유기상 효소반응을 이용한 메틸글루코시드 아크릴레이트 합성

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

화학공학의 이론과 응용 제8권 제1호 2002년

김지석, 박대원, 함승주, 김우식

연세대학교 화학공학과

Enzymatic synthesis of □-methylglucoside acrylate in organic media

Jiseok Kim, Dae-Won Park, Seungjoo Haam, Woo-Sik Kim

Department of Chemical Engineering, Yonsei University

Introduction

Research on sugar esters synthesized from renewable agricultural resources has received keen attention with the prospect of the production of less pollutant molecules. Sugar esters constitute an interesting group of non-ionic, biocompatible and biodegradable surfactants, which are widely used in the cosmetic, pharmaceutical, detergent and food industries [1].

Recently, there has been a great interest in finding biodegradable surface active compounds to replace pollutant chemical surfactants. They can be enzymatically synthesized from renewable starting substrates, in mild reaction conditions [2]. Moreover, these enzymatic methods can be performed in less toxic solvents compared with those of chemical synthesis. Lipase has been often used in reactions for the formation of sugar esters due to a high regioselectivity and stereoselectivity [3,4], and these reactions lead to defined product usable without any hazards in the food, cosmetic and medical industries.

Several researchers have investigated the lipase-catalyzed synthesis of sugar containing acrylic esters and biodegradable polymers due to their biomedical applicability [5,6,7]. Acrylic acid has a high hydrophilic characteristic due to its carboxylic group capable of esterifying with the hydroxyl group of sugars. Moreover, it has a vinyl group that can be polymerized. Therefore acrylic esters are expected to be used as monomers for sugar polymers having hydrophilicity and biocompatibility.

The aim of this study was to investigate the esterification of carboxylic acid with □- methylglucoside using Candida antartica lipase, Novozym 435. The effects of reaction parameters such as enzyme amount, molar ratio of substrates, initial □-methylglucoside concentration and reaction temperature were also studied.

Experimental

Esterification reaction was carried out in screw capped test tubes. □-Methylglucoside prepared in specific concentrations was added in 10 mL t-butanol. Reactions were performed by adding Novozym 435 with magnetic stirring. The enzyme concentrations were 1, 3 and 5 % (w/v), and as were the cases for the molar ratios of □-methylglucoside:acrylic acid were 1:3, 1:5, 1:10 and 1:15. The initial concentrations of □-methylglucoside and reaction temperatures were varied in the range of 30∼60 g/L and 45∼60 ℃, respectively. Reactions were monitored by HPLC using a carbohydrate column (Nova-Pak, 250 × 4.6 mm, Waters) with detection by RI. For the quantification for the □-methylglucoside, the eluant was acetonitrile/water (80:20, v/v) at the flow rate of 1.2 ml/min.

Results and Discussion

Optimal molar ratio of □-methylglucoside/acrylic acid

The synthesis of □-methylglucoside acrylate in t-butanol with Novozym 435 at different molar ratio of □-methylglucoside and acrylic acid was shown in Figure 1. The degree of esterification increased with the overall range of molar ratio, and the resulting maximum conversions of □-methylglucoside at 1:3, 1:5, 1:10 and 1:15 after 48 h were 26, 35, 45 and 55 %, respectively. The use of the molar ratio of □-methylglucoside/acrylic acid over 1:15 might increase the reactivity. However, the excess use of acrylic acid could lead to the high cost for removing non-reacted acrylic acid. Therefore the molar ratio used in a process should be decided on a cost basis, depending mainly on the easy seperation of □-methylglucoside acrylate and acrylic acid. Moreover, the increase in acidity in reaction system might bring about the water stripping of enzyme surface, and then denaturation of enzyme. Therefore the molar ratio of 1:15 was used for all subsequent experiments.

Fig. 1. Effect of □-methylglucoside/acrylic acid molar ratio on the synthesis of

□-methylglucoside acrylate

Optimal concentration of enzyme

Figure 2 shows the effect of enzyme concentrations on the esterification of □- methylglucoside with acrylic acid. The conversion of □-methylglucoside after 48 h obviously depended on the amount of enzyme. The resulting maximum conversion was obtained at 5 % (w/v) enzyme. However, there were not much difference in the maximum value between 3 and 5 % (w/v) enzyme. Moreover it was expected that the final conversion of □- methylglucoside would be independent of the use of higher enzyme concentrations. Therefore the concentration of enzyme over 5 % (w/v) was not used.

Fig. 2. Effect of enzyme concentration on the esterification of □-methylglucoside with acrylic acid

Initial concentration of □-methylglucoside

Table 1 illustrates the effect of the initial concentration of substrate on the synthesis of □- methylglucoside acrylate. The conversions of □-methylglucoside after 48 h incubation reached the maximum value at 40 g/L, and then showed low rate of esterification at 60 g/L. However, the conversion at 40 g/L was slightly higher than that at 50 g/L, and the use of 50 g/L would seem to be more effective compared with 40 g/L in the view point of product formation. Therefore initial concentration of 50 g/L was used in subsequent experiments.

Table 1. Effect of initial concentration of □-methylglucoside

Initial concentration of □-methylglucoside (g/L)	30	40	50	60
Conversion (%)	49.2	57.9	57.1	48.9

Optimal reaction temperature

The effect of temperature on the acylation reaction was shown in Figure 3. The optimal temperature was observed at 50 ℃ and the reaction rate reduced subsequently over 55 ℃. Novozym 435 was known as heat-tolerant enzyme which maintains its activity even at 90 ℃. Therefore, it was considered that the decrease in reaction rate was caused not by rising temperature but by increasing water activity at higher temperature. Water activity can influence the reaction rate as well as the enzyme activity in organic media. The water transfer (J) was determined by equation (1) which was proportional to the diffusion coefficient (Daw) for water.

[eqn: J``=``D_{a`w}{PARTIAL`a_w}over{PARTIAL`x}]    (mol/m2s)                                              (1)

The diffusion coefficient is temperature dependent parameter, and the increase in diffusion coefficient and water transfer by rising temperature consequently caused the water activity in reaction system to increase. Therefore high water activity due to increasing temperature was considered to be the major factor to have the reaction rate decreased.

Fig. 3. Effect of reaction temperature on the esterification of □-methylglucoside with acrylic acid

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