Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials.
A separation procedure for the analysis of the enzyme components of the hyperthermophilic bacterium Thermotoga maritima involved in cellulose and xylan degradation was developed. Resolution of the enzymes was achieved by a combination of fast protein liquid chromatography anion exchange and hydrophobic interaction chromatography. Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), beta-glucan, laminarin, xylan, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, p-nitrophenyl-alpha-L-arabinofuranoside, and 4-O-methyl-glucuronosyl-xylotriose. The activities of two cellulases, one laminarinase, one xylanase, two putative beta-D-xylosidases, alpha-D-glucuronidase, and alpha-L-arabinosidase were identified. Because of their selective retardation on a Superdex gel filtration column, the two cellulases could be purified to homogeneity. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular masses of 27 and 29 kDa, respectively, were determined for cellulase I and cellulase II. Maximal activities of both enzymes were observed at 95 degree C between pH 6.0 and 7.5. In the presence of 2.5 M NaC1 the purified enzymes retained about 90% of their initial activities after a 6-h incubation at 80 degree C. On the basis of its activity towards CMC, cellulase I was classified as endo-beta-1,4-glucanase. Cellulase II was able to attack Avicel in addition to CMC, beta-glucan, and p-nitrophenyl-beta-D-cellobioside. It releases cellobiose and cellotriose from Avicel. The latter product is further cleaved into glucose and cellobiose. Cellulase II may therefore be classified as exo-beta-1,4-glucanase.
Bronnenmeier
K
K
Institut für Mikrobiologie, Technische Universität München, Federal Republic of Germany.
Kern
A
A
Liebl
W
W
Staudenbauer
W L
WL
D001673:Biodegradation, Environmental; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000302:isolation & purification / Q000378:metabolism
D014990:Xylans; D002482:Cellulose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase