A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization

Yong-Suk  Lee; Jae Bok  Heo; Je-Hoon  Lee; Yong-Lark  Choi

doi:10.4014/jmb.1402.02033

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Research article

Biocatalysis and Fermentation Technology

A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization

Yong-Suk Lee ¹, Jae Bok Heo ², Je-Hoon Lee ¹ and Yong-Lark Choi ^1*

¹Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea, ²Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Republic of Korea

Received: February 18, 2014; Accepted: March 27, 2014

J. Microbiol. Biotechnol. 2014; 24(7): 925-935

Published July 28, 2014 https://doi.org/10.4014/jmb.1402.02033

Abstract

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the α/β-class of protein consisted of a central six-stranded β-sheet flanked by eight α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were 15°C and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or 60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na+ and Mg2+ ions but was strongly inhibited by Cu+ and Hg2+ ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM and a kcat of 1.9 s-1. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

Keywords

Cold-adapted enzyme, carbohydrate esterase, Microbulbifer thermotolerans, purification, esterase, marine bacterium

References

Alalouf O, Balazs Y, Volkinshtein M, Grimpel Y, Shoham G, Shoham Y. 2011. A new family of carbohydrate esterases is represented by a GSDL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus. J. Biol. Chem. 86: 41993-42001.
Al Khudary R, Venkatachalam R, Katzer M, Elleuche S, Antranikian G. 2010. A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization. Extremophiles 14: 273-285.
Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
Aurilia V, Parracino A, Saviano M, Rossi M, D’Auria S. 2007. The psychrophilic bacterium Pseudoalteromonas halosplanktis TAC125 possesses a gene coding for a cold-adapted feruloyl esterase activity that shares homology with esterase enzymes from γ-proteobacteria and yeast. Gene 397: 51-57.
Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO. 2011. Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1. Protein Expr. Purif. 80: 74-79.
Babu J, Ramteke PW, Thomas G. 2008. Cold active microbial lipases: some hot issues and recent developments. Biotechnol. Adv. 26: 457-470.
Biely P. 2012. Microbial carbohydrate esterases deacetylating plant polysaccharides. Biotechnol. Adv. 30: 1575-1588.
Bitto E, Bingman CA, McCoy JG, Allard STM, Wesenberg E, Phillips Jr GN. 2005. The structure at 1.6 Å resolution of the protein product of the At4g34215 gene from Arabidopsis thaliana. Acta Crystallogr. D Biol. Crystallogr. D61: 1655-1661.
Blum DL, Li XL, Chen H, Ljungdahl LG. 1999. Characterization of an acetyl xylanesterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Appl. Environ. Microbiol. 65:3990-3995.
Burgess JG. 2012. New and emerging analytical techniques for marine biotechnology. Curr. Opin. Biotechnol. 23: 29-33.
Burton SG, Cowan DA, Woodley JM. 2002. The search for the ideal biocatalyst. Nat. Biotechnol. 20: 37-45.
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The Carbohydrate-Active Enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37: D233-D238.
Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. 2002. Low-temperature extremophiles and their applications. Curr. Opin. Biotechnol. 13: 253-261.
Collins T, De Vos D, Hoyoux A, Savvides SN, Gerday C, van Veeumen J, Feller G. 2005. Study of the active site residues of a glycoside hydrolase family 8 xylanase. J. Mol. Biol. 354: 425-435.
Darlymple BP, Cybinski DH, Layton I, McSweeney CS, Xue GP, Swadling YJ, Lowry JB. 1997. Three Neocallimastix partiarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology 143: 2605-2614.
Dheeman DS, Henehan GTM, Frias JM. 2011. Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavor ester synthesis. Bioresour. Technol. 102: 3373-3379.
Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, et al. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18: 103-107.
Gupta Udtha DBRK, Kouskoumvekaki I, Solsson L, Panagiotou G. 2011. The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases. Biotechnol. Adv. 29: 94-110.
Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
Jiang X, Huo Y, Cheng H, Zhang X, Zhu X, Wu M. 2012. Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T. Extremophiles 16: 427-435.
