lipase catalyzed sugar esters synthesis in ionic liquids

In this study, the synthesis of sugars esters by using lipase-catalyzed esterification with fatty acids in ionic liquids mixtures by using supersaturated sugar solution with the combinat

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합성

LIPASE-CATALYZED SUGAR ESTERS

SYNTHESIS IN IONIC LIQUIDS

합성

LIPASE-CATALYZED SUGAR ESTERS

SYNTHESIS IN IONIC LIQUIDS

LIPASE-CATALYZED SUGAR ESTERS SYNTHESIS IN IONIC LIQUIDS

By

NGUYEN MINH HIEP

A THESIS submitted to the faculty of

INHA UNIVERSITY

In partial fulfillment of the requirements

For the degree of MASTER OF SCIENCE

Department of Marine Science and Biological Engineering

February 2009

TRƯỜNG ĐẠI HỌC KỸ THUẬT INHA

Luận văn thạc sĩ

NGUYỄN MINH HIỆP

TỔNG HỢP SUGAR ESTERS TRONG IONIC LIQUIDS

DƯỚI SỰ XÚC TÁC CỦA LIPASE

Cán bộ hướng dẫn

GS TS YOON-MO KOO

Khoa Hải Dương Học và Công Nghệ Sinh Học

Tháng 2 năm 2009

이 論文을 NGUYEN MINH HIEP 의 碩士學位 論文으로 認定함

2009 年 2 月

主審

副審

委員

LIPASE-CATALYZED SUGAR ESTERS SYNTHESIS IN IONIC LIQUIDS

By NGUYEN MINH HIEP

Supervised by

Dr YOON-MO KOO

Department of Marine Science and Biological Engineering

February 2009

ACKNOWLEDGEMENT

In the first place, I would like to express my gratitude to Professor Koo Yoon-Mo, who has given me the opportunity to live and work in Korea I will never forget the useful lessons he has taught me and I am grateful for this

I also extend my thanks to all professors in Department of Marine Science and Biological Engineering, Inha University for their lectures together with their cherishing and support

I really appreciate Doctor Ha Sung Ho and Doctor Lee Sang Hyun for their help and guidance I am grateful to all of the members in Bionanoprocess laboratory, who are not only friendly but also enthusiastic to help me during the time I lived and studied in Korea These will become the nice memories about the period I was in Korea

I wish to thank ERC organization for supporting me in research and trips to the conference

at Cheju island of Korea and Dalian of China

Warm thanks also go to all of the Vietnamese and Korean friends at Inha University who have shared and helped me in my living and work

Finally, I would like to express my biggest thank to my family, my partner – Nguyen Thi Huynh Nga as well as her family for their constant help, availability and motivation, thus allowing me to be more optical and confident when things were not going on so well

ABSTRACT

Fatty acid sugar esters known as bio-sufactants can be produced from renewable resources They are tasteless, odorless, nontoxic, non-irritant, non-ionic and cover a wide range of hydrophilic-lipophilic balance which explain well their increasing importance in numerous areas such as food, cosmetic, detergent and pharmaceutical industry While most

of these compounds are chemically synthesized, the enzymatic synthesis of sugar esters has garnered considerable interest by reasons of milder and simpler conditions, higher selectivity, and greener process Nowadays, ionic liquids (ILs) are known as ‘green’ alternatives to volatile organic solvents (VOCs) in electrochemical, synthetic, separation and chemical processes because of their characteristics such as non-volatility, non-flammability, solvating power for dissolving a wide range of organic, inorganic and polymeric materials and their excellent chemical and thermal stability In addition, ILs have been described as ‘designer solvents’ since their physicochemical properties such as polarity, hydrophobicity, density and viscosity can be adjusted to suit the requirements for a particular process by altering the anions, and (or) cations Besides, ultrasound irradiation is known as a useful tool for strengthening mass transfer of liquid-liquid heterogeneous systems In this study, the synthesis of sugars esters by using lipase-catalyzed esterification with fatty acids in ionic liquids mixtures by using supersaturated sugar solution with the combination of ultrasound irradiation was investigated

Firstly, the lipase-catalyzed synthesis of glucose fatty acid esters using ionic liquid mixtures was investigated Although the activity of Novozym 435 in a 1-butyl-3-

1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide ([Bmim][Tf2N]) mixture (1:1 v/v) was somewhat lower than that in pure [Bmim][TfO] which showed the highest enzyme

activity, the stability of Novozym 435 was significantly increased Specifically, the activity

glucose solution in this mixture, compared with reaction using saturated solution After 5 times reuse of Novozym 435, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36% Therefore, the productivity obtained by using IL mixtures was higher than those in pure ILs

