Abstract
The synergism between the endo/exocellulase, Cel9A, and β-glucosidase (βgl) of Thermobifida fusca was investigated. Wild type βgl or S319C, a βgl mutant with significantly improved cellobiase activity, were added to Cel9A. Both wild type and mutant βgl enhanced the Cel9A hydrolysis of carboxymethyl cellulose (CMC) and filter paper by 50–100% compared to Cel9A alone. No enhancement occurred with addition of E388A, an inactive form of βgl. HPLC analysis showed that, with Cel9A alone, the resulting hydrolysate of glucose and cellobiose contained about half glucose; after addition of equimolar amounts of either wild type βgl or mutant S319C to Cel9A, the hydrolysate contained more than 85% glucose. βgl thus acted synergistically with Cel9A by converting cello-oligomers to glucose; this reduced the soluble sugar accumulation during hydrolysis of cellulose.
Similar content being viewed by others
References
Bae HJ, Turcotte G, Kim YS, Vézina LP, Laberge S (2004) Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose. FEMS Microbial Lett 233:325–331
Barr BK, Hsieh YL, Ganem B, Wilson DB (1996) Identification of two functionally different classes of exocellulases. Biochemistry 35:586–592
Bravo V, Páez MP, Aoulad El-Hadj M, Reyes A, García AI (2002) Hydrolysis of carboxymethylcellulose with mixtures of cellulase and beta-1,4-glucosidase. J Chem Technol Biotechnol 77:15–20
Din N, Damude HG, Gilkes NR, Miller RC, Warren RA Jr, Kilburn DG (1994) C1-Cx revisited: intramolecular synergism in a cellulase. Proc Natl Acad Sci USA 91:11383–11387
Escovar-Kousen JM, Wilson D, Irwin D (2004) Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca. Appl Biochem Biotechnol 113–116:287–397
Fujii M, Shimizu M (1986) Synergism of endoenzyme and exoenzyme on hydrolysis of soluble cellulose derivatives. Biotechnol Bioeng 28:878–882
Irwin DC, Spezio M, Walker LP, Wilson DB (1993) Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects. Biotechnol Bioeng 42:1002–1013
Irwin DC, Shin DH, Zhang S, Barr BK, Sakon J, Karplus PA, Wilson DB (1998) Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis. J Bacteriol 180:1709–1714
Jeoh T, Wilson DB, Walker LP (2002) Cooperative and competitive binding in synergistic mixtures of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A. Biotechnol Prog 18:760–769
Jeoh T, Baker JO, Ali MK, Himmel ME, Adney WS (2005) Beta-d-glucosidase reaction kinetics from isothermal titration microcalorimetry. Anal Biochem 347:244–253
Jeoh T, Wilson DB, Walker LP (2006) Effect of cellulase mole fraction and cellulose recalcitrance on synergism in cellulose hydrolysis and binding. Biotechnol Prog 22:270–277
Kim E, Irwin DC, Walker LP, Wilson DB (1998) Factorial optimization of a six-cellulase mixture. Biotechnol Bioeng 58:494–501
Kosugi A, Arai T, Doi RH (2006) Degradation of cellulosome-produced cello-oligosaccharides by an extracellular non-cellulosomal beta-glucan glucohydrolase, BglA, from Clostridium cellulovorans. Biochem Biophys Res Commun 349:20–23
Kruus K, Andreacchi A, Wang WK, Wu JH (1995) Product inhibition of the recombinant CelS, an exoglucanase component of the Clostridium thermocellum cellulosome. Appl Microbiol Biotechnol 44:399–404
Li Y, Irwin DC, Wilson DB (2007) Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol 73:3165–3172
Lynd LR, Weimer PJ, Zyl WHV, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577
Reese ET, Siu RG, Levinson HS (1950) The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J Bacteriol 59:485–497
Sakon J, Irwin D, Wilson DB, Karplus PA (1997) Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. Nat Struct Biol 4:810–818
Sánchez MM, Irwin DC, Javier Pastor FI, Wilson DB, Pilar Diaz (2004) Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca. Biotechnol Bioeng 87:161–169
Spiridonov NA, Wilson DB (2001) Cloning and biochemical characterization of BglC, a beta-glucosidase from the cellulolytic actinomycete Thermobifida fusca. Curr Microbiol 42:295–301
Teeri TT, Koivula A, Linder M, Wohlfahrt G, Divne C, Jones TA (1998) Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose? Biochem Soc Trans 26:173–178
Watson DL, Wilson DB, Walker LP (2002) Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A on BMCC and Avicel. Appl Biochem Biotechnol 101:97–111
Wilson DB (2004) Studies of Thermobifida fusca plant cell wall degrading enzymes. Chem Rec 4:72–82
Woodward J, Lima M, Lee NE (1988) The role of cellulase concentration in determining the degree of synergism in the hydrolysis of microcrystalline cellulose. Biochem J 255:895–899
Yue Z, Bin W, Baixu Y, Peiji G (2004) Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase. Sci China, Ser C Life Sci 47:18–24
Zhou W, Irwin DC, Escovar-Kousen J, Wilson DB (2004) Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes. Biochemistry 43:9655–9663
Acknowledgment
This research was supported by the National Science Council of Taiwan.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Chir, JL., Wan, CF., Chou, CH. et al. Hydrolysis of cellulose in synergistic mixtures of β-glucosidase and endo/exocellulase Cel9A from Thermobifida fusca . Biotechnol Lett 33, 777–782 (2011). https://doi.org/10.1007/s10529-010-0500-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-010-0500-9