1444 微生物学通报 2008, Vol.35, No.9, β- [1] [2], [3 5] [6] [7] [8] [9] [10] [11] [12] [13], 1992, Nakamura T [14], 2003, [15] Subtilisin DFE, 16S rdna, (Mi

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1 微生物学通报 SEP 20, 2008, 35(9): 1443~1449 Microbiology 2008 by Institute of Microbiology, CAS 研究报告纤溶酶产生菌藤黄微球菌的筛选鉴定及纤溶酶基因的克隆 1, 2 肖璐 2 张仁怀 2* 张义正 ( ) ( ) 摘要 : 通过血粉培养基富集纤维蛋白培养基筛选, 从自然环境中分离到一株纤溶酶产生菌, 命名为 ML909 利用传统细菌分类鉴定方法对其形态和生理生化特征进行了研究, 发现其与藤黄微球菌的特征相符, 16S rdna 序列分析的结果也表明它与藤黄微球菌的 16S rdna 序列的同源性高达 99%, 因此该菌在系统分类学上属于微球菌属 (Micrococcus Cohn) 藤黄微球菌 (Micrococcus luteus) 通过 PCR 方法对其纤溶酶的编码区序列进行了克隆和序列分析, 基因登录 GenBank, 登录号为 EU 关键词 : 纤溶酶, 基因克隆, 16S rdna, 藤黄微球菌 Isolation of a Micrococcus luteus Strain with Producing Fibrinolysin and Cloning of Fibrinolitic Enzyme Gene XIAO Lu 1,2 ZHANG Ren-Huai 2 ZHANG Yi-Zheng 2* (1. College of Life Science, Hunan University of Science and Technology, Xiangtan ) (2. College of Life Science, Sichuan University, Sichuan Key Laboratary of Molecular Biology and Biotechnology, Chengdu ) Abstract: A strain, producing a fibrinolytic enzyme, was isolated from the nature environment by using blood powder plates gathering and fibrin plates screening, and was named ML909. All results from morphological, physiological and biochemical characterization, the 16S rdna sequence and phylogenetic assay identified that the strain belongs to Micrococcus luteus. The DNA fragment encoding mature peptide of fibrinolytic enzyme was cloned by PCR and sequenced. The gene sequence was registered in GenBank of NCBI and the accession number is EU Keywords: Fibrinolytic enzyme, Gene cloning, 16S rdna, Micrococcus luteus,,,,,,,, * 通讯作者 :Tel: ; : yizzhang@scu.edu.cn 收稿日期 : ; 接受日期 :

2 1444 微生物学通报 2008, Vol.35, No.9, β- [1] [2], [3 5] [6] [7] [8] [9] [10] [11] [12] [13], 1992, Nakamura T [14], 2003, [15] Subtilisin DFE, 16S rdna, (Micrococcus luteus), 1 材料与方法 1.1 材料和培养基试剂 : ; Sigma ; pmd18-t Taq DNA TaKaRa ; 富集培养基 : 2, 1, NaCl 5, 2, ph 纤维蛋白培养基 : 2, 10, NaCl 0.5, 2, ph 菌株 DC-4,, ML909, Escherichia coli DH5α 1.3 菌种的分离 [16],,,, 1.4 纤溶酶活性的测定 [17] 1.5 形态及理化鉴定 [18] 1.6 分离菌株 G+C mol DNA [19], DNA 0.1 SSC Tm G+C mol 0.1 SSC G+C = 2.44(Tm-53.9) [20] S rdna 和纤溶酶基因的克隆与测序 S rdna 的扩增和序列测定 : 16S rdna 5 -ATGGATCCGAGAGTTTGATCCTGGCTCAG-3 5 -TATCTGCAGTGGTGTGACGGGC GGTGT-3 50 μl 0.2 mmol/l dntps, DNA 100 ng, 0.2 mmol/l, Taq DNA 聚合 1.5 U, 1/10 10 PCR, 1.5 mmol/l MgCl 2 PCR 94 5 min; s, 54 1 min, 72 1 min, 30 ; 72 8 min PCR 20 PCR 0.7,, 16S rdna PCR pmd18-t, Kit Ⅰ 5 μl, PCR 1 μl, pmd18-t 0.5 μl, 10 μl, h~16 h DH5α,,,,, PCR,, M13(+) M13( ), 纤溶酶基因的克隆与测序 : 5 -CGCCATGGCGCAGTCCGTGCCT TAC-3, 5 -GTCGGATCCTTACTGAGCT GCCGCCTGTAC-3, PCR 94 5 min; s, s, s, 30 ; 72 8 min PCR 20 PCR GenBank, Blast MEGA4, (neighbor-joining method) 2 结果 2.1 形态学特征,

: 1445 ML909,,, 0.5 μm~2 μm,,,, 2.2 生理生化特征,,, 1 99 16S rdna, RPD, GenBank,, (Micrococcus luteus), ML909 表 1 ML909 菌株的生理生化特征与藤黄微球菌的比较 Table 1 Characteristics comparison of isolated ML909 and M.

luteus Colour of colony Producing acid Glucose Glycerin Mannose Lactose Hydrolyzing gelatin + + Nitrate reduction Growth Inorganic nitrogen agar + + Citric acid agar 7.

