Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris.
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 (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)). Here, we report the functional analysis of Delta(1-90)Cel16, 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(1-90)Cel16 in a pure form. The molecular mass of Delta(1-90)Cel16, 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(1-90)Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta(1-90)Cel16 had a pH optimum of 6.0. The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.
Mølhøj
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Biotechnology Group and Center for Molecular Plant Physiology (PlaCe), Danish Institute of Agricultural Sciences, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Ulvskov
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P
Dal Degan
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D000595:Amino Acid Sequence; D000910:Antibodies, Heterophile; D029688:Brassica napus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002118:Calcium / Q000494:pharmacology; D002266:Carboxymethylcellulose Sodium; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
D000910:Antibodies, Heterophile; D008565:Membrane Proteins; D010940:Plant Proteins; D002482:Cellulose; D004492:Edetic Acid; C440492:Cel16 protein, Brassica napus; D002480:Cellulase; D008274:Magnesium; D015032:Zinc; D002266:Carboxymethylcellulose Sodium; D002118:Calcium