Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

Abstract

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.

FIG. 1.

Elution profile of ABN and ABF from a phenyl-Sepharose column. The conditions for chromatography are described in Materials and Methods. ○, ABF activity on p-nitrophenyl-α-l-arabinofuranoside; •, ABN activity on debranched arabinan; ▵, ABN activity on arabinan.

FIG. 2.

SDS-PAGE of purified ABN-TS. Lane 1, marker proteins; lane 2, purified ABN-TS (1 μg).

FIG. 3.

Effect of temperature on stability (A) and reaction rate (B) of ABN-TS. (A) Thermostabilities of ABN-TS and PPase-C. Residual ABN activities were measured at 70°C and pH 6.0 after incubation at 70°C (ABN-TS) (▪), 75°C (ABN-TS) (□), or 65°C (PPase-C) (○), at various times. (B) Relationship between temperature and activity of ABN-TS (Arrhenius plots).

FIG. 4.

Protopectinase activity of PPase-C and ABN-TS. Sugar beet protopectin (20 mg) was incubated with ABN-TS (5.9 U) and PPase-C (0.59 U) in 1 ml of 100 mM sodium acetate buffer (pH 6.0) at 50°C. Samples were taken out and filtered, and released pectic substance was measured by the carbazole-sulfuric acid method. •, ABN-TS; ○, PPase-C.

FIG. 5.

Determination of apparent K_m and V_max values of ABN-TS at 70°C from a Lineweaver-Burk plots. ○, debranched arabinan; □, arabinan.

FIG. 6.

HPLC analysis of products generated from debranched arabinan by ABN-TS and PPase-C. Debranched arabinan (0.1%) was incubated with ABN-TS (1.26 U) and PPase-C (1.18 U) in 1 ml of 20 mM sodium acetate buffer (pH 6.0) at 70 and 50°C, respectively. At 0 min (a), 1 min (b), 2 min (c), 5 min (d), 30 min (e), and 2 h (f), reaction mixtures were removed and boiled for 5 min to stop the reaction. Twenty microliters of each samples was subjected to HPLC analysis as described in Materials and Methods. The elution times of the standards arabinose (1), arabinobiose (2), arabinotriose (3), and arabinotetraose (4) are indicated.