3 : 1445 ML909,,, 0.5 μm~2 μm,,,, 2.2 生理生化特征,,, S rdna, RPD, GenBank,, (Micrococcus luteus), ML909 表 1 ML909 菌株的生理生化特征与藤黄微球菌的比较 Table 1 Characteristics comparison of isolated ML909 and M. luteus Characteristics ML909 Strain ML909 M. luteus Colour of colony Producing acid Glucose Glycerin Mannose Lactose Hydrolyzing gelatin + + Nitrate reduction Growth Inorganic nitrogen agar + + Citric acid agar 7.5% NaCl Nutrient agar containing 7.5% NaCl 图 1 16S rdna 的 PCR 扩增产物的琼脂糖凝胶电泳 Fig. 1 Agarose gel electrophoretogram of 16S rdna PCR product 1: DL2000 DNA marker; 2: 1.4 kb PCR product of 16S rdna 2.3 分离菌株的 G+C mol% 含量 ML909 Tm 81.8 DNA G+C mol 68 (Micrococcus Cohn,1872) mol G+C S rdna 的克隆及序列分析 DNA, 16S rdna PCR, 1.4 kb ( 1), pmd18-t, DNA, BamH I Sal I, 1.4 kb, ( 2) ( 3), ML909 16S rdna 1386 bp Blastn ML909 16S rdna GenBank 16S rdna,, 图 2 克隆 16S rdna 的重组子酶切电泳鉴定 Fig. 2 Agarose gel electrophoretogram of restriction endonuclease digested recombinant plasmid 1: DL2000 DNA marker; 2: Recombinant plasmid; 3: Fragments of recombinant plasmid digested by BamH I and Sal I 2.5 菌株 ML909 的系统发育分析 ML909 16S rdna 16S rdna MEGA4, N-J ML909,, ML 纤溶酶的克隆与序列分析, ML909 DNA PCR,

4 菌株 ML909 的系统发育学地位 The phylogenetic position of strain ML909 DC-4, ML909 DNA 828 bp( 5), (DFE), PCR pmd18-t, ( 6), ( 7), 828 bp, 275,, ML-FE, 99

4 1446 微生物学通报 2008, Vol.35, No.9 图 3 菌株 ML909 的 16S rdna 基因序列 Fig. 3 The 16S rdna gene sequence of strain ML909 T C,, 72 Val Ala; 456 A G,, GenBank EU 图 4 Fig. 4 菌株 ML909 的系统发育学地位 The phylogenetic position of strain ML909 DC-4, ML909 DNA 828 bp( 5), (DFE), PCR pmd18-t, ( 6), ( 7), 828 bp, 275,, ML-FE, 99 2 ( 7 ), 215 图 5 溶栓酶 PCR 扩增产物的琼脂糖凝胶电泳 Fig. 5 Agarose gel of PCR products of fibrinolytic enzyme from the strain ML909 1: λdna/ecor I+Hind III marker; 2: PCR products of fibrinolytic enzyme from the strain ML909; 3: PCR products of fibrinolytic enzyme from the strain DC-4

5 : 1447 图 6 重组质粒的酶切电泳鉴定 Fig. 6 Agarose gel electrophoretogram of restriction endonuclease digested recombinant plasmid 1: PCR products of fibrinolytic enzyme from the strain ML909; 2: λdna/ecor I+Hind III marker; 3: The recombinant plasmid digested by Nco I+BamH I; 4: The recombinant plasmid 11, BA-DFE [21] BS-DFE [22],, 80,,, MLFE BA-DFE K-54, NAT BS-DFE QK,,,, Fig. 7 图 7 ML909 纤溶酶成熟肽编码序列和推测的氨基酸序列 Coding sequence of mature peptide of fibrinolytic enzyme from the stain ML909 and its amino acid sequence

