J. Gen. Appl. Microbiol., 45, 149–153 (1999) Production of 2-phenylethylamine by decarboxylation of L-phenylalanine in alkaliphilic Bacillus cohnii Koei Hamana* and Masaru Niitsu1 School of Health Sciences, Faculty of Medicine, Gunma University, Maebashi 371–8514, Japan 1Faculty of Pharmaceutical Sciences, Josai University, Sakado 350–0290, Japan (Received February 22, 1999; Accepted August 16, 1999) Cellular polyamine fraction of alkaliphilic Bacillus species was analyzed by HPLC. 2-Phenylethyl- amine was found selectively and ubiquitously in the five strains belonging to Bacillus cohnii within 27 alkaliphilic Bacillus strains. A large amount of this aromatic amine was produced by the decar- boxylation of L-phenylalanine in the bacteria and secreted into the culture medium. The production of 2-phenylethylamine may serve for the chemotaxonomy of alkaliphilic Bacillus. Key Words——alkaliphilic Bacillus; phenylethylamine; polyamine In the course of our study on polyamine distribution sequence data of bacilli belonging to the genera Bacil- profiles as a chemotaxonomic marker, we have shown lus, Sporolactobacillus, and Amphibacillus, including that diamines such as diaminopropane, putrescine, various neutrophilic, alkaliphilic, and acidophilic and cadaverine, and a guanidinoamine, agmatine, species (Nielsen et al., 1994, 1995; Yumoto et al., sporadically spread within gram-positive bacilli (Hama- 1998). Therefore alkaliphilic members of Bacillus are na, 1999; Hamana et al., 1989, 1993). Mesophilic phylogenetically heterogeneous. In the present study, Bacillus species, including some alkaliphilic strains, we describe the distribution of this amine and the de- and Brevibacillus, Paenibacillus, Virgibacillus, Sporo- carboxylase activity for phenylalanine to produce this lactobacillus, and halophilic Halobacillus species con- amine within newly validated alkaliphilic Bacillus tained spermidine as the major polyamine and lacked species. spermine (Hamana, 1999; Hamana et al., 1989). Spermine was found as a major polyamine in Materials and Methods mesophilic Aneurinibacillus, thermophilic Bacillus and Amphibacillus species, and acidothermophilic Alicy- Alkaliphilic bacilli were aerobically grown in 0.5% clobacillus and Sulfobacillus (Hamana, 1999; Hamana peptone-0.1% yeast extract-0.5% glucose (PYG) et al., 1989, 1993). On the other hand, the occurrence medium, 0.5% peptone-0.1% yeast extract (PY) of an aromatic amine, 2-phenylethylamine (b- medium, 0.5% glucose-0.1% yeast extract (GY) phenethylamine), in an alkaliphilic Bacillus sp. YN- medium, and nutrient broth (NB), adjusted at pH 10 by 2000 was reported by Hamasaki et al. (1993). Newly Na2CO3. Nutrient agar supplemented with 0.5% uric validated 12 alkaliphilic Bacillus species, B. agarad- acid (NA-U), pH 7.2, was for Bacillus fastidiosus. Two haerens, B. alcalophilus, B. clarkii, B. clausii, B. polyamine-free synthetic media, Eagle MEM and 199 cohnii, B. gibsonii, B. halodurans, B. halmapalus, B. (Nissui Pharmaceutical Co., Tokyo, Japan), adjusted horikoshii, B. horti, B. pseudalcalophilus, and B. pseu- at pH 10 or 7 were also used. The 199 medium con- dofirmus, formerly belonging to the Bacillus firmus- tained 0.2–0.4 mML-phenylalanine, L-lysine, L-argi- Bacillus lentus complex (Fritze et al., 1990; Gordon nine, L-tyrosine, and other usual amino acids, but not and Hyde, 1982; Nielsen et al., 1994, 1995; Spanka L-ornithine. Culture media and growth temperatures and Fritze, 1993; Yumoto et al., 1998), widely spread are listed in Table 1. Cells at stationary phase were within the phylogenetic tree derived from 16S rRNA harvested by centrifugation at 10,000ϫg for 10 min. An extracellular secretion of amines was expected to be dominant at the stationary phase. The amines se- * Address reprint requests to: Dr. Koei Hamana, School of Health Sciences, Faculty of Medicine, Gunma University, 3–39–15 Showa- creted into the culture supernatant, the amines ex- machi, Maebashi 371–8514, Japan. tracted into cold 0.5 M perchloric acid (PCA) from the 150 HAMANA and NIITSU Vol. 45 Table 1. Cellular amines of alkaliphilic Bacillus. Polyamines (mmol/g wet cell) Medium °C Put Cad Spd Spm Agm Pea Bacillus