Kam DK, Jun HS, Ha JK, Forsberg CW. 2005. Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan. Can. J. Microbiol. 51: 821-832.
Kelly LA, Sternberg MJE. 2009. Protein structure prediction of the web: a case study using the PHYRE server. Nat. Protoc. 4: 363-371.
Kim SH, Park IH, Lee SC, Lee YS, Zhou Y, Kim CM, et al. 2008. Discovery of three novel lipase (lipA1, lipA2, and lipA3) and lipase-specific chaperone (lipB) genes present in Acinetobacter sp. DYL129. Appl. Microbiol. Biotechnol. 77:1041-1051.
Kulakova L, Galkin A, Nakayama T, Esaki N. 2004. Coldactive esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly → Pro substitution near the active site on its catalytic activity and stability. Biochim. Biophys. Acta 169: 59-65.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Lemak S, Tchigvintsev A, Petit P, Flick R, Singer AU, Brown G, et al. 2012. Structure and activity of the coldactive and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica. Biochem. J. 445: 193-203.
Liang WQ, Wang ZY, Li H, Wu PC, Hu JM, Luo N, et al. 2005. Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11. J. Agric. Food Chem. 53: 7415-7420.
Lopez-Cortes N, Reyes-Duarte D, Beloqui A, Polaina J, Ghazi I, Golishina OV, et al. 2007. Catalytic role of conserved HQGE motif in the CE6 carbohydrate esterase family. FEBS Lett. 581: 4657-4662.
Margesin R, Feller G. 2010. Biotechnological applications of psychrophiles. Environ. Technol. 31: 835-844.
Miyazaki M, Nogi Y, Ohta Y, Hatada Y, Fujiwara Y, Ito S, Horikoshi K. 2008. Microbulbifer agarlyticus sp. nov. and Microbulbifer thermotolerans sp. nov., agar-degrading bacteria isolated from deep-sea sediment. Int. J. Syst. Evol. Microbiol. 58: 1128-1133.
Ohgiya S, Hoshino T, Okuyama H, Tanaka S, Ishizaki K. 1999. Biotechnology of enzymes from cold-adapted microorganisms, pp. 17-34. In Margesin R, Schinner F (eds.). Biotechnological Applications of Cold-Adapted Organisms, Heidelberg, SpringerVerlag.
Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K. 2004. Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbiferlike isolate. Appl. Microbiol. Biotechnol. 66: 266-275.
Ohta Y, Nogi Y, Miyazaki M, Li Z, Hatada Y, Ito S, Horikoshi K. 2004. Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from the novel marine isolate, JAMB-A94. Biosci. Biotechnol. Biochem. 68: 1073-1081.
Park IH, Kim SH, Lee YS, Lee SC, Zhou Y, Kim CM, et al. 2009. Gene cloning, purification, and characterization of a cold-adapted lipase produced by Acinetobacter baumannii BD5. J. Microbiol. Biotechnol. 19: 128-135.
Peterson TN, Brunak S, von Heijne G, NielsenH. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Rao L, Zhao X, Pan F, Li Y, Xue Y, Ma Y, Lu JR. 2009. Solution behavior and activity of a halophilic esterase under high salt concentration. PLoS One 4: e6980.
Salameh MA, Wiegel J. 2007. Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl. Environ. Microbiol. 73: 7725-7731.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning:A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Suzuki T, Nakayama T, Kurihara T, Nishino T, Esaki N. 2002. A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization. J. Mol. Catal. B Enzym. 16: 255-263.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: 4876-4882.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.
Wang BZ, Guo P, Hang BJ, Li L, He J, Li SP. 2009. Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product. Appl. Environ. Microbiol. 75: 5496-5500.
Weisburg WG, Barns SM, Pelletire DA, Lane DJ. 1991. 16S ribosomal DNA amplication for phylogenetic study. J. Bacteriol. 173: 697-703.
Wei Y, Schottel JL, Derewenda U, Swenson L, Patkar S, Derewenda S. 1995. A novel variant of the catalytic triad in the Streptomyces scabies esterase. Nature 2: 218-223.
Winkler UK, Stuckman M. 1979. Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J. Bacteriol. 138: 663-679.
Yoshida S, Mackie RI, Cann IKO. 2010. Biochemical and domain analyses of FSUAxe6B, a modular acetyl xylan esterase, identify a unique carbohydrate binding module in Fibrobacter succinogenes S85. J. Bacteriol. 192: 483-493.
Zhai Y, Li K, Song JL, Shi YH, Yan YC. 2012. Molecular cloning, purification and biochemical characterization of a novel pyrethroid-hydrolyzing carboxylesterase gene from Ochrobactrum anthropi YZ-1. J. Hazard. Mater. 221-222: 206212.