Secondly, the application of ultrasound irradiation in enhancing lipase activity in the synthesis of sugar ester using ionic liquids was investigated In the lipase-catalyzed esterifications of glucose with vinyl laurate or lauric acid in [Bmim][TfO], 2.4 and 4.7 times higher activities, respectively, were obtained by using supersaturated solution under ultrasound irradiation than those in conventional method In the lipase-catalyzed

ultrasound irradiation

Finally, the optimization of the synthesis of glucose fatty acid ester in IL mixtures was investigated by using supersaturated glucose solution The effect of IL mixtures ratio, substrate ratio, water content, lipase content, and temperature on the activity and stability of lipase was also studied The highest yield of sugar ester was obtained in a [Bmim][TfO]

successfully carried out with optimized reaction conditions After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained

Bio-surfactant 로 알려져 있는 Fatty acid sugar esters 는 재생자원으로부터 생산가능한 물질입니다 이것들은 무미, 무취, 무독, 비자극, 비이온성이며 넓은 범위에서 hydrophilic-lipophilic balance 를 조절할 수 있어서 식품업, 화장업, 세제업, 의약품산업에서 관심이 증가하고 있습니다 Sugar ester 대부분이 화학적 방법으로 생산되지만, 이보다 더 온화한 조건에서 더 높은 선택도를 가지며 생산할 수 있는 효소적 생산방법에 대한 관심이 높아지고 있습니다 이온성액체는 휘발성이 낮고, 화학적으로나 열적으로 안정하며, 다양한 유기물질, 무기물질, 고분자물질을 녹일

수 있다는 특성 때문에 전자/합성/분리/화학공정에서 기존의 휘발성 유기 용매를 대체할 친환경 용매로 각광받고 있습니다 그리고 음이온과 양이온의 종류를 바꿔주는 것만으로 극성, 소수성, 밀도, 점도 등 어떤 공정에서 원하는 물리적 특성의 용매를 만들 수 있어 “디자이너 용매”라고도 불립니다 초음파는 액체-액체 이상계에서 물질전달 효과를 높일 수 있는 훌륭한 방법으로 알려져 있습니다 본 연구에서는 과포화된 sugar 용액과 지방산이 녹아있는 혼합 이온성액체를 가지고 lipase 를 촉매로 한 suger ester 의 합성에 초음파 장치를 이용한 결과를 알아보았습니다

첫째로, 혼합 이온성액체에서 lipase 를 촉매로 한 glucose fatty acid esters의

혼합물의 경우에는 활성도가 이보다 좀 낮았을 뿐이고 효소 안정성은 극적으로 증대되었습니다 특히, Novozym 435의 활성도는 sugar 포화용액에 비해

과포화용액을 사용했을 때 1.1 에서 2.9 mmol min-1 g-1 로 증가하였습니다 Novozym 435를 5회 재사용한 결과 순수한[Bmim][TfO] 에서는 초기 활성도에 비해 36%의 활성도를 유지하였지만 혼합이온성액체에서는 86%나 유지되었습니다 그러므로 혼합이온성액체를 이용한 경우 더 높은 생산성을 얻을 수 있습니다

둘째로, 이온성 액체에서 sugar ester 합성에 있어 초음파 장치 사용이 lipase 의 활성도에 미치는 영향에 관해 연구하였습니다 [Bmim][TfO]에서 lipase 를

방법을 사용했을 때보다 과포화용액과 초음파 장치를 사용했을 때 각각 2.4 배와

transesterification 경우에도 효소활성은 5.8 배 증가 되었습니다

마지막으로 혼합 이온성액체 내에서 과포화 glucose 용액을 이용한 glucose fatty acid ester 합성의 최적화에 대한 연구를 수행하였습니다 이온성액체의 혼합비, 기질의 비율, 수분함량, 효소함량의 영향과 온도에 대한 lipase 의 활성과 안정에

부피비의 mixture 에서 Novozym 435 는 최적의 안정성과 활성도를 보였습니다 최적의 생산 조건에서 lipase 와 이온성액체의 재사용도 잘 수행되었습니다 Novozym

