J. Gen. Appl. Microbiol., 45, 149–153 (1999)

Production of 2-phenylethylamine by decarboxylation of L-phenylalanine in alkaliphilic 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 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 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 10,000g 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 10Phe 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 (0.005). 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. dzu, Kyoto, Japan) after heptafluorobutyrization of the cohnii (Fig. 1). Besides spermidine and agmatine as samples (Hamana et al., 1993). The identity of amines the major polyamine, this amine was detected in the was confirmed by gas chromatography-mass spec- PCA extracts from the packed cells grown in 199 and trometry (GC-Mass) on a JMS-DEX 300 (JEOL, PYG media (Figs. 1 and 2). Cadaverine and spermine Tokyo, Japan) (Hamana et al., 1993). corresponding to two minor peaks found in the organ- For determination of amino acid decarboxylase ac- isms grown in PYG medium were incorporated from tivities, exponentially growing cells in PYG-pH 10 the medium; however, the two were not found in the medium were harvested and washed with 0.8% NaCl. organisms cultivated in the polyamine-free 199 The packed cells were suspended in two volumes of medium. The aromatic amine, 2-phenylethylamine, was detected in the PCA extract of B. cohnii also by 50 mM Tris-HCl buffer (pH 7.2) containing 5 mM dithio- threitol, then disrupted by an ultrasonic vibrator (Hama- GC and identified by GC-Mass, but it was not de- na and Matsuzaki, 1992). The crude homogenate thus tected in the 6 M HCl-hydrolysate (at 110°C for 24 h) of the cell residue after the PCA extraction. These re- obtained was incubated at 30°C for 30 min with 10 mM sults suggest that the major part of 2-phenylethyl- amino acids in the presence of 0.1 M acetate buffer, pH 6.25, or 2.5% Na CO , pH 10. After the incubation, amine is secreted into the culture medium by the bac- 2 3 teria. Intracellular this amine is completely extracted PCA was added finally at 0.5 M to the reaction mixture. The increase in amine content in the PCA soluble with 0.5 M PCA. A part of agmatine was extracellularly fraction was analyzed by HPLC. secreted, but spermidine was not found in the culture medium of B. cohnii (Fig. 1). In facultatively alkaliphilic B. cohnii NCIMB 10327, the extracellular and intracellular 2-phenylethylamine 152 HAMANA and NIITSU Vol. 45

Fig. 1. HPLC analysis of the culture supernatant of B. cohnii NCIMB 10327 grown at pH 7 (A-7), at pH 10 (A-10), and at pH 10 in the presence of 10 mML-phenylalanine (A-10-Phe), and the PCA Fig. 2. HPLC analysis of the amino acid decarboxylase reac- extract of the packed cells grown at pH 7 (B-7), at pH 10 (B-10), tion mixture from B. cohnii NCIMB 10327 (A) and B. pseudofirmus and at pH 10 in the presence of 10 mML-phenylalanine (B-10-Phe) NCIMB 10283 (B) grown in PYG-pH 10 medium. of B. cohnii NCIMB 10327 cultivated in 199 medium. The mixture was incubated in the absence () or presence () of The supernatant (30 ml/100 ml culture) and the PCA extract 10 mM of L-2,4-diamino-n-butyric acid, L-ornithine, L-lysine, L-argi- (10 ml/200 ml extract of the packed cells obtained from 100 ml cul- nine, L-phenylalanine, and L-tyrosine in an acetate buffer system. ture) were analyzed. Abbreviations for amines are shown in Table 1. Abbreviations for amines are shown in Table 1. levels were higher in the culture at pH 10 than at pH 7 1998). Bacillus fastidiosus and Bacillus megaterium, (Fig. 1). When 10 mML-phenylalanine were supple- which are the next phylogenetic relatives of B. cohnii mented into the 199 medium, 