Gene cloning, sequencing, and characterization of a family 9 endoglucanase (CelA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009.
A gene encoding a beta-1,4-glucanase (CelA) belonging to subfamily E1 of family 9 of glycoside hydrolases was cloned and sequenced from the gram-positive thermoacidophile Alicyclobacillus acidocaldarius strain ATCC27009. The translated protein contains an immunoglobulin-like domain but lacks a cellulose-binding domain. The enzyme, when overproduced in Escherichia coli and purified, displayed a temperature optimum of 70 degrees C and a pH optimum of 5.5. CelA contained one zinc and two calcium atoms. Calcium and zinc are likely to be important for temperature stability. The enzyme was most active against substrates containing beta-1,4-linked glucans (lichenan and carboxy methyl cellulose), but also exhibited activity against oat spelt xylan. A striking pattern of hydrolysis on p-nitrophenyl-glycosides was observed, with highest activity on the cellobioside derivative, some on the cellotetraoside derivative, and none on the glucoside and cellotrioside derivatives. Unmodified cellooligosaccharides were also hydrolyzed by CelA. No signal peptide for transport across the cytoplasmic membrane was detected. This, together with the substrate specificity displayed, near neutral pH optimum and irreversible inactivation at low pH, suggests a role for CelA as a cytoplasmic enzyme for the degradation of imported oligosaccharides.
Eckert
K
K
Humboldt-Universität zu Berlin, Institut für Biologie/Bakterienphysiologie, Chausseestrasse 117, Germany.
Zielinski
F
F
Lo Leggio
L
L
Schneider
E
E
D000143:Acids / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D000143:Acids; D002482:Cellulose; C074775:CelA endoglucanase; D002480:Cellulase