435 와 이온성액체를 5 회 재사용했을 경우 초기에 비해 78%의 활성도를 유지하였습니다

TABLE OF CONTENTS

ACKNOWLEDMENT i

ABSTRACT ii

TABLE OF CONTENTS vi

LIST OF FIGURES ix

LIST OF TABLES x

ABBREVIATIONS xi

1 INTRODUCTION 1

1.1 Fatty acid sugar esters 1

1.2 Ionic liquids 2

1.3 Ionic liquids in biocatalysis 4

1.4 Supersaturated solution 6

1.5 Ultrasound irradiation 8

1.5.1 Mechanical effect 9

1.5.2 Thermal effect 9

1.5.3 Cavitation effect 9

2 OBJECTIVES 11

3 EXPERIMENTAL 12

3.1 Materials 12

3.2 Supersaturated sugar solution by water-mediated method 12

3.3 Solubility of lauric acid 13

3.4 Apparatus 13

3.5 Reaction with saturated glucose solution 14

3.5.1 In transesterification reaction 14

3.5.2 In direct esterification reaction 15

3.6 Reaction with supersaturated glucose solution 16

3.6.1 In transesterification reaction 16

3.6.2 In direct esterification reaction 16

3.7 Reaction with the combination of stirring with ultrasound irradiation 17

3.8 Reuse of ILs and enzyme after reaction 17

3.9 Structure determination 18

3.10 HPLC analysis 18

4 RESUTS AND DISCUSSION 19

4.1 Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquid mixtures 19

4.1.1 Dissolved glucose concentration, enzyme activity, and residual enzyme activity in IL and IL mixtures 19

4.1.2 Time courses for the lipase-catalyzed esterification of glucose with vinyl laurate in pure ILs and IL mixtures 24

4.1.3 Residual activity of Novozym 435 after reuse in pure ILs and IL mixtures 25

4.2 Ultrasound-enhanced lipase activity in the synthesis of sugar ester using ionic liquids 27

4.2.1 Dissolution rate of glucose in [Bmim][TfO] under ultrasound irradiation 27

4.2.2 Transesterification of glucose with vinyl laurate 28

4.2.3 Residual acitivity of enzyme in ILs under ultrasound irradiation 32

4.3 Optimization of lipase-catalyzed glucose ester synthesis in ILs 33

4.3.1 Supersaturated glucose solution in ILs 33

4.3.2 Transesterification of glucose with vinyl laurate 36

4.3.3 Effect of substrate ratio on enzyme activity 38

4.3.4 Effect of water content on enzyme activity 40

4.3.5 Effect of enzyme load on enzyme activity 42

4.3.6 Effect of temperature on enzyme activity 43

4.3.7 Reuse of enzyme and ILs in optimized reaction conditions 44

4.3.8 Synthesis of other glucose esters 46

5 CONCLUSIONS 48

6 REFERENCES 49

LIST OF FIGURES

Figure 1 Some typical cation/anion combinations in ILs 3

Figure 2 System of the combination of ultrasound irradiation and stirring 14

Figure 3 Time course for the lipase-catalyzed esterification of glucose with vinyl laurate in pure ILs and IL mixtures 25

Figure 4 Residual activity of Novozym 435 after reuse in pure ILs and IL mixtures 27

Figure 5 Dissolution rate of glucose crystal in [Bmim][TfO] at 40°C 28

Figure 6 Lipase-catalyzed transesterification of glucose in hydrophilic ILs 30

Figure 7 Lipase-catalyzed transesterification of glucose in hydrophobic ILs 31

Figure 8 Stability of enzyme under ultrasound irradiation 32

Figure 9 Dissolution rate of glucose crystal and crystallization rate of supersaturated glucose solution in [Bmim][TfO] at 30°C 36

Figure 10 Enzyme activity and stability in IL mixtures 38

Figure 11 Effect of substrate ratio on enzyme activity 40

Figure 12 Effect of water content on enzyme activity 41

Figure 13 Effect of enzyme content on enzyme activity 43

Figure 14 Effect of temperature on enzyme activity 44

Figure 15 Time course for the lipase-catalyzed esterification of glucose with lauric acid in optimized reaction conditions 45

Figure 16 Reusability of ILs mixture and enzyme 46

LIST OF TABLES

Table 1 Dissolved glucose concentration, enzyme activity, and residual

enzyme activity in IL and IL mixtures 21

Table 2 Enzyme activity in IL and IL mixtures 22 Table 3 Residual enzyme activity in ILs and IL mixtures 23

