Plant Physiol. Illumina
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (30)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mølhøj, M.
Right arrow Articles by Dal Degan, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mølhøj, M.
Right arrow Articles by Dal Degan, F.
Agricola
Right arrow Articles by Mølhøj, M.
Right arrow Articles by Dal Degan, F.

Plant Physiol, October 2001, Vol. 127, pp. 674-684

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris1

Michael Mølhøj,2 Peter Ulvskov,* and Florence Dal Degan3

Biotechnology Group and Center for Molecular Plant Physiology (PlaCe), Danish Institute of Agricultural Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta -glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-beta -glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine1-lysine70), a hydrophobic transmembrane domain (isoleucine71-valine93), and a periplasmic catalytic core (lysine94-proline621). Here, we report the functional analysis of Delta 1-90Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Delta 1-90Cel16 in a pure form. The molecular mass of Delta 1-90Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Delta 1-90Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta 1-90Cel16 had a pH optimum of 6.0. The activity of Delta 1-90Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta 1-90Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1right-arrow3),(1right-arrow4)-beta -D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta 1-90Cel16-hydrolyzed carboxymethylcellulose showed that Delta 1-90Cel16 is a true endo-acting glucanase.

1 This work was supported by a grant from the Danish National Research Foundation.

2 Present address: Department of Molecular and Cell Biology, University of Connecticut, 75 North Eagleville Road, Storrs, CT 06269.

3 Present address: M&E Biotech A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark.

* Corresponding author; e-mail p.ulvskov{at}dias.kvl.dk; fax 45-35-28-25-89.

© 2001 American Society of Plant Physiologists


This article has been cited by other articles:


Home page
 Plant Cell PhysiolHome page
J. Takahashi, U. J. Rudsander, M. Hedenstrom, A. Banasiak, J. Harholt, N. Amelot, P. Immerzeel, P. Ryden, S. Endo, F. M. Ibatullin, et al.
KORRIGAN1 and its Aspen Homolog PttCel9A1 Decrease Cellulose Crystallinity in Arabidopsis Stems
Plant Cell Physiol., June 1, 2009; 50(6): 1099 - 1115.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. S. Kang, J. Frank, C. H. Kang, H. Kajiura, M. Vikram, A. Ueda, S. Kim, J. D. Bahk, B. Triplett, K. Fujiyama, et al.
Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus
PNAS, April 15, 2008; 105(15): 5933 - 5938.
[Abstract] [Full Text] [PDF]

Home page
 J Exp BotHome page
S. Lu, L. Li, X. Yi, C. P. Joshi, and V. L. Chiang
Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress
J. Exp. Bot., February 16, 2008; (2008) erm350v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. R. Urbanowicz, C. Catala, D. Irwin, D. B. Wilson, D. R. Ripoll, and J. K. C. Rose
A Tomato Endo-beta-1,4-glucanase, SlCel9C1, Represents a Distinct Subclass with a New Family of Carbohydrate Binding Modules (CBM49)
J. Biol. Chem., April 20, 2007; 282(16): 12066 - 12074.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
K. Yoshida and K. Komae
A Rice Family 9 Glycoside Hydrolase Isozyme with Broad Substrate Specificity for Hemicelluloses in Type II Cell Walls
Plant Cell Physiol., November 1, 2006; 47(11): 1541 - 1554.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
S. Robert, A. Bichet, O. Grandjean, D. Kierzkowski, B. Satiat-Jeunemaitre, S. Pelletier, M.-T. Hauser, H. Hofte, and S. Vernhettes
An Arabidopsis Endo-1,4-{beta}-D-Glucanase Involved in Cellulose Synthesis Undergoes Regulated Intracellular Cycling
PLANT CELL, December 1, 2005; 17(12): 3378 - 3389.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
H. Aspeborg, J. Schrader, P. M. Coutinho, M. Stam, A. Kallas, S. Djerbi, P. Nilsson, S. Denman, B. Amini, F. Sterky, et al.
Carbohydrate-Active Enzymes Involved in the Secondary Cell Wall Biogenesis in Hybrid Aspen
Plant Physiology, March 1, 2005; 137(3): 983 - 997.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
M. Molhoj, S. Pagant, and H. Hofte
Towards Understanding the Role of Membrane-bound Endo-{beta}-1,4-glucanases in Cellulose Biosynthesis
Plant Cell Physiol., December 15, 2002; 43(12): 1399 - 1406.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
M. S. Doblin, I. Kurek, D. Jacob-Wilk, and D. P. Delmer
Cellulose Biosynthesis in Plants: from Genes to Rosettes
Plant Cell Physiol., December 15, 2002; 43(12): 1407 - 1420.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
C. S. Gillmor, P. Poindexter, J. Lorieau, M. M. Palcic, and C. Somerville
{alpha}-Glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis
J. Cell Biol., March 18, 2002; 156(6): 1003 - 1013.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
L. Peng, Y. Kawagoe, P. Hogan, and D. Delmer
Sitosterol-beta -glucoside as Primer for Cellulose Synthesis in Plants
Science, January 4, 2002; 295(5552): 147 - 150.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ASPB Publications PLANT PHYSIOLOGY® THE PLANT CELL
Copyright © 2001 by the American Society of Plant Biologists