Article Tools

Article

Research article

J. Microbiol. Biotechnol. 2014; 24(7): 925-935

Published online July 28, 2014 https://doi.org/10.4014/jmb.1402.02033

A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization

Yong-Suk Lee ¹, Jae Bok Heo ², Je-Hoon Lee ¹ and Yong-Lark Choi ^1*

¹Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea, ²Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Republic of Korea

Received: February 18, 2014; Accepted: March 27, 2014

Abstract

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6,
was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids
with the first 22 serving as a secretion signal peptide. The calculated molecular mass and
isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic
triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG,
QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST
belongs to the α/β-class of protein consisted of a central six-stranded β-sheet flanked by eight
α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the
characterization showed its optimal temperature and pH were 15°C and 8.0, respectively.
Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or
60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results
suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was
increased by Na+ and Mg2+ ions but was strongly inhibited by Cu+ and Hg2+ ions, at all ion
concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM
and a kcat of 1.9 s-1. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192,
and His281) and Asp278 were essential for the enzyme activity.

Keywords: Cold-adapted enzyme, carbohydrate esterase, Microbulbifer thermotolerans, purification, esterase, marine bacterium

Abstract

References

Alalouf O, Balazs Y, Volkinshtein M, Grimpel Y, Shoham G, Shoham Y. 2011. A new family of carbohydrate esterases is represented by a GSDL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus. J. Biol. Chem. 86: 41993-42001.
Al Khudary R, Venkatachalam R, Katzer M, Elleuche S, Antranikian G. 2010. A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization. Extremophiles 14: 273-285.
Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
Aurilia V, Parracino A, Saviano M, Rossi M, D’Auria S. 2007. The psychrophilic bacterium Pseudoalteromonas halosplanktis TAC125 possesses a gene coding for a cold-adapted feruloyl esterase activity that shares homology with esterase enzymes from γ-proteobacteria and yeast. Gene 397: 51-57.
Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO. 2011. Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1. Protein Expr. Purif. 80: 74-79.
Babu J, Ramteke PW, Thomas G. 2008. Cold active microbial lipases: some hot issues and recent developments. Biotechnol. Adv. 26: 457-470.
Biely P. 2012. Microbial carbohydrate esterases deacetylating plant polysaccharides. Biotechnol. Adv. 30: 1575-1588.
Bitto E, Bingman CA, McCoy JG, Allard STM, Wesenberg E, Phillips Jr GN. 2005. The structure at 1.6 Å resolution of the protein product of the At4g34215 gene from Arabidopsis thaliana. Acta Crystallogr. D Biol. Crystallogr. D61: 1655-1661.
Blum DL, Li XL, Chen H, Ljungdahl LG. 1999. Characterization of an acetyl xylanesterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Appl. Environ. Microbiol. 65:3990-3995.
Burgess JG. 2012. New and emerging analytical techniques for marine biotechnology. Curr. Opin. Biotechnol. 23: 29-33.
Burton SG, Cowan DA, Woodley JM. 2002. The search for the ideal biocatalyst. Nat. Biotechnol. 20: 37-45.
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The Carbohydrate-Active Enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37: D233-D238.
Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. 2002. Low-temperature extremophiles and their applications. Curr. Opin. Biotechnol. 13: 253-261.
Collins T, De Vos D, Hoyoux A, Savvides SN, Gerday C, van Veeumen J, Feller G. 2005. Study of the active site residues of a glycoside hydrolase family 8 xylanase. J. Mol. Biol. 354: 425-435.
Darlymple BP, Cybinski DH, Layton I, McSweeney CS, Xue GP, Swadling YJ, Lowry JB. 1997. Three Neocallimastix partiarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology 143: 2605-2614.
Dheeman DS, Henehan GTM, Frias JM. 2011. Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavor ester synthesis. Bioresour. Technol. 102: 3373-3379.
Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, et al. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18: 103-107.
Gupta Udtha DBRK, Kouskoumvekaki I, Solsson L, Panagiotou G. 2011. The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases. Biotechnol. Adv. 29: 94-110.
Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
