Naoki Tahara

Degree of Polymerization of Cellulose from Acetobacter xylinum BPR2001 Decreased by Cellulase Produced by the Strain

Acetobacter xylinum produces both cellulase and bacterial cellulose, but some report believed that this cellulase activity does not decrease the degree of polymerization (DP) of bacterial cellulose during cultivation. A. xylinum subsp. sucrofermentans BPR2001 produces two enzymes that hydrolyze CM-cellulose and cellotriose, respectively. We examined the effect of the two cellulase activities on the DP of bacterial cellulose when bacterial cells were cultured with agitation at pH 4, where little cellulase is produced, and at pH 5, where much cellulase is produced. The weight-average degree of polymerization (DPw) of bacterial cellulose remained in the range of 14,000 of 16,000 during cultivation at pH 4, but at pH 5, the DPw decreased from 16,800 to 11,000. The mechanical strength of a sheet prepared from the bacterial cellulose produced at pH 4 was higher than those of BC produced at pH 5. These results suggest that the two cellulase activities cause the decrease in DP and deterioration of physical properties of bacterial cellulose seen during cultivation.

Two types of cellulase activity produced by a cellulose-producing Acetobacter strain

Cellulase activities were detected in a culture supernatant concentrate (designated EX) and a cell extract (designated IN) of Acetobacter xylinum subsp. sucrofermentans BPR2001. IN and EX each hydrolyzed carboxymethylcellulose (CMC) strongly, but neither hydrolyzed bacterial cellulose (BC), Avicel or cellobiose. However, they did decrease the degree of polymerization of BC. By contrast, IN hydrolyzed cellotriose (G3). From the results of TLC assays and pH stability tests, it is suggested that this organism produces not only CMCase but also another cellulase, G3ase.

Purification and characterization of exo-1,4-β-glucosidase from Acetobacter xylinum BPR2001

Abstract An exo-1,4-β-glucosidase (EC 3.2.1.74; G3ase) was obtained from the supernatant of cultured Acetobacter xylinum subsp. sucrofermentans BPR2001 and purified to homogeneity by ammonium sulfate precipitation, cation-exchange, gel-filtration and hydrophobic interaction chromatography. The enzyme migrated to a position corresponding to 81.2 kDa on SDS-polyacrylamide gel electrophoresis under both non-reducing and reducing conditions, suggesting that this enzyme is a monomer polypeptide. The isoelectric point was 6.0. N-Bromosuccinimide inhibited the activity of exo-1,4-β-glucosidase completely, whereas sulfhydryl reagents did not. The Km and Vmax for the hydrolysis of cellotriose as substrate were 3.7 mM and 7.4 μmol/min/mg, respectively. The enzyme specifically cleaved the non-reducing ends of β-glucosyl linkages of cellotriose or larger cello-oligosaccharides, 4-methylumberiferryl- and p-nitrophenyl-β- d -glucosides, but cellobiose was hydrolyzed only slightly and salicin not at all. The enzyme catalyzes the hydrolysis of glucosidic linkages in such a manner that the product retains the anomeric configuration of the substrate.

Subsite structure of exo-1,4-β-glucosidase from Acetobacter xylinum BPR2001

Abstract The Michaelis constant ( K m ) and molecular activity ( k 0 ) of an exo-1,4-β-glucosidase (EC 3.2.1.74) from Acetobacter xylinum subsp. sucrofermentans BPR2001 for hydrolysis of cello-oligosaccharides (G2–G6) were determined by steady-state kinetic analysis. The 1 K m and k 0 values for G2 were much lower than those for G3–G6. The enzyme was competitively inhibited by glucono-δ-lactone and conduritol-β epoxide. Based on the theory of Hiromi et al. (Biochim. Biophys. Acta, 302: 362–375, 1973), the subsite affinities (A i , i=1–6) and the intrinsic hydrolysis rate constant for substrate linkage in a productive complex ( k int ) of the enzyme were kinetically estimated: A 1 =2.46 kcal/mol, A 2 =−0.44 kcal/mol, A 3 =3.70 kcal/mol, A 4 =0.33 kcal/mol, A 5 =0.27 kcal/mol, A 6 =0.06 kcal/mol, and k int =33.4 s −1 . The subsite affinities were different from those for the β-glucosidase from Aspergillus niger and for the exo-1,4-β-glucosidase from Torulopsis wickerhamii , in that the A 2 value for the Acetobacter enzyme was negative. These results suggest that the enzyme possesses subsite affinities which have never previously been reported among exo-type glucosidases.