Table 4 Dissolved glucose concentration and lauric acid in ILs and IL

mixtures 34

Table 5 Enzyme activity in ILs and IL mixtures 35

Table 6 Conversion of various sugar esters in mixture of [Bmim][TfO] and

ABBREVIATIONS

[Bmim][TfO]: 1-Butyl-3-methylimidazolium trifluoromethanesulfonate

1 INTRODUCTION

1.1 Fatty acid sugar esters

Fatty acid sugar esters, well known as bio-sufactants are produced from renewable

resources [1] They are tasteless, odorless, nontoxic and non-irritating, non-ionic and their

high biodegradability explains their increasing importance in numerous areas such as pharmaceuticals, cosmetics, detergents, and the food industry [2, 3] Their physicochemical properties can be tailored to suit potential applications by varying the sugar head group size and the length and number of alkyl chains [1] They offer a full range of hydrophile/lipophile balance (HLB) values from 1 to 16, with all grades displaying exceptionally good surfactant properties [4] One of the important fatty acid derivatives are the fatty acid esters of fructose which exhibit interesting surface properties and higher interfacial tension values compared with commercial sucrose esters [5] In addition, fructose esters can be used as antibacterial agents that suppress the cell growth of Streptococcus mutans, a causative organism of dental decay [6] While most of these compounds are chemically synthesized which have a lot of disadvantages such as being mainly performed at high temperatures in the presence of alkaline catalyst, high energy consumption, coloring of products and low selectivity, the enzymatic synthesis of sugar esters has garnered considerable interest by reason of milder and simpler conditions, higher selectivity, and greener processes [7, 8] In addition, the choice of solvent for the enzymatic esterifications between underivatized sugars and free fatty acids is very difficult because one reactant is polar and the other is nonpolar Many years ago, water was used as a poor solvent in preparative organic chemistry because most such compounds are insoluble in water, and the use of water frequently gave rise to unwanted side reactions such degradation of common organic reagents and was sometimes very difficult to recover

product from this medium Many of these problems might be overcome by switching from water to an organic solvent, which could supply a good environment for the enzymes, and make them more stable Organic solvents also exhibit new behaviour such as “molecular

including substrate, stereo-, regio- and chemoselectivity than water [9] However, most enzymes are quickly inactivated under hydrophilic organic solvents (e.g., pyridine, dimethyl sulfoxide, and dimethyl formamide) which are able to dissolve high concentrations of both sugars and fatty acids [10, 11]

1.2 Ionic liquids

Ionic liquid (ILs) are a new class of solvents by their non-molecular nature They

(RTIL) The term IL covers inorganic as well as organic molten salts [13] The first

or tetraalkylphosphonium cations, while the anions are either organic or inorganic,

“task-specific” solvents based on the various combinations of the large number of cations and anions that allow a wide range of physical and chemical characteristics to be achieved, including polarity, hydrophobicity, solvent miscibility behavior as well as density,

refractive index and viscosity [15, 16, 17] While thermal stability and miscibility mainly depend on the anion, others, such as viscosity, surface tension and density depend on the length of the alkyl chain in the cation and/or shape or symmetry [18, 19]

Some typical cation/anion combinations comprising the main families of ILs are illustrated

in Figure 1

Figure 1 Some typical cation/anion combinations in ILs

Recently, ionic liquids have been extensively evaluated as environmentally friendly or

“green” alternatives to conventional volatile organic solvents (VOCs) in many applications such as electrochemistry, synthesis, biochemical, and biological process [17, 20, 21, 22] because they possess exciting properties such as:

ILs exhibit negligible vapour pressure, which make them desirable alternatives for volatile organic compounds The use of ILs to replace VOCs can reduce fire and explosion hazards

ILs possess non-flammability properties, the ability to dissolve a wide range of organic, iorganic and polymeric materials and exhibit excellent chemical and thermal stability Their water miscibility can also be controlled Therefore, reactions carried out in these media can have enhanced reaction rates, higher yields and improved selectivities Although polar organic solvents inactivate enzymes, surprisingly 1-alkyl-3-methylimidazolium ionic liquids which are polar solvent do not This feature extends enzyme-catalyzed reactions to a solvent polarity range that was previously inaccessible [23]

They exhibit Bronsted, Lewis and Franklin acidity, as well as superacidity [22]

1.3 Ionic liquids in Biocatalysis

Biocatalysis can be defined as utilization catalysts, such as protein enzymes, that perform chemical transformations on organic compounds In organic synthesis, the term

“selectivity” is used popularly, which is necessary to obtain a high yield of a specific product The major reasons why synthetic chemists have become interested biocatalysis is because these enzymes can display chemoselectivity, regioselectivity and enantioselectivity

In addition, another important advantage of biocatalysts are that they are environmentally acceptable, being completely degraded in the environment Furthermore, the enzymes act under mild conditions, which minimizes problems of undesired side-reactions such as decomposition, isomerization, racemization and rearrangement, which often plague

traditional methodology [24, 25]

