are highly conserved. Alignment of 28 nattokinase sequences retrieved from the current NCBI Protein Search Database showed that they shared more than 90% sequence identity. For example, three mature peptides from B. subtilis, nattokinase (GenBank no. AHZ12722.1), nattokinase (GenBank no. ABA29609.1), and nattokinase (GenBank no. ABM97611.1) shared 93% sequence identity (Supplementary Fig. S1). In this study, a strain isolated from Chinese traditional fermented soybean food (Yongchuan, China) by our group was found to secrete fibrinolytic enzyme and it was confirmed that this strain belongs to the B. subtilis by 16S rDNA analysis. To identify whether the enzyme is similar to nattokinase, rapid-amplification of cDNA ends (RACE) assay was carried out. Chromosomal DNA of the strain which is able to secrete fibrinolytic enzyme was isolated for use as the PCR template. On the basis of the submitted sequence of nattokinase (GenBank no. AHZ12722.1), two primers were designed as follows: P1, 5′-CGGGATCCGCACAGAGCGTGCCGTAC-3′, P2, 5′-CCGCTCGAGTTATTGTGCTGCTGCCTGGAC-3′. The cloned gene of the enzyme was inserted into the pMD19-T vector. Finally, the T vector was transformed into TOP10 to obtain the sequence of the enzyme. The amino acid sequence deduced from RACE result showed that it shares 99% sequence identity with the nattokinase (GenBank no. AHZ12722.1), indicating that the proteases derived from this strain is a nattokinase-like protease named as NK-01 (Supplementary Fig. S2). The strain was cultured as previously reported  and harvested by centrifugation. The supernatant was substituted with 10 mM sodium phosphate buffer (pH 5.6) by ultrafiltration using the vivaflow 200 (Sartorius, Germany), and then loaded onto a SP Sepharose Fast Flow column (15 ml, GE Healthcare, Fairfield, CT, USA) at a flow rate of 1 ml/min. The bound protein was eluted with a gradient ranging from 0 M to 0.1 M NaCl. As shown in Fig. 1A, purification by ion exchange chromatography resulted in one peak. Then, the protein was further purified by gel filtration chromatography on a HiPrepTM 16/60 SephacrylTM S-200 HR column (120 ml, GE Healthcare, Fairfield, CT, USA) using 20 mM Tris (pH 8.0), 150 mM NaCl as the running buffer. Only one elution peak was observed and the molecular weight was ~27 kDa based on the elution volume (Fig. 1B). The fractions corresponding to the peak were collected and dialyzed against water to remove NaCl before lyophilization. The purity of enzyme was evaluated by Tricine-SDS-PAGE and Coomassie Blue R-250 staining. Unexpectedly, as shown in Fig. 1C, multiple bands appeared on the gel. Figure 1. View largeDownload slide Purification of the enzyme (A) Elution profile of ion exchange chromatography with Sepharose SP Fast Flow column (15 ml). (B) The gel filtration chromatogram of the enzyme using the 16/60 SephacrylTM S-200 HR column. The inset figures demonstrate the correlation between the molecular weight (MW) and elution volume. Chitosanase OU01 (30 kDa), PreScission protease (43 kDa), bovine serum albumin (BSA, 66 kDa), and lysozyme (14 kDa) were used as the standard. The MW of the purified enzyme of NK-01 was estimated to be 27 kDa. (C) Tricine-SDS-PAGE analysis of the enzyme after gel filtration (M: Protein marker, lane 1: enzyme. The labels 1–5 on the right are the gel bands corresponding to the PMF data in Table 1). Figure 1. View largeDownload slide Purification of the enzyme (A) Elution profile of ion exchange chromatography with Sepharose SP Fast Flow column (15 ml). (B) The gel filtration chromatogram of the enzyme using the 16/60 SephacrylTM S-200 HR column. The inset figures demonstrate the correlation between the molecular weight (MW) and elution volume. Chitosanase OU01 (30 kDa), PreScission protease (43 kDa), bovine serum albumin (BSA, 66 kDa), and lysozyme (14 kDa) were used as the standard. The MW of the purified enzyme of NK-01 was estimated to be 27 kDa. (C) Tricine-SDS-PAGE analysis of the enzyme after gel filtration (M: Protein marker, lane 1: enzyme. The labels 1–5 on the right are the gel bands corresponding to the PMF data in Table 1). To further confirm the purity of this enzyme, the pooled fractions from gel filtration were concentrated to 5 mg/ml for crystallization trials. Finally, crystals were collected and further characterized by Tricine-SDS-PAGE. The result showed that even after crystallization, it still contained several fragments (Supplementary Fig. S3), suggesting that this purified NK-01 was different from any other reported nattokinases. Peptide mass fingerprinting (PMF) assay was carried out to identify whether these fragments belonged to nattokinase. As shown in Table 1, the result demonstrated that four major bands possibly belonged to the degradation products of nattokinase while one band did not. This finding suggested that the purified enzyme actually contained multiple fragments derived from nattokinase, which seemed inconsistent with the ion exchange and gel filtration chromatography results (Fig. 