Enzymatic synthesis and characterization of hydroquinone galactoside using kluyveromyces lactis lactase

9492

J. Agric. Food Chem. 2010, 58, 9492–9497
DOI:10.1021/jf101748j

Enzymatic Synthesis and Characterization of Hydroquinone Galactoside Using Kluyveromyces lactis Lactase
GO-EUN KIM,† JIN-HA LEE,‡ SUN-HWA JUNG,† EUN-SEONG SEO,§ SHENG-DE JIN,§ GHAHYUN J. KIM,¥ JAEHO CHA, EUI-JOONG KIM,^ KI-DEOK PARK,# AND DOMAN KIM*,†,§
† Interdisciplinary Program of Graduate School for Bioenergy andBiomaterials, Chonnam National University, Gwangju 500-757, Republic of Korea, ‡Dental Science Research Institute, 2nd Stage of Brain Korea 21 for School of Dentistry, Chonnam National University, Gwangju 500-757, Republic of Korea, § School of Biological Sciences and Technology and Research Institute for Catalysis, Chonnam National University, Gwangju 500-757, Republic of Korea, Department ofMicrobiology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea, ^GenoFocus Inc., Daejeon 305-811, Republic of Korea, #Gwangju Center, Korea Basic Science Institute, Gwangju 500-757, Republic of Korea, and ¥Department of Biology, University of California;San Diego, La Jolla, California 92093 )

Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agentwas synthesized by the reaction of lactase (β-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans, and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K. lactis lactase showed a higher conversion ratio to HQ-Gal (60.27%). HQ-Gal was purified using butanol partitioning and silica gelcolumn chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance, and the ionic product was observed at m/z 295 (C12H16O7Na)þ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-β-D-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K. lactis determined using responsesurface methodology were 50 mM HQ, 60 mM lactose, and 20 U mL-1 lactase. These conditions produced a yield of 2.01 g L-1 HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicrylhydrazyl scavenging activity was 3.31 mM, indicating a similar antioxidant activity compared to β-arbutin (IC50 = 3.95 mM). The Ki value of HQ-Gal (0.75 mM) against tyrosinase was smaller than that ofβ-arbutin (Ki = 1.97 mM), indicating its superiority as an inhibitor. HQ-Gal inhibited (23%) melanin synthesis without being significantly toxic to the cells, while β-arbutin exhibited only 8% reduction of melanin synthesis in B16 melanoma cells compared with the control. These results indicate that HQ-Gal may be a suitable functional component in the cosmetics industry.
KEYWORDS: Kluyveromyces lactis;lactase; hydroquinone; acceptor reaction; galactosylation; DPPH assay; Tyrosinase inhibition; MTT assay

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INTRODUCTION

Hydroquinone (HQ) is a constituent of β-arbutin. The compound is a good general-purpose inhibitor of tyrosinase (one of the key enzymes in melanin synthesis), a stabilizer against light or oxidation and an antioxidant agent (1). HQ can function as a skin-whitening agent,but it has the potential to cause skin irritation and contact dermatitis. The United States Food and Drug Administration recently issued a proposal to ban HQcontaining over-the-counter and prescription products. The prospect of the curtailed availability of HQ has spurred intense interest in the cosmetics industry in alternatives, including naturally occurring compounds, for the inhibition ofmelanin synthesis (2).
*To whom correspondence should be addressed. E-mail: dmkim@ jnu.ac.kr. Phone: 82 62 530 1844. Fax: 82 62 530 1949.

Transglycosylation catalyzed by enzymes from various bacteria has been used to improve physiochemical properties such as water solubility and oxidative stability of various compounds. Glucansucrase-mediated glycosylation of HQ potently obviates lipid oxidation...