On the other side, many years ago, water was used as a poor solvent in preparative organic chemistry because most such compounds are insoluble in water Water also

frequently gave rise to unwanted side reactions such as degradation of common organic reagents and is sometimes very difficult to recover the product form this medium Most of these problems might be overcome by switching from water to an organic solvent, which could supply a good environment for the enzymes, and make them more stable Organic solvent also exhibit new behaviour such as “molecular memory” Moreover, organic solvent can yield the higher enzyme selectivity, including substrate, stereo-, regio- and chemoselectivity than water Some enzyme-catalyzed reactions in organic solvents and even in supercritical fluids during the gas phase have already been commercialized [26] However, the use of organic solvent in bioprocess operations possesses a number of further problems The main concerns are the toxicity of the solvent to both process operators and the environment, and the volatile and flammable nature of these solvents which make them

a potential explosion hazard [27] The use of ILs as replacements for organic solvents in

biocatalytic processes could potentially overcome these problems and open up some exciting new opportunities

Ionic liquids with their unique properties are receiving increasing attention as solvents for organic synthesis in general and catalytic processes in particular Depending on their structure, they can be immiscible with water or e g alkanes, which render them useful for performing catalytic reactions in biphasic media, thus facilitating catalyst recovery and recycling Most studies have involved the use of 1, 3-dialkylimmidazolium salts, e.g 1-

example of biotransformation ILs was reported by Lye et al in 2000 It involved the use of

3-cyanobenzamide and 3-cyanobenzoic acid catalysed by whole cells of Rhodococcus R312

The ILs essentially act as a reservoir for the substrate and product, thereby, decreasing the substrate and product inhibition observed in water, and hence, increasing the catalyst

activity and productivity [28] The feasibility of using isolated enzyme in IL media has also

been demonstrated Erbeldinger et al reported the thermolysin-catalysed synthesis of

reaction media for chemical and biocatalytic reaction have been reported [30, 31] For

enzyme displayed a higher stability in an IL medium It was further shown that the small

advantages, such as increased regioselectivity and stability of the enzymes, have been reported when ionic liquids are used instead of organic solvents in enzyme-catalyzed

reactions [32, 33] The use of ILs in catalytic systems is also generally regarded as polar but

weakly coordinating This means that, although the catalysts are very soluble in the IL phase, a non-coordinating anion will mean that the active site is very accessible to organic substrates Furthermore, ILs are generally chemically inert towards both catalyst and reactive intermediates, meaning that catalyst stability is not a problem [30]

ILs are also used in the separation In the process extraction of n-butanol into the IL phase followed by recovery from the IL by pervaporation constitutes an attractive alternative to conventional, energy intensive separation from water by distillation [28]

1.4 Supersaturated solution

There is considerable current interest in the utilization of carbohydrates as readily available, relatively inexpensive and renewable feedstocks for the chemical and related industries The transformation of underivatised carbohydrates is still quite challenging due

to their low solubility in almost any solvent but water The few exceptions, such as DMF and DMSO, have many undesirable characteristics and are not compatible with many intended applications of carbohydrate-derived products Especially, in the synthesis of fatty acid sugar esters through use in non-aqueous media, a major problem is the selection of an

etc., hydrophilic organic solvents are the preferred reaction media However, most enzymes are quickly inactivated under hydrophilic organic solvents Therefore, a partially dissolved

or a solid-phase system or a metastable supersaturated solution can be used for the enzymatic reaction in less harmful organic solvents such as acetonitrile, acetone, t-butanol, and 2-methyl [34, 35, 36] Alternatively, the solubility can be increased by using protected glucoses or alkyl glycosides However, the use of these substrates requires extra steps;

otherwise the products show different properties compared to non-derivatized glucose esters [37]

attractive alternatives for volatile organic solvents Moreover, ILs exhibit excellent physical characteristics including the ability to dissolve polar and non-polar organic, inorganic, and polymeric compounds based on the combination of cations and anions The best results were obtained in 1-methoxyethyl-3-methylimidazolium tetrafluoroborate, [MOEMIm][BF4], which dissolved 5 g/l of glucose at 55oC [38] Another group of researchers reported the dissolution of high concentrations of a -cyclodextrin (350 g/l) in 1-methoxymethyl-3-methylimidazolium bromide [39].Spear et al reported the high solubility

of various mono and disaccharides in ILs containing [Cl]- [40] However, ILs containing [Cl]- cannot be used to carry out enzyme reactions due to the inactivation of most enzymes [41] Only ILs containing the dicyanamide ([dca]) anion have been reported to be good

