Abstract
A family 26, glycoside hydrolase (GH26) named lichenase of the molecular size 30 kDa fromClostridium thermocellum previously cloned into an expression vector pET21a was over-expressed inEscherichia coli BL-21 cells and was purified to homogeneity by a single step purification method using immobilised metal ion affinity-chromatography. The purified enzyme showed no activity towards soluble substrates such as carboxymethyl cellulose, hydroxymethyl cellulose, laminarin, galactomannan or glucomannan. However, unlike other members of the family GH26, the enzyme lichenase showed high activity towards lichenan and β-glucan and was highly specific endo acting β-1,3-1,4-glucanase. Quantitative affinity-gel electrophoresis on native-PAGE showed that lichenase binds only glucomannan (with aKa of 41.3 C−1) and not lichenan or β-glucan.
This is a preview of subscription content, log in via an institution to check access.
References
Araki T., Hashikawa S., Morishita T. (2000). Cloning, sequencing and expression inEscherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium,Vibrio sp. strain XY-214. Appl. Environ. Microbiol., 66: 1741–1743.
Braithwaite K.L., Black G.W., Hazlewood G.P., Ali B.R., Gilbert H.J. (1995). A non-modular endo-beta-1,4-mannanase fromPseudomonas fluorescens subspeciescellulosa. Biochem. J., 305: 1005–1010.
Dias F.M.V., Vincent F., Pell G., Prates J.A.M., Centeno M.S.J., Tailford L.E., Ferreira L.M.A., Fontes C.M.G.A., Davies G.J., Gilbert, H.J. (2004). Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics ofCellvibrio mixtus mannosidase 5A. J. Biol. Chem., 279: 25517–25526.
Goyal A., Carvalho A.L., Prates J.A.M., Bolam D.N., Gilbert H.J., Pires V.M.R., Ferreira L.M.A., Planas A., Romao M.J., Fontes C.M.G.A. (2004) The family 11 carbohydrate-binding module ofClostridium thermocellum Lic26ACel5E accommodates ß-1,4 and ß-1,3-1,4-mixed linked glucans at a single binding site. J. Biol. Chem., 279: 34785–34793.
Goyal A., Taylor E.J., Guerreiro C.I.P.D., Prates J.A.M., Money V., Ferry N., Morland C., Planas A., Macdonald J.A., Stick R.V., Gilbert H.J., Fontes C.M.G.A., Davies G.J. (2005). How family 26 glycoside hydrolases orchestrate catalysis on different polysaccharides? Structure and activity of aClostridium thermocellum lichenase, CtLic26A. J. Biol. Chem., 280: 32761–32767.
Henrissat B. (1991). A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J., 280: 309–316.
Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680–685.
Okazaki F., Tamaru Y., Hashikawa S., Li Y.T., Araki T. (2002). Novel carbohydrate binding module of beta-1,3-xylanase from a marine bacterium,Alcaligenes sp. strain XY-234. J. Bacteriol., 184: 2399–2403.
Takeo K. (1984). Affinity electrophoresis: Principles and applications. Electrophoresis, 5: 187–195.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ahmed, S., Fontes, C.M.G.A. & Goyal, A. Recombinant lichenase fromClostridium thermocellum binds glucomannan but not to lichenan: Analysis by affinity electrophoresis. Ann. Microbiol. 58, 723–725 (2008). https://doi.org/10.1007/BF03175581
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF03175581