Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials

K. Bronnenmeier, A. Kern, W. Liebl, W. L. Staudenbauer

Research output: Contribution to journalArticlepeer-review

114 Scopus citations

Abstract

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), β-glucan, laminarin, zylan, p-nitrophenyl-β-D-glucoside, p-nitrophenyl-β-D-cellobioside, p- nitrophenyl-β-D-xyloside, p-nitrophenyl-α-L-arabinofuranoside, and 4-O- methyl-glucuronosyl-xylotriose. The activities of two cellulases, one laminarinase, one zylanase, two putative β-D-xylosidases, α-D- glucuronidase, and α-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°C between pH 6.0 and 7.5. In the presence of 2.5 M NaCl the purified enzymes retained about 90% of their initial activities after a 6-h incubation at 80°C. On the basis of its activity towards CMC, cellulase I was classified as endo-β-1,4-glucanase. Cellulase II was able to attack Avicel in addition to CMC, β-glucan, and p- nitrophenyl-β-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-β-1,4-glucanase.

Original languageEnglish
Pages (from-to)1399-1407
Number of pages9
JournalApplied and Environmental Microbiology
Volume61
Issue number4
DOIs
StatePublished - 1995

Fingerprint

Dive into the research topics of 'Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials'. Together they form a unique fingerprint.

Cite this