solvents for sugar dissolution and enzyme reactions, but Candida Antarctica lipase B irreversibly deactivated in [Bmim][dca] after reaction [42, 43] Recently, pure [Bmim][BF4] and [Bmim][PF6] were used as reaction media in the lipase-catalyzed transesterification of glucose with a fatty acid vinyl ester However, the solubility of glucose in these ILs is very low [37] To overcome this problem, supersaturated sugar solutions in ILs were recently used to carry out enzymatic synthesis of sugar esters [44] The supersaturated solution, a solution which contains more dissolved solute than the solubility limit, is usually prepared

by dissolving excess solute at high temperature and then slowly cooling to low temperature [45] However, considerable time is required to dissolve sugars in viscous ILs and high temperature can cause degradation of sugars Thus, an alternative dissolution process that

concentrations in the supersaturated [Emim][TfO] and [Bmim][TfO] were 19 and 10 times higher, respectively, than the solubilities (6.1 and 4.8 g/l) of glucose in the ILs at 25oC Furthermore, the supersaturated glucose solutions in ILs were maintained over a long period of time without any significant loss of glucose because of the high viscosity of ILs Therefore, the conversion increased from 8% to 96% at 1 day of the reaction by using a supersaturated solution in [Bmim][TfO] which had a dissolved glucose concentration of 400% higher than its solubility, compared with the reaction using a saturated glucose solution [44]

1.5 Ultrasound irradiation

Ultrasound, defined as sound of a frequency beyond that to which the human ear can respond, is generally considered to lie between 20 kHz to beyond 100 MHz When ultrasound propagates in sound bearing media, it usually can cause some effects on this

media including mechanical effect, thermal effect and cavitation effect [46]

1.5.1 Mechanical effect

The mechanical effect consists of vibration effect and acoustic streaming effect The vibration with high frequency induced by ultrasound causes effective agitation in liquid media, which increases fluid mixing, diffusion and mass transfer of substrate [47] The boundary layer of stagnant fluid adjacent to a solid surface creates a resistance to the transport of small molecules to the surface The high-frequency vibration reduces the thickness of this boundary layer, thus increasing convection transport When ultrasound propagates in liquid media, it can create unidirectional and constant sound radiation pressure, which causes the liquid to flow in the direction of sound propagation This phenomenon is defined as acoustic streaming, and it increases with ultrasound intensity Therefore, any amount of ultrasound in a liquid produces additional convection transport from acoustic streaming [48] Vibration effect and acoustic streaming effect both belong to mechanical effect of ultrasound These two effects can improve the mass transfer and fluid mixing through the acceleration of convection transport

1.5.2 Thermal effect

In the process of ultrasound propagation, the media can absorb the energy of ultrasound and convert it into thermal energy However, in exposure systems such as enzyme solutions and cell suspensions with efficient heat transfer and narrow temperature control, thermal effect would make only a marginal contribution to enhanced bio-reaction induced by ultrasound [49]

1.5.3 Cavitation effect

Cavitation is a particular phenomenon of ultrasound in liquid media The molecules

of pure liquid have a very high strength of extension Actually, some minute gas bubbles can enter into the practical liquid for various reasons, which produce the “weak link” of the liquid The cycles of low and high acoustic pressure causes the gas bubbles to expand and shrink, which in turn creates shear flow around the oscillating bubbles This process of expansion, shrinkage and collapse of gas bubbles induced by ultrasound is called ultrasonic cavitation, and the minute gas bubbles are called cavitation bubbles [50] Based on the different behaviors of cavitation bubbles, cavitation can be classified as instantaneous cavitation and stable cavitation The instantaneous cavitation, which occurs when ultrasonic

effect and mechanical effect are widely believed to be the main factors contributing to the ultrasonic enhancement of biochemical reactions Low intensity ultrasound can stimulate

enzyme activity For example, Barton et al (1996) compared the activities of some

glycosidase enzymes in the presence and absence of ultrasound [51] Results showed that ultrasonication made no significant difference when the substrate concentration was low; however, at higher substrate concentration (sucrose solutions was visibly viscous), a marked increase in invertase activity toward sucrose was observed in the presence of ultrasound Therefore, they concluded that ultrasonication could produce efficient mixing

of solutions, which would achieve a more homogeneous reaction mixture and facilitate diffusion to and from the active sites of the enzymes, reducing the depression of secondary metabolite to enzyme activity Recent studies have demonstrated the similar mechanism of enzyme activity stimulation Lin et al (1997) reported that the activity of inulinase could achieve a 60% rise in the presence of ultrasound of 20W [52] Zong et al (2000) observed

a more significant increase in activity in the presence of ultrasound for immobilized enzymes than for free enzymes [53]

