Abstract
A bacterium hydrolyzing carboxymethylcellulose, isolated from Antarctic sea ice, was identified as Pseudoalteromonas sp. based on 16S rDNA gene sequences and named as Pseudoalteromonas sp. AN545. The extracellular endo-1,4-β-glucanase AN-1 was purified successively by ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of AN-1 was estimated to be 47.5 kDa utilizing SDS-PAGE and gel chromatography analysis. AN-1 could hydrolyze caboxymethylcellulose, avicel and β-glucan, but not cellobiose, xylan and p-Nitrophenyl-β-D-glucopyranoside. The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30°C and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0–6.5 and temperatures below 30°C for 1 h. Moreover, the specific activity was enhanced by Ca2+ and Mg2+, and inhibited by Cu2+. The kinetic parameters Michaelis constant (K m) and maximum velocity (V max) of AN-1 were 3.96 mg/mL and 6.06×10−2 mg/(min·mL), respectively.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ausubel F M, Brent R, Kingston R E, Moort D D, Seidman J G, Smith J A, Struhl K eds. Ma X J, Shu Y L Trans. 2005. Short Protocols in Molecular Biology (4th ed.). Bei**g: Science Press. (in Chinese)
Bozal N, Tudela E, Rosselló-Mora R, Lalucat J, Guinea J. 1997. Pseudoalteromonas antarctica sp. nov., isolated from an Antarctic coastal environment. Int. J. Syst. Bacteriol., 47(2): 345–351.
Bradford M M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal. Biochem., 72: 248–254.
Castellano I, Ruocco M R, Cecere F, Maro A D, Chambery A, Michniewicz A, Parlato G, Masullo M, De Vendittis M. 2008. Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis. BBA-Proteins & Proteomics, 1 784(5): 816–826.
Chen M, Qin Y Q, Liu Z Y, Liu K, Wang F S, Qu Y B. 2010. Isolation and characterization of a β-glucosidase from Penicillium decumbens and improving hydrolysis of corncob residue by using it as cellulase supplementation. Enzym. Microb. Tech., 46(6): 444–449.
Ekborg N A, Morrill W, Burgoyne A M, Li L, Distel D L. 2007. CelAB, a multifunctional cellulase encoded by Teredinibacter turnerae T7902T, a culturable symbiont isolated from the wood-boring marine bivalve Lyrodus pedicellatus. Appl. Environ. Microbiol., 73(23): 7 785–7 788.
Garsoux G, Lamotte J, Gerday C, Feller G. 2004. Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis. Biochem. J., 384: 247–253.
Hakamada Y, Endo K, Takizawa S, Kobayashi T, Shirai T, Yamane T, Ito S. 2002. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans. BBA-General Subjects, 1 570(3): 174–180.
Han Y J, Chen H Z. 2007. Synergism between corn stover protein and cellulose. Enzym. Microb. Tech., 41: 638–645.
Iyo A H, Forsberg C W. 1996. Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain. Can. J. Microbiol., 42(9): 934–943.
Iyo A H, Forsberg C W. 1999. A cold-active glucanase from the ruminal Bacterium Fibrobacter succinogenes S85. Appl. Envi. Micro., 5: 995–998.
Kaur J, Chadha B S, Kumar B A, Saini H S. 2007. Purification and characterization of two endoglucanase from Melanocarpus sp. MTCC 3922. Bioresour. Technol., 98: 74–81.
Khudary R A, Stößer N I, Qoura F, Antranikian G. 2008. Pseudoalteromonas arctica sp. nov., an aerobic, psychrotolerant, marine bacterium isolated from Spitzbergen. Int. J. Syst. Evol. Microbiol., 58: 2 018–2 024.
Kim D, Baik K S, Park S C, Kim S J, Shin T S, Jung S J, Oh M J, Seong C N. 2009. Cellulase production from Pseudoalteromonas sp. NO3 isolated from the sea squirt Halocynthia rorentzi. J. Ind. Microbiol. Biotechnol., 36: 1 375–1 382.
Kumar R, Dahiya J S, Singh D, Nigam P. 2000. Production of endo-1,4-β-glucanase by a biocontrol fungus Cladorrhinum foecundissimum. Bioresour. Technol., 75(1): 95–97.
Lee Y J, Kim B K, Lee B H, Jo K I, Lee N K, Chung C H, Lee Y C, Lee J W. 2008. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour. Technol., 99(2): 378–386.
Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56: 658–666.
Miller G L, Blum R, Glennon W E, Burton A L. 1960. Measurement of carboxymethylcellulase activity. Anal. Biochem., 2: 127–132.
Murashima K, Nishimura T, Nakamura Y, Koga J, Moriya T, Sumida N, Yaguchi T, Kono T. 2002. Purification and characterization of new endo-1,4-β-D-glucanases from Rhizopus oryzae. Enzyme Microb. Technol., 30: 319–326.
Oikawa T, Tsukagawa Y, Soda K. 1998. Endo-beta-glucanase secreted by a psychrotrophic yeast: purification and characterization. Biosci. Biotechnol. Biochem., 62(9): 1 751–1 756.
Pérez-Avalos O, Sánchez-Herrera L M, Salgado L M, Ponce-Noyola T. 2008. A bifunctional endoglucanase/endoxylanase from Cellulomonas flavigena with potential use in industrial processes at different pH. Curr. Microbiol., 57(1): 39–44.
Rastogi G, Bhalla A, Adhikari A, Bischoff K M, Hughes S R, Christopher L P, Sani R K. 2010. Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresour. Technol., 101(22): 8 798–8 806.
Singh J, Batra N, Sobti R C. 2004. Purification and characterisation of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1. J. Ind. Microbiol. Biotechnol., 31: 51–56.
Ueda M, Goto T, Nakazawa M, Miyatake K, Sakaguchi M, Inouye K. 2010. A novel cold-adapted cellulase complex from Eisenia foetida: Characterization of a multienzyme complex with carboxymethylcellulase, β-glucosidase, β-1,3 glucanase, and β-xylosidase. Comp. Biochem. Physiol. B: Biochem. Mol. Biol., 157(1): 26–3
Wang C Y, Hsieh Y R, Ng C C, Chan H, Lin H T, Tzeng W S, Shyu Y T. 2009. Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05. Enzyme Microb. Technol., 44(6–7): 373–379.
Zeng R Y, **ong P J, Wen J J. 2006. Characterization and gene cloning of a cold-active cellulase from a deep-sea psychrotrophic bacterium Pseudoalteromonas sp. DY3. Extremophiles, 10: 79–82.
Zeng Y X, Yu Y, Chen B, Li H R. 2005. Screening, classification and growth of bacterium producing coldactive cellulase and its enzymatic characterization. High Tech. Lett., 4: 58–62. (in Chinese with English abstract)
Author information
Authors and Affiliations
Corresponding author
Additional information
Supported by the National High Technology Research and Development Program of China (863 Program) (No. 2007AA091905), the Natural Science Foundation of Shandong Province (No. ZR2010DQ010), and the Fundamental Research Funds for the Central Universities (No. HIT. IBRSEM.2009148)
Rights and permissions
About this article
Cite this article
Shen, J., Kan, G., Shi, C. et al. Purification and characterization of cold-active endo-1,4-β-glucanase produced by Pseudoalteromonas sp. AN545 from Antarctica. Chin. J. Ocean. Limnol. 29, 1086–1092 (2011). https://doi.org/10.1007/s00343-011-0311-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00343-011-0311-4