Alkalophilic Bacillus - Applied and Environmental Microbiology

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An enzyme which catalyzes decarboxylation of phenylalanine and tyrosine is known in animals and bacteria as aromatic L-amino acid decarboxylase (2).

Vol. 59, No. 8

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2720-2722 0099-2240/93/082720-03$02.00/0

An Alkalophilic Bacillus NOBUKO


Produces 2-Phenylethylamine


AND TAIRO OSHIMA1* Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 227,1 Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-02,2 and Department of Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152 Japan

Received 13 October 1992/Accepted 29 May 1993

A large amount of 2-phenylethylamine was produced in cells of alkalophilic Bacilus sp. strain YN-2000. This amine is secreted in the medium during the cell growth. The amounts of 2-phenylethylamine in both cells and medium change upon changing the pH of the medium.

Polyamines are thought to play important roles in cellular physiology, such as protein synthesis and cell division (10). Polyamines also respond to environmental stresses and cellular polyamine composition changes upon change of external conditions (7, 10). In the course of our study on the distribution of polyamines in microorganisms under extreme conditions, we detected an unidentified basic compound in an extract of alkalophilic Bacillus sp. strain YN-2000 (3, 5). In this report, we describe the chemical structure of the unknown compound. Alkalophilic bacteria were aerobically grown at 37°C in a nutrient medium (3) supplemented with 1.5 g of Na2HPO4- 12H20 and 0.1 g of MgCl2. 6H20 in 1,000 ml of distilled water. The pH of the medium was kept at 9.5 during growth by adding solid Na2CO3. In some experiments, we attempted to grow strain YN-2000 in a synthetic medium and found that this strain requires methionine as an essential amino acid. Some vitamins were also essential for the growth. A synthetic medium used in this study is composed of 20 g of sucrose, 20 g of sodium glutamate, 50 mg of methionine, 1.0 g of Na2HPO4. 12H20, 2.0 g of NaCl, 100 ,tg of biotin, 1 mg of thiamine, 0.1 g of MgCl2- 6H20, and 0.025 g of CaCl2. 2H20 in 1,000 ml of distilled water. MgCl2 and CaCl2 were added after the autoclaving. This alkalophile can grow on glucose, maltose, or sucrose as a carbon source and sodium glutamate as a nitrogen source in the presence of methionine, vitamins, and minerals. Amines extracted from the cells or from the culture medium were analyzed as described previously (6) with a slight modification. Amines were detected by using a spectrofluorometer (Hitachi 204-A) after reaction with an ophthalaldehyde solution (4). The unknown compound was eluted after spermine was eluted under the analytical conditions. The unknown amine was purified by repeated chromatographic separation with a column (1.6-cm diameter by 20 cm high) of Dowex 5OW-X4 (H+ form; Dow Chemical Co.). The fractions containing the unknown amine were collected and evaporated to dryness. Since the unknown amine was secreted into the culture medium, one-step isolation of this amine from both the cells and the medium was devised for a large scale preparation. To the culture medium of late log phase of the alkalophile was added 50% trichloroacetic acid to make a final concen-

tration of 5%. After the mixture was centrifuged, solid sodium hydroxide was added to the supernatant to make its pH 12.5. The amine was extracted with ethyl acetate for three times from the medium and then extracted with 1 N hydrochloric acid from the ethyl acetate. The colorless aqueous layer was applied to the cation exchange resin column as described above. 1H- and proton decoupled 13C-nuclear magnetic resonance (NMR) spectra were measured with a JEOL FX-90Q (90MHz) spectrometer. Infrared spectra were recorded on a Hitachi 260-10 spectrophotometer. UV absorption was measured with Beckman DU-40 after the sample was dissolved in 0.1 N hydrochloric acid. Gas chromatography-mass spectra were recorded on a JEOL JMS-DX300 mass spectrometer, and samples were introduced after being derivatized with heptafluorobutyric anhydride. The amine composition of alkalophile YN-2000 grown in a nutrient medium at pH 9.5 and 37°C and harvested at the middle log phase is shown in Table 1. The cells contained spermidine as the major polyamine and putrescine as a minor component. A small amount of spermine was also detected, and the bacterium is able to synthesize this polyamine since spermine was detected in the cells grown in a synthetic medium. In addition to these polyamines, the alkalophile produces a large amount of an unknown compound. The unknown compound was purified and analyzed by physicochemical measurements. In the 'H-NMR spectrum, three signals were observed at 3.0 ppm (triplet), 3.2 ppm (triplet) and 7.4 ppm (broad), respectively. Six signals found in the 13C-NMR spectrum and their possible assignments (6, TABLE 1. Polyamine and 2-phenylethylamine contents of various alkalophilic Bacillus spp. Amt detected (nmol/g [W/wt])b


Bacillus sp. YN-2000 Bacillus sp. YN-1 B. alkalophilus B. firmus RAB RA-1 B. finnus OF-4





25 tr tr 23 29

2,100 109 248 1,223 2,167

65 4 107 307 467


a Strains YN-2000 and YN-1 were provided by Y. Nosoh,


University, and B. alkalophilus (ATCC27647), B. firmus RAB RA-1 (Sm'),

and its homologous strain OF-4 were provided by Y. Imae, Nagoya UniversitK. *

Corresponding author.

ND, not detected. For other abbreviations, see the legend to Fig. 3.


VOL. 59, 1993







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