alcalophilus JCM 5262T NB-pH 10 30 0.01 0.01 0.60 0.06 1.10 — PYG-pH 10 30 0.04 0.12 0.90 0.12 0.24 — (a) Eagle-pH 10 37 0.26 — 0.18 — 0.01 — (a) PYG-pH 10 37 0.48 0.20 0.89 0.08 0.92 — Bacillus clausii NCIMB 10309T PYG-pH 10 30 0.05 0.22 2.10 0.07 0.26 — JCM 9137 PYG-pH 10 30 0.02 0.04 1.50 0.02 0.04 — JCM 9138 PYG-pH 10 30 0.02 0.04 1.55 0.04 0.04 — JCM 9139 NB-pH 10 37 0.04 0.04 2.50 0.10 0.70 — PYG-pH 10 37 — 0.01 2.10 0.02 0.60 — Bacillus cohnii IFO 15565T PY-pH 10 30 — 0.02 2.45 0.06 0.04 0.62 PYG-pH 10 30 0.02 0.01 1.37 0.02 0.08 0.80 199-pH 10 30 — — 2.20 — 0.08 0.40 NCIMB 9218 PYG-pH 10 30 — 0.05 2.15 0.02 0.02 0.10 199-pH 10 30 — — 2.50 — 0.04 0.02 NCIMB 10284 PYG-pH 10 30 — — 1.02 0.02 0.04 0.20 199-pH 10 30 — — 1.20 — 0.02 0.05 NCIMB 10318 PYG-pH 10 30 — 0.01 1.55 0.04 0.07 0.37 NCIMB 10327 PYG-pH 10 30 — — 1.50 0.07 0.17 0.90 199-pH 10 30 — — 1.30 — 0.02 0.05 199-pH 10ϩPhe 30 — — 1.50 0.04 0.04 0.19 199-pH 7 30 — — 1.95 0.02 0.02 — Bacillus gibsonii NCIMB 11495 NB-pH 10 30 0.01 — 0.62 — 0.04 — NB-pH 7 30 — — 1.25 — 0.02 — 199-pH 10 30 — — 0.54 — — — 199-pH 7 30 0.15 — 1.52 0.06 — — Bacillus halodurans ATCC 27557T (b) PYG-pH 10 37 0.01 — 1.65 0.34 0.21 — (b) 199-pH 10 37 0.06 — 2.10 — 0.21 — JCM 9148 PY-pH 10 37 0.02 0.10 0.85 0.01 0.05 — PYG-pH 10 37 0.05 0.10 1.25 0.15 1.00 — 199-pH 10 37 0.04 — 1.40 — 1.10 — Bacillus horti JCM 9943T PYG-pH 10 30 0.02 — 0.90 0.02 0.02 — Bacillus pseudofirmus NCIMB 10283T PYG-pH 10 30 0.03 0.02 1.80 0.04 0.45 — 199-pH 10 30 — — 2.45 — 0.24 — JCM 9141 PYG-pH 10 30 — — 1.80 0.15 0.19 — JCM 9144 PYG-pH 10 30 — — 0.75 0.07 0.07 — Bacillus sp. N-4 JCM 9156 (a) Eagle-pH 10 37 0.04 — 2.02 — 0.01 — (a) PYG-pH 10 37 1.73 1.76 4.16 0.10 0.07 — Bacillus sp. W-1 JCM 2888 (a) Eagle-pH 10 37 0.13 — 3.39 — 0.10 — (a) PYG-pH 10 37 0.40 0.19 1.92 0.21 0.05 — Bacillus sp. TX-3 JCM 9162 PY-pH 10 55 — — 1.17 1.20 — — Bacillus sp. IC JCM 9158 PY-pH 10 50 — — 1.00 0.11 — — Bacillus sp. N-1 JCM 9140 PYG-pH 10 30 0.10 0.15 2.10 0.14 0.27 — Bacillus sp. 17-1 JCM 9142 PYG-pH 10 30 — — 1.60 0.05 0.25 — Bacillus sp. 38-2 JCM 9143 PYG-pH 10 30 — — 1.20 0.02 0.05 — 1999 Phenylethylamine in alkaliphilic Bacillus 151 Table 1. (Continued) Polyamines (mmol/g wet cell) Medium °C Put Cad Spd Spm Agm Pea Bacillus sp. 13 JCM 9145 PYG-pH 10 30 — — 2.15 0.03 0.40 — Bacillus sp. 135 JCM 9146 PYG-pH 10 30 — 0.07 1.30 0.15 0.04 — Bacillus sp. 169 JCM 9147 PYG-pH 10 30 — — 2.20 0.04 0.05 — Amphibacillus xylans JCM 7361T (b) GY-pH 10 40 — — 0.82 0.50 — — Bacillus megaterium IAM 1166 (a) PYG-pH 7 37 0.01 0.01 0.10 — 0.01 — (a) Eagle-pH 7 37 0.01 — 0.18 — 0.02 — Bacillus fastidiosus NCIMB 10016 NA-U-pH 7.2 30 0.01 0.04 0.70 — 0.17 — NCIMB 11326T NA-U-pH 7.2 30 — 0.02 0.51 — 0.04 — Bacillus sp. YN-2000 (c) NB-pH 10 37 0.03 — 2.10 0.07 ND 0.80 Bacillus sp. YN-1 (c) NB-pH 10 37 — — 0.11 — ND — Bacillus alcalophilus ATCC 27647T (c) NB-pH 10 37 — — 0.25 0.11 ND — Put, putrescine; Cad, cadaverine; Spd, spermidine; Spm, spermine; Agm, agmatine; Pea, 2-phenylethylamine; JCM, Japan Collection of Microorganisms, Institute of Physical and Chemical Research (RIKEN), Wako, Japan; IFO, Institute for Fermentation, Osaka, Japan; IAM, IAM Culture Collection, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan; ATCC, American Type Culture Collection, Manassas, VA, U.S.A.; NCIMB, The National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, U.K.; NB, nutrient broth; T, type strain; ND, not determined; —, not detectable (Ͻ0.005). a, cited from Hamana et al. (1989). b, cited from Hamana (1999). c, cited from Hamasaki et al. (1993). packed cells, and the amines released into the hy- Results and Discussion drolysate of the cell residue after the hydrolysis with Diamines, triamines, tetraamines, pentaamines, 6 M HCl were analyzed by high-performance liquid chromatography (HPLC) on a column of cation-ex- guanidinoamines, and aromatic amines are separately change resin (Hitachi 2619F), as described previously detected in the HPLC. An amine peak corresponding (Hamana et al., 1993). Gas chromatography (GC) was to 2-phenylethylamine in HPLC was found in the cul- performed on a GC-9A gas chromatograph (Shima- ture medium after the growth of the five strains of B.