A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates

BMC Biotechnology

Yunxia Li¹  Yingjie Pan¹  Qunxin She²  Lanming Chen¹ 

[1] Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), China Ministry of Agriculture, Engineering Centre for Quality Control and Risk Assessment of Aquatic Products, College of Food Science and Technology, Shanghai Ocean University, 999 Hu Cheng Huan Road, Shanghai 201306, People's Republic of China

[2] Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen DK-2200N, Denmark

关键词: Aqueous environment;    Protease;    Paenibacillus;    CtpA;   

Others  :  835232
DOI  :  10.1186/1472-6750-13-89

 received in 2013-08-12, accepted in 2013-10-17,  发布年份 2013

【 摘 要 】

Background

Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated.

Results

Screening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser₃₀₉-Lys₃₃₄) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase.

Conclusions

Our work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications.

【 授权许可】

   
2013 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Satoh K, Yamamoto Y: The carboxyl-terminal processing of precursor D1 protein of the photosystem II reaction center. Photosynth Res 2007, 94(2–3):203-215.
• [2]Inagaki N, Maitra R, Satoh K, Pakrasi HB: Amino acid residues that are critical for in vivo catalytic activity of CtpA, the carboxyl-terminal processing protease for the D1 protein of photosystem II. J Biol Chem 2001, 276(32):30099-30105.
• [3]Liao DI, Qian J, Chisholm DA, Jordan DB, Diner BA: Crystal structures of the photosystem II D1 C-terminal processing protease. Nature Struct Biol 2000, 7(9):749-753.
• [4]Hara H, Yamamoto Y, Higashitani A, Suzuki H, Nishimura Y: Cloning, Mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3. J Bacteriol 1991, 173(15):4799-4813.
• [5]Silber KR, Keiler KC, Sauer RT: Tsp: A tail-specific protease that selectively degrades proteins with nonpolar C termini. Proc Natl Acad Sci USA 1992, 89(1):295-299.
• [6]Keiler KC, Silber KR, Downard KM, Papayannopoulos IA, Biemann K, Sauer RT: C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease. Protein Sci 1995, 4(8):1507-1515.
• [7]Oelmuller R, Herrmann RG, Pakrasi HB: Molecular studies of CtpA, the carboxyl-terminal processing protease for the D1 protein of the photosystem II reaction center in higher plants. J Biol Chem 1996, 271(36):21848-21852.
• [8]Shestakov SV, Anbudurai PR, Stanbekova GE, Gadzhiev A, Lind LK, Pakrasi HB: Molecular cloning and characterization of the ctpA gene encoding a carboxyl-terminal processing protease: analysis of a spontaneous photosystem II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 1994, 269(30):19354-19359.
• [9]Wang CY, Wang SW, Huang WC, Kim KS, Chang NS, Wang YH, Wu MH, Teng CH: Prc contributes to Escherichia coli evasion of classical complement-mediated serum killing. Infect Immun 2012, 80(10):3399-3409.
• [10]Kumru OS, Bunikis I, Sorokina I, Bergstrom S, Zuckert WR: Specificity and role of the Borrelia burgdorferi CtpA protease in outer membrane protein processing. J Bacteriol 2011, 193(20):5759-5765.
• [11]Dong J, Signo KS, Vanderlinde EM, Yost CK, Dahms TE: Atomic force microscopy of a ctpA mutant in Rhizobium leguminosarum reveals surface defects linking CtpA function to biofilm formation. Microbiology 2011, 157(11):3049-3058.
• [12]Bandara AB, Sriranganathan N, Schurig GG, Boyle SM: Carboxyl-terminal protease regulates Brucella suis morphology in culture and persistence in macrophages and mice. J Bacteriol 2005, 187(16):5767-5775.
• [13]Bandara AB, DeShazer D, Inzana TJ, Sriranganathan N, Schurig GG, Boyle SM: A disruption of CtpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice. Microb Pathog 2008, 45(3):207-216.
• [14]Yamamoto Y, Inagaki N, Satoh K: Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein: regulation of enzyme-substrate interaction by molecular environments. J Biol Chem 2001, 276(10):7518-7525.
• [15]Rao MB, Tanksale AM, Ghatge MS, Deshpande VV: Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998, 62(3):597-635.
• [16]Krik O, Borchert TV, Fuglsang CC: Industrial enzyme applications. Curr Opin Biotechnol 2002, 13(4):345-435.
• [17]Sharmaa R, Chistib Y, Banerjee UC: Production, purification, characterization, and applications of lipases. Biotechnol Adv 2001, 19:627-662.
• [18]Shi Y, Pan Y, Li B, He W, She Q, Chen L: Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents. BMC Biotechnol 2013, 13:13. BioMed Central Full Text
• [19]Lal S, Tabacchioni S: Ecology and biotechnological potential of Paenibacillus polymyxa: a minireview. Indian J Microbiol 2009, 49(1):2-10.
• [20]Choi KK, Park CW, Kim SY, Lyoo WS, Lee SH, Lee JW: Polyvinyl alcohol degradation by Microbacterium barkeri KCCM 10507 and Paeniblacillus amylolyticus KCCM 10508 in dyeing wastewater. J Microbiol Biotechnol 2004, 14:1009-1013.