2 OBJECTIVES

In this thesis, enzymatic synthesis of sugar esters by using lipase-catalyzed reaction

in ILs with supersaturated sugar solution prepared by water- mediated method under effects

of ultrasound irradiation was investigated This investigation includes three parts:

1 Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquid mixtures

2 Ultrasound-enhanced lipase activity in the synthesis of sugar ester using ionic liquids

3 Optimization of lipase-catalyzed glucose esters synthesis in ionic liquids

3 EXPERIMENTAL

3.1 Materials

Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) was

and had a residual chloride content of less than 30 ppm ILs were dried in a vacuum oven at 60°C for 3 days before use Glucose, vinyl laurate, n-caprylic acid, n-lauric acid, and n-palmitic acid were purchased from Sigma (St Louis, USA) All other chemicals used in this work were of analytical grade and used without further purification

3.2 Supersaturated sugar solution by water-mediated method

Twenty mg of glucose was dissolved in water (0.3 ml) ILs or IL mixtures (0.5 ml) were then added to these solutions at room temperature After clear solutions were obtained, the water in the mixtures was removed by vacuum evaporation for 12 hr at 60°C The supersaturated fructose solutions were slowly cooled and incubated for 2 hr at low temperature After centrifugation, the supernatant was obtained to analyze glucose concentration The fructose concentration in ILs was determined by the dinitrosalicylic acid

process was also carefully investigated and no influence of ILs on the DNS methods in the measured concentration range 0.1 g/l – 1.0 g/l was observed The residual water content in the supersaturated sugar solution was measured from the weight difference of the solution and confirmed to be lower than 0.5% (w/w) by Karl-Fischer Titration (831 KF Coulometer, Metrohm, Switzerland) using HYDRANAL-Coulomat AG-H reagent

3 3 Solubility of lauric acid

Lauric acid (0.3 mmol) was added to glass vials containing ILs or IL mixtures (1 ml) The suspension was stirred for 12 hr at 50°C After centrifugation at 14000 rpm for 5 min, the IL-rich phase was carefully obtained and dissolved lauric acid was extracted with

n-hexane The concentration of lauric acid was quantified by a CP 9001 gas

0.25mm i.d.; film thickness 0.25 mm; J&W Scientific, USA) The oven temperature was kept at 150°C for 2 min, raised to 250°C at 10°C/min, and held for 3 min The temperatures

of the injector and detector were set at 270°C and 275°C, respectively Pure nitrogen was used as a carrier gas

3 4 Apparatus

In the investigation of the effects of ultrasound irradiation to enzymatic reaction, the reactions were carried out in an ultrasonic cleaning bath (model 5210, Branson, USA) The temperature of the water in the bath was controlled by circulator with an accuracy of

±1°C A magnetic stirrer was submerged to the bottom of the bath

Figure 2 System of the combination of ultrasound irradiation and stirring

3.5 Reaction with saturated glucose solution

3.5.1 In transesterification reaction

Glucose (0.111 mmol) and vinyl laurate (0.222 mmol) were added to 0.5 ml of ILs

or IL mixtures and stirred at 40°C to allow the dissolution of sugar After 12 hr, Novozym

435 (50 mg) was added to the mixture At the end of the reaction, deionized water (1 ml) was added to the reaction vials in order to remove any unreacted glucose The supernatant was obtained after centrifugation of the reaction media The concentration of unreacted glucose in the supernatant was determined by the DNS method As the solubilities of product in water and IL mixtures are lower than 0.01 g/l at 25°C, glucose concentrations measured by DNS method were not influenced by the product dissolved in the supernatant The conversion was calculated from the concentration of consumed glucose The precipitated product was dissolved in tetrahydrofuran and the concentration was determined

by HPLC analysis to calculate product yield and confirm the conversion determined by

remained glucose The differences of conversion values which were determined by the concentration of unreacted glucose and produced ester, respectively, were less than 5% The reaction rate was calculated on the basis of conversion at 6 hr of reaction The enzyme activity was defined as mmol product formed per minute per g of immobilized lipase

