Abstract
Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl, respectively. This indicates that NaCl can improve enzyme thermostability. The K m and k cat values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL−1 and 26 s−1, while the K m and k cat values of CelB without NaCl were 6.6 mg mL−1 and 2.1 s−1. Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides a new model to study salt effects on the structure of protein.
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Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402
Au KS, Chan KY (1987) Purification and properties of the endo-1,4-β-glucanase from Bacillus subtilis. J Gen Microbiol 133:2155–2162
Aygan A, Arikan B (2008) A new halo-alkaliphilic, thermostable endoglucanase from moderately halophilic Bacillus sp. C14 isolated from Van soda lake. Int J Agric Biol 10:369–374
Cendrin F, Chroboczek J, Zaccai G, Eisenberg H, Mevarech M (1993) Cloning, sequencing, and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui. Biochemistry 32(16):4308–4313
Cho KM, Hong SJ, Math RK, Islam SM, Kim JO, Lee YH, Kim H, Yun HD (2008) Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison with cel 44C-man 26A. J Basic Microbiol 48(6):464–472
Dey PM, Del Campillo E (1984) Biochemistry of the multiple forms of glycosidases in plants. Adv Enzymol Relat Areas Mol Biol 56:141–249
Elcock AH, McCammon JA (1998) Electrostatic contributions to the stability of halophilic proteins. J Mol Biol 280(4):731–748
Ghauri MA, Khalid AM, Grant S, Heaphy S, Grant WD (2003) Phylogenetic analysis of different isolates of Sulfobacillus spp isolated from uranium-rich environments and recovery of genes using integron-specific primers. Extremophiles 7(5):341–345
Hansen CK, Diderichsen B, Jorgensen PL (1992) CelA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-β-1,4-glucanase. J Bacteriol 174(11):3522–3531
Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280(Pt 2):309–316
Hirasawa K, Uchimura K, Kashiwa M, Grant WD, Ito S, Kobayashi T, Horikoshi K (2006) Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens. Antonie van Leeuwenhoek 89(2):211–219
Horikoshi K (1971) Production of alkaline enzymes by alkalophilic microorganisms. Part I. Alkaline protease produced by Bacillus no. 221. Agric Biol Chem 36:1407–1414
Hu Y, Zhang GM, Li AY, Chen J, Ma LX (2008) Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach. Appl Microbiol Biotechnol 80(5):823–830
Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F (2002) Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 99(1):63–78
Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66(1):10–26
Liu L, Feng Y, Duan CJ, Pang H, Tang JL, Feng JX (2009) Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World J Microbiol Biotechnol 25:1035–1042
Ma Y, Xue Y, Grant WD, Collins NC, Duckworth AW, Van Steenbergen RP, Jones BE (2004) Alkalimonas amylolytica gen nov., sp. nov., and Alkalimonas delamerensis gen. nov., sp. nov., novel alkaliphilic bacteria from soda lakes in China and East Africa. Extremophiles 8(3):193–200
Mevarech M, Frolow F, Gloss LM (2000) Halophilic enzymes: proteins with a grain of salt. Biophys Chem 86(2–3):155–164
Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of carboxymethyl cellulase activity. Anal Biochem 2:127–132
Mosbah A, Belaich A, Bornet O, Belaich JP, Henrissat B, Darbon H (2000) Solution structure of the module X2 1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum. J Mol Biol 304(2):201–217
Ogawa A, Sumitomo N, Hakamada Y, Saeki K, Ozaki K, Kobayashi T (2009) Nucleotide sequence of a Paenibacillus endoglucanase gene and characterization of the recombinant enzyme. J Appl Glycosci 56:253–259
Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N, Kuhara S, Horikoshi K (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28(21):4317–4331
Voget S, Steele HL, Streit WR (2006) Characterization of a metagenome-derived halotolerant cellulase. J Biotechnol 126:26–36
Vanfossen AL, Ozdemir I, Zelin SL, Kelly RM (2011) Glycoside hydrolase inventory drives plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. Biotechnol Bioeng 108(7):1559–1569
Wang CY, Hsieh YR, Ng CC, Chan H, Lin HT, Tzeng WS, Shyu YT (2009) Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05. Enzym Microb Technol 44:373–379
Zhang GM, Hu Y, Zhuang YH, Ma LX, Zhang XE (2006) Molecular cloning and expression in Pichia pastoris of a xylanase gene from Bacillus pumilus HBP8. Biocatal Biotransformation 24(5):371–379
Acknowledgments
We thank Dr. Guo Reyting for his critical review of this manuscript and helpful discussions. This research project was supported by the National Natural Science Foundation of China (31170068), the Ministry of Sciences and Technology of China (973 programs 2007CB707801 and 2012CB721000, 863 programs 2006AA020201 and 2007AA021306).
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Communicated by M. da Costa.
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Zhang, G., Li, S., Xue, Y. et al. Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10. Extremophiles 16, 35–43 (2012). https://doi.org/10.1007/s00792-011-0403-2
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DOI: https://doi.org/10.1007/s00792-011-0403-2