2-phenylethylamine lev- but not alkaliphilic (Nielsen et al., 1994), lacked this els were increased (Fig. 1 and Table 1). amine, as shown in Table 1. This amine has never Cellular distribution of 2-phenylethylamine within 27 been found in various neutrophilic species, including strains of alkaliphilic bacilli are shown in Table 1. some strains of B. firmus and B. lentus and various Since the production of 2-phenylethylamine in B. acidophilic species (Hamana, 1999). Therefore the cohnii was dominant in the complex media PYG and detection of this aromatic amine might help in the PY, these complex media were used for the cultivation identification of strains of B. cohnii and may serve as of other alkaliphilic bacilli. This aromatic amine was a chemotaxonomic marker for classification of alka- selectively and ubiquitously distributed in the five liphilic members of Bacillus. strains of B. cohnii, but not in the culture supernatant Spermine was detected as a major polyamine in a or in cells of the other six alkaliphilic Bacillus species, moderately thermophilic alkaliphile, Bacillus sp. TX-3, B. alcalophilus, B. clausii, B. gibsonii, B. halodurans, and a slightly thermophilic alkaliphile, Amphibacillus B. horti, and B. pseudofirmus. Neither was it distrib- xylans. Selective distribution of spermine in ther- uted in an alkaliphilic Amphibacillus species, A. mophilic strains within the genus Bacillus has been re- xylans, nor in 10 alkaliphilic bacilli in the genus Bacil- ported (Hamana, 1999). lus isolated by Horikoshi and Akiba (1982). The phylo- Although 12 phylogenetically validated species of genetic position of Bacillus sp. YN-2000 isolated by alkaliphilic Bacillus were examined for various physio- Ohta et al. (1975), producing this amine (Hamasaki et logical and biochemical characters, including deami- al., 1993), has never been described; furthermore; five nation of phenylalanine (positive in B. pseudofirmus new alkaliphilic Bacillus species, B. agaradhaerens, B. and negative in the other 11 species), amino acid de- clarkii, B. halmapalus, B. horikoshii, and B. carboxylase activities have never been established pseudalcalophilus, unavailable in the present study, (Nielsen et al., 1995; Yumoto et al., 1998). As shown were proposed (Nielsen et al., 1995; Yumoto et al., in Fig. 2, L-arginine and L-phenylalanine supple- 1999 Phenylethylamine in alkaliphilic Bacillus 153 mented into the reaction mixture were decarboxylated Ghenghesh, K. S. and Drucker, D. B. (1989) Gas liquid chromatog- to produce agmatine and 2-phenylethylamine, respec- raphy of amines produced by the Enterobacteriaceae. Braz. J. tively, by the crude homogenate of B. cohnii NCIMB Med. Biol. Res., 22, 653–665. Gordon, R. E. and Hyde, J. L. (1982) The Bacillus firmus-Bacillus 10327 and of NCIMB 10284. The decarboxylase activ- lentus complex and pH 7.0 variants of some alkalophilic strains. ities were detected in the two reaction buffer systems, J. Gen. Microbiol., 128, 1109–1116. however, being dominant in the acetate buffer system. Hacisalihoglu, A., Jongejan, J. A., and Duine, J. A. (1997) Distribu- D-Phenylalanine was not decarboxylated. L-Arginine tion of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine ox- was decarboxylated, but L-phenylalanine was not in B. idase from Klebsiella oxytoca. Microbiology, 143, 505–512. pseudofirmus NCIMB 10283 or in B. alcalophilus JCM Hamana, K. (1999) Polyamine distribution catalogues of clostridia, 5262 and B. clausii NCIMB 10309. These decarboxy- acetogenic anaerobes, actinobacteria, bacilli, heliobacteria and lase activities were not detected in the supernatants of haloanaerobes within Gram-positive eubacteria: Distribution of the cultures, indicating