Enzymatic synthesis of benzyl alcohol galactoside using Escherichia coli β-galactosidase

대장균 β-galactosidase를 이용한 benzyl alcohol galactoside의 합성 연구

Abstract

Recently, it has been reported that benzyl alcohol (BzOH) as an additive in cosmetics, food, and medicine lead to toxicity and allergy problem. Then, to circumvent this hurdle, we carried out the synthesis of benzyl alcohol galactoside (BzO-gal). Previously, it was confirmed that BzO-gal was synthesized by transgalactosylation reaction using Escherichia coli (E. coli) ${\beta}$-galactosidase (${\beta}-gal$). Meanwhile, in this study, two peaks of BzO-gal as sodium adduct ion (m/z=293.1004) and protonated ion (m/z=271.1180) were detected in the reaction mixture by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). In addition, the amount of ${\beta}-gal$ and BzOH concentration, temperature, pH, and lactose concentration, respectively, were optimized (${\beta}-gal$, 0.75 U/mL; BzOH, 185 mM; temperature, $40^{\circ}C$, pH, 7.5; lactose, 350 g/l). Under these optimal conditions, 185 mM BzOH was converted into about 131 mM BzO-gal, in which the conversion yield was about 72%. In the future, BzO-gal will be applicable as a substitute for BzOH as a less toxic preservative for the cosmetic, pharmaceutical, and food industries, and we are planning to investigate the characteristics of BzO-gal as a preservative.

최근에 화장품, 식품, 그리고 의약용품의 첨가제로 사용되어지는 benzyl alcohol (BzOH)에 대한 독성 문제와 피부 알러지 문제가 보고되고 있다. 그래서, 본 연구에서는 이러한 첨가제의 문제점을 해결하기 위하여 galactose 한 분자를 BzOH 분자에 결합시킨 benzyl alcohol galactoside (BzO-gal)를 합성하여 이러한 문제를 해결하려는 시도를 수행하였다. 이미 선행연구에서 대장균의 ${\beta}$-galactosidase (${\beta}-gal$)를 이용하여 BzOH로부터 BzO-gal이 transgalactosylation 반응으로 합성된다는 것을 확인하였다. 본 연구에서는 먼저 대장균의 ${\beta}-gal$을 이용하여 BzOH로부터 BzO-gal의 합성을 반응액의 액체 크로마토그래피/질량 분석을 이용하여 BzO-gal-sodium adduct ion (m/z=293.1004)과 BzO-gal의 protonated ion (m/z=271.1180)의 검출로 확인할 수 있었다. 그리고, BzOH로부터 BzO-gal로의 합성반응을 실시 할 때, 최적의 ${\beta}-gal$ 양, BzOH 양, 반응 온도, 반응 pH, lactose 농도 등 반응의 최적 조건을 확인하는 실험을 수행하였다. 그 결과 0.75 U/ml ${\beta}-gal$, 185 mM BzOH, 온도 $40^{\circ}C$, pH 7.5, 350 g/l lactose의 조건이 가장 많은 양의 BzO-gal이 합성되는 최적의 조건을 확인하였다. 또한, BzO-gal의 최적 합성 조건에서 36시간 동안 ${\beta}-gal$에 의하여 185 mM BzOH로부터 약 131 mM BzO-gal이 합성되었고, 이 때, 전환 수율(conversion, %)은 약 72%로 확인되었다. 본 연구를 통하여 보다 안전한 식품, 화장품, 그리고 의약품용 첨가제의 개발을 기대하고 있으며, BzO-gal의 특성 분석 등 추가적인 연구를 계획하고 있다.

Fig. 1. Enzymatic synthesis of BzOH-gal using β-gal.

Fig. 2. TLC analysis for BzOH-gal synthesis using β-gal, in which zero- and 12-h samples were analyzed. BzOH, Lac, Glu, and Gal represent 1% standards of benzyl alcohol, lactose, glucose, and galactose, respectively. Arrow A and B indicate BzOH and BzOH-gal, respectively.

Fig. 3. High resolution ESI mass spectrum data of purified BzOH-gal. The mass spectrum was obtained in positive-ion mode. [M+Na]⁺ and [M+H]⁺ indicate sodium adduct ion and protonated form of BzOH-gal, respectively. The preferred structure of BzOH-gal are shown in the mass spectrum.

Fig. 4. Effects of (A) the amount of β-gal, (B) BzOH concentration, (C) temperature, (D) pH, and (E) lactose concentration on BzOH-gal synthesis. The samples collected at 18 h were used for TLC analysis. The reaction conditions are shown within the box in each chromatogram. Dashed boxes indicate the synthesized BzOH-gal. BzOH, Lac, Glu, and Gal represent 1% standards of benzyl alcohol, lactose, glucose, and galactose, respectively.

Fig. 5. Optimal conditions for BzOH-gal synthesis by β-gal. Values of relative BzOH-gal synthesis were calculated on the basis that the maximum synthesis of BzOH-gal was 1.0 under each condition, in which BzOH-gal syntheses were measured as scanned TLC spot areas. (A) Optimization of the amount of β-gal. (B) Optimization of BzOH concentration. (C) Temperature optimization. (D) pH optimization. (E) Optimization of lactose concentration. All measurements were conducted three times (n=3) using the same sample, and the average and standard deviation were calculated.

Fig. 6. TLC analysis of BzO-gal synthesis under optimal conditions. BzOH standard (0.25-2.0%, v/v) was used. Lac, Glu, Gal indicate 1% standards of lactose, glucose, and galactose, respectively.

Fig. 7. (A) Time-course profiles of BzOH and BzO-gal under optimal conditions for BzO-gal synthesis using β-gal. BzOH and BzO-gal were analyzed by TLC-image analysis of Fig. 6. (B) Percent conversion of BzOH to BzO-gal. All measurements were performed three times (n=3) using the same sample, and the average and standard deviation were calculated.