2015 ; Vol.25-1: 89~97
|Author||Seon-Ju Jeong, Kyeong Heo, Ji Yeong Park, Kang Wook Lee, Jae-Yong Park, Sang Hoon Joo, Jeong Hwan Kim|
|Place of duty||Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea|
|Title||Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176|
J. Microbiol. Biotechnol.2015 ;
|Abstract||Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a
Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was
cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid
pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein
and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR
was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after
ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first
using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest
activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity
of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (kcat/Km)
of M179 was not different from the wild-type enzyme owing to reduced substrate affinity.
Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h
at 45°C, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the
176th residue of M179, threonine, was located near the cation-binding site compared with the
wild type. This probably caused tight binding of M179 with Ca2+, which increased the
thermostability of M179.|
|Key_word||Bacillus subtilis, fibrinolytic enzyme, error-prone PCR, thermostability|
Agrebi R, Haddar A, Hmidet N, Jellouli K, Manni L, Nasri M. 2009. BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: purification, biochemical and molecular characterization. Process Biochem. 44: 1252-1259.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Bryan P, Alexander P, Strausberg S, Schwarz F, Lan W, Gilliland G, Gallagher DT. 1992. Energetics of folding subtilisin BPN’. Biochemistry 31: 4937-4945.
Bryan PN. 2000. Protein engineering of subtilisin. Biochim. Biophys. Acta 1543: 203-222.
Cadwell RC, Joyce GF. 1994. Mutagenic PCR. Genome Res. 3:S136-S140.
Dower WJ, Miller JF, Ragsdale CW. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16: 6127-6145.
Estell DA, Graycar TP, Wells JA. 1985. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. J. Biol. Chem. 260: 6518-6521.
Ghasemi Y, Dabbagh F, Ghasemmian A. 2012. Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli. Mol. Biotechnol. 52: 1-7.
Heo K, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim JH. 2013. Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning. J. Microbiol. Biotechnol. 23: 974-983.
Jaouadi B, Aghajari N, Haser R, Bejar S. 2010. Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Biochimie 92: 360-369.
Jeong S -J, Kwon G-H, Chun J, Kim JS, Park C-S, Kwon DY, Kim JH. 2007. Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from cheonggukjang and its expression in protease-deficient Bacillus subtilis strain. J. Microbiol. Biotechnol. 17: 1018-1023.
Jeong S-J, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim JH. 2014. Overexpression of aprE2, a fibrinolytic enzyme gene from Bacillus subtilis CH3-5, in Escherichia coli and the properties of AprE2. J. Microbiol. Biotechnol. 24: 969-978.
Jo H-D, Kwon G-H, Park J-Y, Cha J, Song Y-S, Kim JH. 2011. Cloning and overexpression of aprE3-17 encoding the major fibrinolytic protease of Bacillus licheniformis CH 3-17. Biotechnol. Bioproc. Eng. 16: 352-359.
Khurana J, Singh R, Kaur J. 2011. Engineering of Bacillus lipase by directed evolution for enhanced thermal stability:effect of isoleucine to threonine mutation at protein surface. Mol. Biol. Rep. 38: 2919-2926.
Killer M, Ladurner G , Kunz A B, K raus J . 2010. C urrent endovascular treatment of acute stroke and future aspects. Drug Discov. Today 15: 640-647.
Kim GM, Lee AR, Lee KW, Park J-Y, Chun J, Cha J, et al. 2009. Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J. Microbiol. Biotechnol. 19: 997-1004.
Kim S-H, Choi N-S. 2000. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from doen-jang. Biosci. Biotechnol. Biochem. 64: 1722-1725.
Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, et al. 2006. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, tempeh. J. Ind. Microbiol. Biotechnol. 33: 436-444.
Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, et al. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus s p. s train C K 11-4 s creened f rom chungkook-jang. Appl. Environ. Microbiol. 62: 2482–2488.
Kim YW, Choi JH, Kim JW, Park C, Kim JW, Cha H, et al. 2003. Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Appl. Environ. Microbiol. 69: 4866-4874.
Kwon GH, Lee HA, Park JY, Kim JS, Lim J, Park CS, et al. 2009. Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang. Int. J. Food Microbiol. 129: 282-287.
Lee S-Y, Yu S-N, Choi H-J, Kim K-Y, Kim S-H, Choi Y-L, et al. 2013. Cloning and characterization of a thermostable and alkaline fibrinolytic enzyme from a soil metagenome. Afr. J. Biotechnol. 12: 6389-6399.
Liu B, Zhang J, Fang Z, Gu L, Liao X, Du G, Chen J. 2013. Enhanced thermostability of keratinase by computational design and empirical mutation. J. Ind. Microbiol. Biotechnol. 40: 697-704.
Martinez R, Jakob F, Tu R, Siegert P, Maurer K-H, Schwaneberg U. 2012. Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution. Biotechnol. Bioeng. 110: 711-720.
McPhalenf CA, James MNG. 1988. Structural comparison of two serine proteinase-protein inhibitor complexes: Eglin-CSubtilisin Carlsberg and CI-2-Subtilisin Novo. Biochemistry 27: 6582-6598.
Nakamura T, Yamagata Y, Ichishima E. 1992. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem. 56: 1869-1871.
Pantoliano MW, Whitlow M, Wood JF, Rollence ML, Finzel BC, Gilliland GL, et al. 1988. The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case. Biochemistry 27: 8311-8317.
Peng Y, Huang Q, Zhang R-H, Zhang Y-Z. 2003. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp. Biochem. Phys. B 134: 45-52.
Peng Y, Yang XJ, Xiao L, Zhang YZ. 2004. Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens D C -4 in Bacillus subtilis. Res. Microbiol. 155: 167-173.
Schwede T. 2003. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31: 3381-3385.
Stephens DE, Singh S, Permaul K. 2009. Error-prone PCR of a fungal xylanase for improvement of its alkaline and thermal stability. FEMS Microbiol. Lett. 293: 42-47.
Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto; a typical and popular soybean food in the Japanese diet. Experientia 43: 1110-1111.
Tina KG, Bhadra R, Srinivasan N. 2007. PIC: protein interactions calculator. Nucleic Acids Res. 35: W473-W476.
Uehara R, Angkawidjaja C, Koga Y, Kanaya S. 2013. Formation of the high-affinity calcium binding site in prosubtilisin E with the insertion sequence IS1 of Pro-Tksubtilisin. Biochemistry 52: 9080-9088.
Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y. 2009. Purification and characterization of nattokinase from Bacillus subtilis Natto B-12. J. Agric. Food Chem. 57: 9722-9729.
Yeo WS, Seo MJ, Kim MJ, Lee HH, Kang BW, Park JU, et al. 2011. Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1. J. Microbiol. 49: 376-380.
Yin LJ, Lin HH, Jiang ST. 2010. Bioproperties of potent nattokinase from Bacillus subtilis YJ1. J. Agric. Food Chem. 58:5737-5742.