• [21]Konishi J, Maruhashi K: 2-(2'-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium Paenibacillus sp. strain A11-2: purification and characterization. Appl Microbiol Biotechnol 2003, 62:356-361.
• [22]Girardin H, Albagnac C, Dargaignaratz C, Nguyen-The C, Carlin F: Antimicrobial activity of foodborne Paenibacillus and Bacillus spp. against Clostridium botulinum. J Food Prot 2002, 65:806-813.
• [23]Lu F, Lu Z, Bie X, Yao Z, Wang Y, Lu Y, Guo Y: Purification and characterization of a novel anticoagulant and fibrinolytic enzyme produced by endophytic bacterium Paenibacillus polymyxa EJS-3. Thromb Res 2010, 126(5):349-355.
• [24]Heyndrickx M, Vandemeulebroecke K, Scheldeman P, Kersters K, De Vos P, Logan NA, Aziz AM, Ali N, Berkeley RCW: A polyphasic reassessment of the genus Paenibacillus, reclassification of Bacillus lautus (Nakamura 1984) as Paenibacillus lautus comb. nov. and of Bacillus peoriae (Montefusco et al. 1993) as Paenibacillus peoriae comb. nov., and emended descriptions of P. lautus and of P. peoriae. Int J Syst Bacteriol 1996, 46:988-1003.
• [25]Mead DA, Lucas S, Copeland A, Lapidus A, Cheng J-F, Bruce DC, Goodwin LA, Pitluck S, Chertkov O, Zhang X, Detter JC, Han CS, Tapia R, Land M, Hauser LJ, Chang Y-J, Kyrpides NC, Ivanova NN, Ovchinnikova G, Woyke T, Brumm C, Hochstein R, Schoenfeld T, Brumm P: Complete Genome Sequence of Paenibacillus strain Y4.12MC10, a Novel Paenibacillus lautus strain Isolated from Obsidian Hot Spring in Yellowstone National Park. Stand Genomic Sci 2012, 6:366-385.
• [26]Zhou R, Kroos L: Serine proteases from two cell types target different components of a complex that governs regulated intramembrane proteolysis of pro-sigmaK during Bacillus subtilis development. Mol Microbiol 2005, 58(3):835-846.
• [27]Mitchell SJ, Minnick MF: A carboxy-terminal processing protease gene is located immediately upstream of the invasion-associated locus from Bartonella bacilliformis. Microbiology 1997, 143(4):1221-1233.
• [28]Ostberg Y, Carroll JA, Pinne M, Krum JG, Rosa P, Bergstrom S: Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi. J Bacteriol 2004, 186(7):2074-2084.
• [29]Hoge R, Laschinski M, Jaeger KE, Wilhelm S, Rosenau F: The subcellular localization of a C-terminal processing protease in Pseudomonas aeruginosa. FEMS Microbiol Lett 2011, 316(1):23-30.
• [30]Jimenez-Flores R, Kang YC, Richardson T: Cloning and sequence analysis of bovine beta-casein cDNA. Biochem Biophys Res Commun 1987, 142(2):617-621.
• [31]Gorodetskii SI, Zakhar'ev VM, Kyarshulite DR, Kapelinskaya TV, Skryabin KG: Cloning and nucleotide sequence of cDNA for bovine alphaS1-casein. Biochem USSR 1987, 51(10-1):1402-1409.
• [32]Taguchi F, Yamamoto Y, Satoh K: Recognition of the structure around the site of cleavage by the carboxyl-terminal processing protease for D1 precursor protein of the photosystem II reaction center. J Biol Chem 1995, 270(18):10711-10716.
• [33]Fabbri BJ, Duff SM, Remsen EE, Chen YC, Anderson JC, CaJacob CA: The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach protein. Pest Manag Sci 2005, 61(7):682-690.
• [34]Rai S, Mukherjee A: Statistical optimization of production, purification and industrial application of a laundry detergent and organic solvent-stable subtilisin-like serine protease (Alzwiprase) from Bacillus subtilis DM-04. Biochem Eng J 2010, 48(2):173-180.
• [35]Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Press; 2001.
• [36]Saran S, Isar J, Saxena RK: A modified method for the detection of microbial proteases on agar plates using tannic acid. J Biochem Biophys Methods 2007, 70(4):697-699.
• [37]Zhang T, Pan Y, Li L, Ou J, Zhang J, Chen Y, Peng X, Chen L: Molecular cloning and antimicrobial activity of enterolysin A and helveticin J of bacteriolysins from metagenome of Chinese traditional fermented foods. Food Control 2013, 31(2):499-507.
• [38]Holt JG: Bergey's Manual of Determinative Bacteriology. Ninth edition. Philedelphia: Lippincott Williams & Wilkins; 1994.
• [39]Weisburg WG, Barns SM, Pelletier DA, Lane DJ: 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991, 173:697-703.
• [40]Larkin MA, Blackshields G, Brown NP, Chenna R, NcGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23:2947-2948.
• [41]Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24(8):1596-1599.
• [42]Kall L, Krogh A, Sonnhammer EL: A combined transmembrane topology and signal peptide prediction method. J Mol Biol 2004, 338(5):1027-1036.
• [43]Spiers A, Lamb HK, Cocklin S, Wheeler KA, Budworth J, Dodds AL, Pallen MJ, Maskell DJ, Charles IG, Hawkins AR: PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide. J Biol Chem 2002, 277(42):39443-39449.
• [44]Kasana RC, Yadav SK: Isolation of a psychrotrophic Exiguobacterium sp. SKPB5 (MTCC 7803) and characterization of its alkaline protease. Curr Microbiol 2007, 54(3):224-229.
• [45]Yamamoto Y, Satoh K: Competitive inhibition analysis of the enzyme-substrate interaction in the carboxy-terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides. FEBS Lett 1998, 430(3):261-265.