Jiang X, Huo Y, Cheng H, Zhang X, Zhu X, Wu M. 2012. Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T. Extremophiles 16: 427-435.
Kam DK, Jun HS, Ha JK, Forsberg CW. 2005. Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan. Can. J. Microbiol. 51: 821-832.
Kelly LA, Sternberg MJE. 2009. Protein structure prediction of the web: a case study using the PHYRE server. Nat. Protoc. 4: 363-371.
Kim SH, Park IH, Lee SC, Lee YS, Zhou Y, Kim CM, et al. 2008. Discovery of three novel lipase (lipA1, lipA2, and lipA3) and lipase-specific chaperone (lipB) genes present in Acinetobacter sp. DYL129. Appl. Microbiol. Biotechnol. 77:1041-1051.
Kulakova L, Galkin A, Nakayama T, Esaki N. 2004. Coldactive esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly → Pro substitution near the active site on its catalytic activity and stability. Biochim. Biophys. Acta 169: 59-65.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Lemak S, Tchigvintsev A, Petit P, Flick R, Singer AU, Brown G, et al. 2012. Structure and activity of the coldactive and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica. Biochem. J. 445: 193-203.
Liang WQ, Wang ZY, Li H, Wu PC, Hu JM, Luo N, et al. 2005. Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11. J. Agric. Food Chem. 53: 7415-7420.
Lopez-Cortes N, Reyes-Duarte D, Beloqui A, Polaina J, Ghazi I, Golishina OV, et al. 2007. Catalytic role of conserved HQGE motif in the CE6 carbohydrate esterase family. FEBS Lett. 581: 4657-4662.
Margesin R, Feller G. 2010. Biotechnological applications of psychrophiles. Environ. Technol. 31: 835-844.
Miyazaki M, Nogi Y, Ohta Y, Hatada Y, Fujiwara Y, Ito S, Horikoshi K. 2008. Microbulbifer agarlyticus sp. nov. and Microbulbifer thermotolerans sp. nov., agar-degrading bacteria isolated from deep-sea sediment. Int. J. Syst. Evol. Microbiol. 58: 1128-1133.
Ohgiya S, Hoshino T, Okuyama H, Tanaka S, Ishizaki K. 1999. Biotechnology of enzymes from cold-adapted microorganisms, pp. 17-34. In Margesin R, Schinner F (eds.). Biotechnological Applications of Cold-Adapted Organisms, Heidelberg, SpringerVerlag.
Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K. 2004. Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbiferlike isolate. Appl. Microbiol. Biotechnol. 66: 266-275.
Ohta Y, Nogi Y, Miyazaki M, Li Z, Hatada Y, Ito S, Horikoshi K. 2004. Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from the novel marine isolate, JAMB-A94. Biosci. Biotechnol. Biochem. 68: 1073-1081.
Park IH, Kim SH, Lee YS, Lee SC, Zhou Y, Kim CM, et al. 2009. Gene cloning, purification, and characterization of a cold-adapted lipase produced by Acinetobacter baumannii BD5. J. Microbiol. Biotechnol. 19: 128-135.
Peterson TN, Brunak S, von Heijne G, NielsenH. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Rao L, Zhao X, Pan F, Li Y, Xue Y, Ma Y, Lu JR. 2009. Solution behavior and activity of a halophilic esterase under high salt concentration. PLoS One 4: e6980.
Salameh MA, Wiegel J. 2007. Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl. Environ. Microbiol. 73: 7725-7731.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning:A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Suzuki T, Nakayama T, Kurihara T, Nishino T, Esaki N. 2002. A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization. J. Mol. Catal. B Enzym. 16: 255-263.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: 4876-4882.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.
Wang BZ, Guo P, Hang BJ, Li L, He J, Li SP. 2009. Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product. Appl. Environ. Microbiol. 75: 5496-5500.
Weisburg WG, Barns SM, Pelletire DA, Lane DJ. 1991. 16S ribosomal DNA amplication for phylogenetic study. J. Bacteriol. 173: 697-703.
Wei Y, Schottel JL, Derewenda U, Swenson L, Patkar S, Derewenda S. 1995. A novel variant of the catalytic triad in the Streptomyces scabies esterase. Nature 2: 218-223.
Winkler UK, Stuckman M. 1979. Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J. Bacteriol. 138: 663-679.
Yoshida S, Mackie RI, Cann IKO. 2010. Biochemical and domain analyses of FSUAxe6B, a modular acetyl xylan esterase, identify a unique carbohydrate binding module in Fibrobacter succinogenes S85. J. Bacteriol. 192: 483-493.
Zhai Y, Li K, Song JL, Shi YH, Yan YC. 2012. Molecular cloning, purification and biochemical characterization of a novel pyrethroid-hydrolyzing carboxylesterase gene from Ochrobactrum anthropi YZ-1. J. Hazard. Mater. 221-222: 206212.

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Research article

A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization

Abstract

Keywords

References

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Article

Research article

A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization

Abstract

References