Journal
PLANT PHYSIOLOGY
Volume 127, Issue 2, Pages 674-684Publisher
AMER SOC PLANT BIOLOGISTS
DOI: 10.1104/pp.127.2.674
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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 I 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 pare 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.
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