Open AccessPurification and Characterization of Thermostable Endo-1,5-α- l -Arabinase from a Strain of Bacillus thermodenitrificans
Author(s) -
Makoto Takao,
Kana Akiyama,
Taku Sakai
Publication year - 2002
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.68.4.1639-1646.2002
Subject(s) - bacillus subtilis , chemistry , sugar beet , enzyme , biochemistry , isoelectric point , molecular mass , pectin , arabinose , enzyme assay , gel electrophoresis , strain (injury) , bacteria , chromatography , fermentation , biology , xylose , genetics , anatomy , horticulture
A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.