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A novel α-L-arabinofuranosidase (Abf22-3) that could biotransform ginsenoside Rc into Rd was obtained from the ginsenoside converting Leuconostoc sp. strain 22-3, isolated from the Korean fermented food kimchi. The gene, termed abf22-3, consisting of 1,527 bp and encoding a protein with a predicted molecular mass of 58,486 Da was cloned into the pMAL-c2x (TEV) vector. A BLAST search using the Abf22-3's amino acid sequence revealed significant homology to that of family 51 glycoside hydrolases. The over-expressed recombinant Abf22-3 in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinofuranoside moiety attached to the C-20 position of ginsenoside Rc under optimal conditions of pH 6.0 and 30 °C. This result indicated that Abf22-3 selectively converts ginsenoside Rc into Rd, but did not catalyze the hydrolysis of glucopyranosyl groups from Rc or other ginsenosides such as Rb1 and Rb2. Over-expressed recombinant enzymes were purified by two steps with amylose-affinity and DEAE-cellulose chromatography and then characterized. The kinetic parameters for α-L-arabinofuranosidase showed apparent Km and Vmax values of 0.95 ± 0.02 μM and 1.2 ± 0.1 μmol min(-1) mg of protein(-1) against p-nitrophenyl-α-L-arabinofuranoside, respectively. Using a purified MBP-Abf22-3 (10 μg/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.

The agar-degrading bacterium GNUM-1 was isolated from the brown algal species Sargassum serratifolium, which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, 0.5-0.6 micrometer wide and 2.0-2.5 micrometer long curved rods with a single polar flagellum, forming nonpigmented, circular, smooth colonies. Cells grew at 20 degrees C- 37 degrees C, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. The DNA G+C content of the GNUM-1 strain was 45.5 mol%. The 16S rRNA sequence of the GNUM-1 was very similar to those of Alteromonas stellipolaris LMG 21861 (99.86% sequence homology) and Alteromonas addita R10SW13 T (99.64% sequence homology), which led us to assign it to the genus Alteromonas. It showed positive activities for agarase, amylase, gelatinase, alkaline phosphatase, esterase (C8), lipase (C14), leucine arylamidase, valine arylamidase, alpha-chymotrypsin, acid phosphatase, naphthol- AS-BI-phosphohydrolase, alpha-galactosidase, beta-galactosidase, beta-glucosidase, catalase, and urease. It can utilize citrate, malic acid, and trisodium citrate. The major fatty acids were summed feature 3 (21.5%, comprising C16:1omega7c/iso- C15:0 2-OH) and C16:0 (15.04%). On the basis of the variations in many biochemical characteristics, GNUM-1 was considered as unique and thus was named Alteromonas sp. GNUM-1. It produced the highest agarase activity in modified ASW medium containing 0.4% sucrose, but lower activity in rich media despite superior growth, implying that agarase production is tightly regulated and repressed in a rich nutrient condition. The 30 kDa protein with agarase activity was identified by zymography, and this report serves as the very first account of such a protein in the genus Alteromonas.

Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 degrees C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca(2+). However, Hg(2+) and Ni(+) ions strongly inhibited the enzyme. The enzyme produced, GlcN(2-4), were the major products from a soluble chitosan.