that the decarboxylases are spermine and agmatine in thermophiles and halophiles. Micro- not extracellularly secreted. The decarboxylase activi- biol. Cult. Coll., 15, 9–28. Hamana, K., Akiba, T., and Matsuzaki, S. (1989) Distribution of ties for L-2,4-diamino-n-butyric acid, L-ornithine, L-ly- spermine in bacilli and lactic acid bacteria. Can. J. Microbiol., sine, and L-tyrosine to produce diaminopropane, pu- 35, 450–455. trescine, cadaverine, and tyramine, respectively, were Hamana, K., Hamana, H., Niitsu, M., Samejima, K., Sakane, T., and not detectable in the five organisms tested by this Yokota, A. (1993) Tertiary and quaternary branched polyamines method. These results indicate that 2-phenylethyl- distributed in thermophilic Saccharococcus and Bacillus. Micro- bios, 75, 23–32. amine is produced from L-phenylalanine mediated by Hamana, K. and Matsuzaki, S. (1992) Polyamines as a chemotaxo- a phenylalanine-specific decarboxylase (phenylala- nomic marker in bacterial systematics. Crit. Rev. Microbiol., 18, nine decarboxylase) in the cells of B. cohnii, then se- 261–283. creted into the culture medium. Hamasaki, N., Shirai, S., Niitsu, M., Kakinuma, K., and Oshima, T. 2-Phenylethylamine production has been reported (1993) An alkaliphilic Bacillus sp. produces 2-phenylethyl- amine. Appl. Environ. Microbiol., 59, 2720–2722. in some gram-negative bacteria belonging to the fam- Hanlon, S. P., Hill, T. K., Flavell, M. A., Stringfellow, J. M., and ily Enterobacteriaceae (Ghenghesh and Drucker, Cooper, R. A. (1997) 2-Phenylethylamine catabolism by Es- 1989). An amine oxidase acting on 2-phenylethyl- cherichia coli K-12: Gene organization and expression. Microbi- amine occurs in the gram-negatives, Escherichia coli ology, 143, 513–518. and Klebsiella oxytoca (Hacisalihoglu et al., 1997; Horikoshi, K. and Akiba, T. (1982) Alkalophilic Microorganisms, Japan Scientific Societies Press, Tokyo, pp. 9–26. Hanlon et al., 1997) and gram-positive Arthrobacter Nielsen, P., Fritze, D., and Priest, F. G. (1995) Phenetic diversity of globiformis (Choi et al., 1995), utilizing the amine as a alkaliphilic Bacillus strains: Proposal for nine new species. Mi- carbon and energy source. The role of the large crobiology, 141, 1745–1761. amount of production of 2-phenylethylamine for the Nielsen, P., Rainey, F. A., Outtrup, H., Priest, F. G., and Fritze, D. growth of B. cohnii is not clear. (1994) Comparative 16S rRNA sequence analysis of some al- kaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus. FEMS Microbiol. Lett., 117, 61–66. We are indebted to IFO, JCM, and NCIMB for supplying bacterial Ohta, K., Kiyomiya, A., Koyama, N., and Nosoh, Y. (1975) The basis strains. of the alkalophilic property of a species of Bacillus. J. Gen. Mi- crobiol., 86, 259–266. References Spanka, R. and Fritze, D. (1993) Bacillus cohnii sp. nov., a new, ob- ligately alkaliphilic, oval spore-forming Bacillus species with or- Choi, Y. H., Matsuzaki, R., Fukui, T., Shimizu, E., Yorifuji, T., Sato, nithine and aspartic acid instead of diaminopimelic acid in the H., Ozaki, Y., and Tanizawa, K. (1995) Copper/topa quinone- cell wall. Int. J. Syst. Bacteriol., 43, 150–156. containing histamine oxidase from Arthrobacter globiformis. Yumoto, I., Yamazaki, K., Sawabe, T., Nakano, K., Kawasaki, K., Molecular cloning and sequencing, overproduction of precursor Ezura, Y., and Shinano, H. (1998) Bacillus horti sp. nov., a new enzyme, and generation of topa quinone cofactor. J. Biol. gram-negative alkaliphilic bacillus. Int. J. Syst. Bacteriol., 48, Chem., 270, 4712–4720. 565–571. Fritze, D., Flossdorf, J., and Claus, D. (1990) of alka- liphilic Bacillus strains. Int. J. Syst. Bacteriol., 40, 92–97.