We characterized a β-L-arabinopyranosidase AbpBL (BLLJ_1823) belonging to the glycoside hydrolase family 27 (GH27) from Bifidobacterium longum subsp. longum JCM1217. The recombinant AbpBL expressed in Escherichia coli hydrolyzed pNP-β-L-arabinopyranoside but not pNP-α-D-galactopyranoside. The enzyme also liberated L-arabinose from the β-L-arabinopyranosyl side chain of larch wood arabinogalactan. However, we could not detect any β-L-arabinopyranosidase activity or remarkable transcriptional induction in cultured cells of B. longum subsp. longum. Mutagenesis experiments revealed that I56D and I56A mutants both exhibited β-L-arabinopyranosidase and α-D-galactopyranosidase activities. AbpBL Ile-56 residue is a critical residue for the specificity of β-L-arabinopyranosidase.
Water-soluble dietary fiber provides numerous health benefits. A novel procedure to efficiently manufacture water-soluble indigestible polysaccharides was developed by heating glucose at 180°C in the presence of activated carbon. Aside from its ability to catalytically assist the polycondensation of saccharides, activated carbon provides the added benefits of being easily separable from the reactants and suppressing coloration of the product. Prior to purification, the indigestible fraction made up over 80% of the reaction mixture. After hydrolysis catalyzed by α-amylase and glucoamylase, and fractionation by ion-exchange chromatography, a total of 99.7% dietary fiber content was attained. This indigestible fraction, termed resistant glucan, was only minimally degraded by upper digestive tract enzymes, similar to the digestibility of polydextrose. Structural analysis by methylation and NMR indicated that the resistant glucan formed a highly branched structure containing α- and β-1,2-, 1,3-, 1,4-, and 1,6-linkages. On an industrial scale, the resistant glucan was obtained from glucose syrup (DE 86) by heating with activated carbon, enzymatic hydrolysis, refining, fractionating, and drying. Our facile method is an efficient means to obtain water-soluble dietary fiber.
Lysozyme was purified from brown eared pheasant (Crossoptilon mantchuricum) egg white using pH treatment and cation exchange chromatography resulting in an 80-fold enhancement of the specific activity. The enzyme exhibited hydrolytic activity toward glycol chitin and chitooligosaccharides [(GlcNAc)n (n=5 and 6)]. The enzyme catalyzed degradation of bacterial cells of not only Micrococcus luteus but also Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 upon treatment with chloroform/tris(hydroxymethyl)aminomethane-HCl. The pH optimum of the glycol chitin hydrolytic reaction was 5.5 at 37°C. The optimal temperature for activity was 53°C in 50 mM sodium acetate buffer (pH 5.5). The enzyme mainly hydrolyzed the fourth glycosidic linkage from the nonreducing end of (GlcNAc)6. The anomeric form of the products indicated it was a retaining enzyme.
Glucose tolerance, an enzymatic performance in the presence of glucose, was improved with Geobacillus thermoglucosidasius oligo-1,6-glicosidase (GTAGL) by site-directed mutagenesis. The quadruple mutant of GTAGL (qGTAGL: M203W/Q216E/G259E/R298I) produced by this work resulted in an increase of the glucose tolerance. Although GTAGL lost its enzyme activity in the presence of 2.2% glucose, qGTAGL retained the activity in the presence of 2.7% glucose. Notably, enzymatic properties including thermostability and optimal temperature were not severely affected by the mutations.