1B). The circular dichroism (CD) spectra of this purified enzyme showed that three obvious negative peaks appeared at 202 nm, 218 nm, and 222 nm, indicating the presence of secondary structure in purified enzyme (Supplementary Fig. S4A). The thermal stability assay for this purified enzyme demonstrated that only one transition temperature of 60°C was detected (Supplementary Fig. S4B). Table 1. Peptide mass fingerprinting of fractions from the purified enzyme Fraction number Sequence Confidence interval % modification Rank result type 1 AQSVPYGISQIKAPALHSQGYTGSNVK, VAVIDSGIDSSHPDLNVR, YPSTIAVGAVNSSNQR 100 Mascot 2 VAVIDSGIDSSHPDLNVR, YPSTIAVGAVNSSNQR, DRLESTATYLGNSFYYGK 100 Mascot 4 HPTWTNAQVR, DRLESTATYLGNSFYYGK 100 Mascot 5 HPTWTNAQVR, DRLESTATYLGNSFYYGK 100 Mascot Fraction number Sequence Confidence interval % modification Rank result type 1 AQSVPYGISQIKAPALHSQGYTGSNVK, VAVIDSGIDSSHPDLNVR, YPSTIAVGAVNSSNQR 100 Mascot 2 VAVIDSGIDSSHPDLNVR, YPSTIAVGAVNSSNQR, DRLESTATYLGNSFYYGK 100 Mascot 4 HPTWTNAQVR, DRLESTATYLGNSFYYGK 100 Mascot 5 HPTWTNAQVR, DRLESTATYLGNSFYYGK 100 Mascot The fibrinolytic activity of the enzyme was evaluated according to the conventional fibrin plate method using urokinase as the positive control . As shown in Fig. 2A, after incubation at 37°C for 18 h, the enzyme was found to result in a larger clear zone around the well compared with the same amount of urokinase. Unlike other plasminogen activators, nattokinase has been found to degrade fibrin directly, which decreases the secondary effects such as platelet activation related to plasmin formation . Therefore, the fibrinogen cleavage pattern assay was conducted .The result showed that the α chain was degraded within a very short time, the β chain was completely degraded after 20 min, and most of the γ chain was not degraded even after 30 min of incubation (Fig. 2B). It can be concluded that the purified enzyme degraded the α chain first, followed by the β chain, while the γ chain was resistant to this enzyme. Figure 2. View largeDownload slide Enzymatic activity assay of the purified enzyme (A) Fibrinolytic activity of the purified enzyme and urokinase based on fibrin plate assay. (a–e) Different concentrations of urokinase (140, 70, 50, 30 μg/ml, and 10 μg/ml, respectively); (f) 10 μg/ml purified enzyme; (g) control 0.9% NaCl. (B) Fibrinogen cleavage pattern analysis of the purified enzyme as a function of time. The degradation product was analyzed by SDS-PAGE (12%). Line 1: control (fibrinogen); lines 2–6: incubation time of 0, 5, 10, 20, and 30 min, respectively. Figure 2. View largeDownload slide Enzymatic activity assay of the purified enzyme (A) Fibrinolytic activity of the purified enzyme and urokinase based on fibrin plate assay. (a–e) Different concentrations of urokinase (140, 70, 50, 30 μg/ml, and 10 μg/ml, respectively); (f) 10 μg/ml purified enzyme; (g) control 0.9% NaCl. (B) Fibrinogen cleavage pattern analysis of the purified enzyme as a function of time. The degradation product was analyzed by SDS-PAGE (12%). Line 1: control (fibrinogen); lines 2–6: incubation time of 0, 5, 10, 20, and 30 min, respectively. In conclusion, we have purified a secreted nattokinase-like enzyme (NK-01) composed of multiple fragments with high-fibrinolytic activity and found that it still maintained its intact structure based on the gel filtration assay and CD spectra. However, the underlying mechanisms for the generation of these fragments remain to be explored in the future. This study may facilitate the understanding of catalytic mechanisms of fibrinolytic agents and the rational design of new fibrinolytic agents. Supplementary Data Supplementary data are available at Acta Biochimica et Biophysica Sinica online. Funding This work was supported by the grants from the National Natural Science Foundation of China (No. 81302684) and the Natural Science Foundation of Shandong Province (No. ZR2013CM044). References 1 Sumi H, Hamada H, Tsushima H, Mihara H. A novel strong fibrinolytic enzyme (Nattokinase) in the vegetable cheese ‘NATTO’. Fibrinolysis 1988, 2: 67. 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Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. N Biotechnol 2011, 28: 196– 202. Google Scholar CrossRef Search ADS PubMed 7 Mander P, Cho SS, Simkhada JR, Yun HC, Jin CY. A low molecular weight chymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624. Process Biochem 2011, 46: 1449– 1455. Google Scholar CrossRef Search ADS 8 Chang CT, Fan MH, Kuo FC, Sung HY. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem 2000, 48: 3210– 3216. Google Scholar CrossRef Search ADS PubMed 9 Bhargavi PL, Prakasham RS. A fibrinolytic, alkaline and thermostable metalloprotease from the newly isolated Serratia sp RSPB11. Int J Biol Macromol 2013, 61: 479– 486. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: email@example.com
Acta Biochimica et Biophysica Sinica – Oxford University Press
Published: Feb 1, 2018
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