7 ML909 纤溶酶活性的检测, ML909 DC-4,, ( 9 10) 图 9 纤维蛋白培养基板上活性检测 Fig.

6 1448 微生物学通报 2008, Vol.35, No.9 Fig. 8 图 8 蛋白酶的核酸 (A) 和氨基酸序列 (B) 聚类分析图 The phylogenetic comparative analysis of the nucleotide (A) and amino acid (B) sequences homology of proteases 2.7 ML909 纤溶酶活性的检测, ML909 DC-4,, ( 9 10) 图 9 纤维蛋白培养基板上活性检测 Fig. 9 Fibrin plate assay for fibrinolitic enzyme of the strain ML909 1: The strain ML909; 2: Bacillus amyloliquefaciens DC-4 图 10 纤维蛋原白平板活性检测 Fig. 10 Fibrinogen plate assay for fibrinolytic activity 1: The supernatant of Bacillus amyloliquefaciens DC-4; 2: The supernatant of the strain ML909 3 讨论,,, DC-4,, DNA,,, K-54,,,,, K-54,,, E 1473, 13, 12, 99%,,,, PCR,, B.amyloliquefaciens DC-4 DNA, 4 ( 5 - ), 5 -

7 : 1449 PCR, ML909, BA-DFE,, MLFE BA-DFE, PCR MLFE,,,,, 参考文献 [1] Jackson KW, Malke H, Gerlach D, et al. Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues. Biochemistry, 1986, 25 (1): [2] Abdel-fattah AF, Ismail AS. Purification and some properties of pure Cochliobolus lunatus fibrinolytic enzyme. Biotech Bioeng, 1984, 26(5): [3] Sumi H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto; a typical and popular soybean food in the Japanese diet. Experientia, 1987, 43(10): [4] Kim W, Choi K, Kim Y, et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK11-4 screened from Chungkook- Jang. Appl Environ Microbiol, 1996, 62(7): [5],.., 2002, 12(2): [6] Egorov NS, Kochetov GA, Khaidarova NV. Isolation and properties of the fibrinolytic enzyme from the Actinomyces thermovulgaris cultural broth. Mikrobiologiia, 1976, 45: [7] El-Aassar SA, El-Badry HM, Abdel-Fattah AF. The biosynthesis of proteases with fibrinolytic activity in immobilized cultures of Penicillium chrysogenum H9. Appl Microbiol Biotechnol, l990, 33(1): [8] Sun T, Liu BH, Li P, et al. New solid-state fermentation process for repeated batch production of fibrinolytic enzyme by Fusarium oxysporum. Process Biochem, 1998, 33(4): [9] El-Aassar SA. Production and properties enzyme in solid state cultures of Fusarium pallidoroseum. Biotechnol Lett, 1995, 17(9): [10] Abdel-Fattah AF, Ismail AS. Purification and some properties of pure Cochliobolus lunatus fibrinolytic enzyme. Biotech Bioeng, 1984, 26(5): [11] Kim JH, Lee HY, Yoo KH, et al. The screening of fibrinolytic activities of extracts from Mushrooms in Mt. Chiak. Kor J Mycol, 1998, 26: [12] Matsubara K, Hori K, Matsuura Y, et al. A fibrinolytic enzyme from a marine green alga, Codium latum. Phytochemistry, 1999, 52(6): [13],,,.., 2002, 23(1): [14] Nakamura T, Yamagata Y, Ichishima E. Nucleotide sequence of the subtilisin NAT, aprn, of Bacillus natto. Biosci Biotech Biochem, 1992, 56(11): [15] Peng Y, Yang XJ, Xiao L, et al. Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis. Res Microbiol, 2004, 155(3): [16] Peng Y, Huang Q, Zhang RH, et al. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B Biochem Mol Biol, 2003, 134(1): [17] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys, 1952, 40(2): [18] RE, NE.... :, 1984, pp [19] Saito H, Miura K. Preparation of transforming DNA by phenol treatment. Biochem Biophys Acta, 1963, [20],,.. :, 1988, pp [21],.., 2002, 8(3): [22],,. DC-2 E. coli., 2002, 18(5):

9 04 Vol. No. Serial No.6 China Brewing Research Report sanning electron microscope SEM g ml = 4 4h UV-800PC 0000r/min 5min SLY-B 0μL - LHS-50SC 7 6h.

9 04 Vol. No. Serial No.6 China Brewing Research Report sanning electron microscope SEM g ml = 4 4h UV-800PC 0000r/min 5min SLY-B 0μL - LHS-50SC 7 6h. 04 6 9 DNAout [].. 5g/L g/l nattokinase NK g/l K HPO 4 0.5g/L KH PO 4 0.g/L [-4] MgSO 4 ph7.0~7. LB 0g/L 5g/L [5-7] 0g/L NaCl 5g/L ph7.0 urokinase UK streptokinase SK tissue-type plasminogenactivator t-pa