Isolation of alliinase-producing microorganism
Appropriately diluted suspension of each soil sample was plated onto a synthetic medium (12% (w/v) Na2HPO4, 0.3% KH2PO4, 0.1% D-glucose, 0.05% NaCl, 0.002% CaCl2, 0.0003% MgSO4·7H2O, and 2% (w/v) agar, pH 6.2), in which (±)-alliin was added at the concentration of 0.1% as a sole nitrogen source. After 7-days incubation at 25°C, colonies formed were isolated as alliin-utilizing strains, and were independently inoculated onto the agar plates for evaluation of odorous compound production. Six strains were chosen, but cells of these strains were poorly grown in the liquid synthetic medium with the above composition. Therefore, alliinase activity was assayed with cells cultivated in the nutrient medium as described in the following section. A strain with the highest activity was chosen and designated strain FERM P-19486, which has been deposited in the National Institute of Advanced Industrial Science and Technology (Tsukuba, Japan).
Identification of isolated strain FERM P-19486
Strain FERM P-19486 was identified on the basis of 16S rDNA sequence in addition to the morphological and physiological properties examined according to the guideline of (Casida (1982)). The genomic DNA was extracted with the DNeasy Tissue Kit (QIAGEN, Valencia, CA). PCR amplification of the 16S rDNA and its sequence analysis were performed with the MicroSeq Full Gene 16S rDNA kit (Applied Biosystems, Foster City, CA).
Enzyme assay
The standard alliinase assay mixture contained 40 mM (±)-alliin, 20 μM pyridoxal 5'-phosphate (PLP), 50 mM sodium phosphate (pH 7.0), and enzyme in a total volume of 1.0 ml. Alliinase activity was determined by colorimetrically measuring pyruvic acid produced in the reaction as described by (Durbin and Uchytil (1971)). One unit of the enzyme activity was defined as the enzyme amount that catalyzed the formation of 1 μmole of pyruvic acid per min at 30°C. Protein concentration was measured according to the method of Bradford using bovine serum albumin as a standard (Bradford, 1976).
Measurement of alliin, allicin, and ammonia
In the reaction with (±)-alliin, the remaining concentrations of (+)-alliin and (-)-alliin were measured by HPLC using a reverse-phase C18 column (YMC-Pack ODS-AM, YMC Co., Kyoto, Japan) at 220 nm. In this HPLC analysis, (+)-alliin and (-)-alliin could be separated by isocratic elution, which was done with 10 mM sodium phosphate buffer (pH 7.5) containing 5 mM tetra n-butylammonium dihydrogenphosphate at a flow rate of 1.0 ml per min. Allicin was also measured by HPLC using the same column except that it was isocratically eluted with a mixture of acetonitrile, H2O, and tetrahydrofuran at a ratio of 30: 67: 3 (v/v) and detected at 240 nm. Ammonia were colorimetrically measured as described by (Mazelis and Creveling (1975)).
Enzyme purification
(i) Preparation of cell free extract
Strain FERM P-19486 was routinely grown in the nutrient medium, which consisted of 3% (w/v) bouillon (Nissui Co., Tokyo, Japan), at 30°C for 3 d with vigorous shaking. Cells from 1,000 ml culture were collected by centrifugation at 7,000 × g for 10 min, washed, and suspended with 0.02 M sodium phosphate buffer, pH 7.0 (buffer A). Cells were then disrupted by ultrasonic treatment at 0°C using a Branson Sonifier 250, and the supernatant obtained after removing cell debris was used as a crude enzyme.
(ii) DEAE-cellulose column chromatography
The supernatant was put on a DEAE-cellulose column (3.0 × 14.0 cm) equilibrated with buffer A. After washing the column with the same buffer, the enzyme was eluted with buffer A containing 0.05 M NaCl.
(iii) Phenyl-sepharose column chromatography
After addition of NaCl to the active fraction at 1.5 M, it was then put on a phenyl-sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) column (1.5 × 3.0 cm) equilibrated with buffer A containing 1.5 M NaCl. After washing the column with the same buffer, the enzyme was eluted with buffer A. The active fractions were combined and dialyzed against buffer A.
(iv) Aminohexyl-sepharose column chromatography
The enzyme was put on an aminohexyl-sepharose (Sigma-Aldrich, St. Louis, MO) column (1.5 × 6.0 cm) equilibrated with buffer A. After the column was washed with the same buffer, the enzyme was eluted with a linear gradient of buffer A to buffer A containing 0.3 M NaCl. The volume of each fraction was 5 ml. The active fractions were combined and concentrated to about 500 μl with an Ultrafree-MC (30,000 NMWL, Millipore, Bedford, MA).
(v) Mono Q column chromatography
The enzyme solution was then applied to a Mono Q (Pharmacia, Uppsala, Sweden) column (5.0 × 50 mm) equilibrated with buffer A. After the column was washed with the same buffer, the enzyme was eluted with a linear gradient of buffer A to buffer A containing 0.2 M NaCl. The flow rate was 1 ml/min. The active fractions were combined and concentrated to about 50 μl with an Ultrafree-MC (30,000 NMWL).
(vi) Gel filtration
The enzyme was then applied to a TSK-GEL (TOSOH, Tokyo, Japan) column (7.8 × 300 mm) equilibrated with buffer A. The flow rate was 1 ml/min. The active fractions were combined and used as the purified enzyme.
Electrophoresis
The purity of the enzyme was examined by native-polyacrylamide gel electrophoresis (PAGE) using 8% polyacrylamide gel at a constant current of 20 mA per gel at 4°C. For detection of alliinase activity, gel slices were cut from another lane with the same sample, and each gel slice was directly incubated in 100 μl of the standard alliinae assay mixture at 30°C for 30 min. Allicin produced in the mixture was measured by HPLC. Sodium dodecyl sulfate (SDS)-PAGE was carried out using 10% (w/v) polyacrylamide gel at a constant current of 20 mA per gel, in which broad-range molecular mass standards (Bio-Rad Laboratories, Tokyo, Japan) were simultaneously run. Proteins were detected by silver staining.
Molecular mass determination
The molecular mass of the native enzyme was estimated by gel filtration using a TSK-GEL column. The operating condition was already described above. The column was calibrated by using the standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (66 kDa). The molecular mass of the enzyme under denaturing conditions was determined by SDS-PAGE.
Measurement of cell viability in medium containing (±)-alliin and P-19486 alliinase
S. cerevisiae W303-1A cells were grown at 30°C for 16 h with vigorous shaking in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose. An overnight-grown culture was diluted with freshly prepared YPD medium to obtain an initial cell density of 107 cells/ml, in which PLP was supplemented at 20 μM. Incubation was then started at 30°C with the addition of various concentrations of (±)-alliin and FERM P-19486 alliinase. Aliquots of the cell suspension were withdrawn, diluted, and spread onto YPD-agar plates to measure the viable cell number as colony-forming units after a 24-h incubation at 30°C.
Chemicals
(±)-Alliin, (+)-alliin, and allicin were products of LKT Laboratories, Inc (St. Paul, MN). S-Methyl-L-cysteine and S-ethyl-L-cysteine were obtained from ICN Pharmaceuticals, Inc (Costa Mesa, CA). S-Methyl-L-cysteine sulfoxide was from Research Organics, Inc (Cleveland, OH).