J. Cosmet. Sci., 59, 33-40 Qanuary/February 2008) Isolation and characterization of Streptomyces hiroshimensis strain TI-C3 with anti-tyrosinase activity TE-SHENG CHANG, MIN TSENG, HSIOU-YU DING, and SORGAN SHOU-KU TAI, Department of Biological Science and Technology, National University of Tainan, 33 Sec. 2, Shu-Lin St., Tainan (T. -S. C.), Bioresources Collection & Research Center, Food Industry Research & Development Institute, Hsinchu ( M. T. ), Department of Cosmetics Science, Chia Nan University of Pharmacy and Science, Tainan (H.-Y.D.), and Department of Biotechnology, National Formosa University, Yunlin (S.S.-K.T.), Taiwan. Accepted for publication August 28, 2007. Synopsis A bacterial strain, TI-C3, was isolated and verified to display 498 U/ml of anti-tyrosinase activity. Based on morphological, physiological, and chemical analysis, gyr B sequences, and DNA-DNA hybridization analy­ sis, the strain TI-C3 was identified as a strain of Streptomyces hiroshimensis. The anti-tyrosinase activity of the strain was improved to 905 U/ml under cultivation, using glucose and malt extract as the sole carbon and nitrogen sources, respectively. INTRODUCTION Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase widely distributed in nature. The enzyme catalyzes the first two reactions of melanin synthesis, the hydrox­ ylation of 1-tyrosine to 3,4-dihydroxyphenylalanine, 1-dopa, and the oxidation of 1- dopa to dopaquinone. This o-quinone is a highly reactive compound and can polymerize spontaneously to form melanin (1). Although the pigment melanin in human skin is a major defense mechanism against the ultraviolet light of the sun, the production of abnormal pigmentation such as melasma, freckles, age-spots, liver spots, and other forms of melanin hyperpigmentation can be a serious aesthetic problem (2). Hence, inhibiting the tyrosinase activity and preventing the abnormal pigmentations has been the subject of many studies (3-7). In our laboratory, we were interested in looking for actinomycetes with anti-tyrosinase activity. We used the high-throughput screening method by employing 48-well mi- Address all correspondence to Te-Sheng Chang. 33

34 JOURNAL OF COSMETIC SCIENCE croplates instead of traditional shake flasks in submerged cultivations. Some targets were isolated by this method (8). The present work reports the isolation and characterization of a novel strain, TI-C3, of Streptomyces hiroshimensis with the highest anti-tyrosinase activity in our screening study. MATERIALS AND METHODS MICROORGANISM Streptomyces hiroshimensis BCRC 12423 used as an indicator for strain identification was obtained from Bioresources Collection and Research Center, Food Industry Research and Development Institute, Taiwan. CHEMICALS Mushroom tyrosinase, L-tyrosine, sodium caseinate, asparagine, sodium propionate, corn steep liquor, maltose, glycerol, sodium nitrate, ammonium sulfate, glucose, nalidixic acid, deoxyribonucleotide triphosphate, and cycloheximide were purchased from Sigma (St Louis, MO). Yeast extract, malt extract, tryptone and agar were obtained from Difeo Laboratories (Detroit, MI). Taq DNA polymerase needed for polymerase chain reaction (PCR) was purchased from Takara Bio (Shiga, Japan). Primers were purchased from MDBio (Taipei, Taiwan). Other reagents and solvents used were commercially available and used as received. IDENTIFICATION OF THE STRAIN TI-C3 The strain TI-C3 was identified according to protocol published by Shirling and Gott­ lieb (9). The melanoid pigment formation was observed on ISP 1, 6, and 7 media. The carbon source utility was determined on basal medium (Pridham-Gottlieb medium) with 1 % (w/v) of the tested sugar. The basal medium contained 2.64 g (NH4)2SO4, 2.38 g KH 2 PO 4 , 5.65 g K 2 H 2 PO4 • 3H2O, 1.00 g MgSO4 • 7H2O, 6.4 mg CuSO4 • 5H2O, 1.1 mg FeSO 4 • 7H 2 O, 7.9 mg MnCl 2 • H 2 O, 1.5 g ZnSO 4 · 7H 2 O, and 18.0 g agar in one liter of distilled water. The pH was adjusted to 6.9 before autoclaving. The biochemical and physiological characteristics of the strain TI-C3 including growth temperature, melanin production, lysozyme resistance, and substrate hydrolysis were determined by the method of Berd (10). The mycelium of the strain was observed with a light microscope. The spore chain and spore surface morphologies were observed with a scanning electron microscope (Hitachi S-420 Hitachi, Ltd., Tokyo). Cell wall composition (DL- and LL-diaminopimelic acid isomer, A 2 pm) was determined by the method of Hasegawa et al. (11). One or two colonies were placed in a cryogenic vial (Evergreen Scientific) with 0.1 ml of 6 N HCl. The vial was autoclaved at 121 °C for 15 minutes. After cooling, 1 µl of the hydrolysate was placed on a thin cellulose plate (microcrystalline cellulose Tokyo Kasei Co., Ltd, Japan). One microliter of 0.01 M DL-A 2 pm (Sigma) was spotted on the same plate as a standard. The plate was developed on the solvent system methanol-distilled water-6N HO-pyridine (80:26:4: 10, v/v) for three to four hours. The plate was then dried and sprayed with Ninhydrin spray reagent