3.5.2 In direct esterification reaction

Glucose (0.111 mmol) and fatty acid (0.111 mmol) were added to 0.5 ml of ILs or

IL mixtures and stirred at 50°C to allow the dissolution of sugar After 12 hr, Novozym 435 (50 mg) was added to the mixture A molecular sieve (4Å, 15 % w/v) was also added to remove the water produced during reaction At the end of the reaction, deionized water (1 ml) was added to the reaction vials in order to remove unreacted glucose The supernatant was obtained after centrifugation of the reaction media The concentration of unreacted glucose in the supernatant was determined by the DNS method As the solubilities of product in water and IL mixtures are lower than 0.01 g/l at 25°C, glucose concentrations measured by DNS method were not influenced by the product dissolved in the supernatant The conversion was calculated from the concentration of consumed glucose The precipitated product was dissolved in tetrahydrofuran and the concentration was determined

by HPLC analysis to calculate product yield and confirm the conversion determined by the remaining glucose The differences of conversion values which were determined by the concentration of unreacted glucose and produced ester, respectively, were less than 5 % The reaction rate was calculated on the basis of conversion at 6 hr of reaction The enzyme activity was defined as mmol product formed per minute per g of immobilized lipase

3.6 Reaction with supersaturated glucose solution

3.6.1 In transesterification reaction

Glucose (0.111 mmol) was dissolved in water (0.2 ml) ILs or IL mixtures (0.5 ml) were then added to these solutions at room temperature After clear solutions were obtained, the water in the mixtures was removed by vacuum evaporation for 12 hr at 60°C Novozym

435 was added to the prepared supersaturated glucose solutions The reactions were initiated by adding vinyl laurate and the mixtures were incubated in a VARIOMAG reaction block (H+P Labortechnik AG, Germany) at 40°C At the end of the reaction, the same procedures previously described were used to analyze the concentrations of unreacted glucose and produced ester To measure residual activity of enzyme after 6 hr of reaction, Novozym 435 was washed with water and tetrahydrofuran and then dried in a desiccator before reuse The residual activity was determined by the relative glucose conversion in the subsequent reaction

3.6.2 In direct esterification reaction

Glucose (0.111 mmol) was dissolved in water (0.2 ml) ILs or IL mixtures (0.5 ml) were then added to these solutions at room temperature After clear solutions were obtained, the water in the mixtures was removed by vacuum evaporation for 12 hr at 60°C Novozym

435 was added to the supersaturated glucose solutions prepared A molecular sieve (4Å,

15 % w/v) was also added The reactions were started by adding fatty acid and the mixtures were incubated in a VARIOMAG reaction block (H+P Labortechnik AG, Germany) at 50°C At the end of the reaction, the same procedures previously described were used to analyze the concentrations of unreacted glucose and produced ester To measure residual activity of enzyme after reaction for 6 hr, Novozym 435 was washed with water and

tetrahydrofuran and then dried in desiccator before reuse The residual activity was determined by the relative glucose conversion in the subsequent reaction

3.7 Reaction with the combination of stirring with ultrasound irradiation

Glucose with supersaturated solution (or saturated solution), vinyl laurate for transesterification reaction (or lauric acid for direct esterification reaction), Novozym 435 (50mg) was prepared in 0.5 ml pure ILs or IL mixtures A molecular sieve (4Å, 15 % w/v) was also added for direct esterification reaction The mixtures were placed in an ultrasound cleaning bath with ultrasound irradiation (47 kHz, 185 W) and the magnetic stirrer was

esterification) At the end of the reaction, the same procedures previously described were used to analyze the concentrations of unreacted glucose and produced ester

3.8 Reuse of ILs and enzyme after reaction

For the reuse experiment, glucose (0.333 mmol) and lauric acid (0.333 mmol) were

(1:1) in the presence of molecular sieve (4Å, 15% w/v) at 50°C After 24 hr of reaction, deionized water (1.5 ml) was added to the reaction vial Enzyme and product were separated by filtration of the reaction mixture and about 80% of product was obtained After centrifugation of the filtered reaction mixture, the precipitated product (about 20%) could be recovered and unreacted glucose concentration was determined For the next reaction, additional glucose finally adjusted to 0.333 mmol was added to the reaction mixture The mixture was then evaporated to remove water for 12 hr at 60°C The new reaction was started by adding the molecular sieve, lauric acid (0.333 mmol), and recovered

enzyme to the prepared supersaturated glucose solution in the IL mixture The concentration of lauric acid was not considered during the reuse experiment

3.9 Structure determination

those in the literature [37, 55] The molecular weight of the product was also confirmed by LC-MS (Varian 1200L, USA)

3.10 HPLC analysis

The concentration of product was measured by HPLC Separation was accomplished using a Shimadzu HPLC system (Model LC-10A, Japan) equipped with a

USA) The mobile phase consisted of methanol : water = 90 : 10 with a flow rate of 1.0 ml/min