Microwave assisted enzymatic synthesis of speciality esters: A mini - review

Accepted Manuscript Title: Microwave assisted enzymatic synthesis of speciality esters: A mini - review Authors: Nishat R. Khan, Virendra K. Rathod PII: DOI: Reference:

S1359-5113(18)30524-5 https://doi.org/10.1016/j.procbio.2018.08.019 PRBI 11430

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

10-4-2018 16-7-2018 16-8-2018

Please cite this article as: Khan NR, Rathod VK, Microwave assisted enzymatic synthesis of speciality esters: A mini - review, Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.08.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Microwave assisted enzymatic synthesis of speciality esters: A mini - review

Nishat R. Khan & Dr. Virendra K. Rathod

Department of Chemical Engineering,

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Institute of Chemical Technology, Matunga, Mumbai-400019, India. *Corresponding Author: Dr.Virendra K. Rathod

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E-mail: [email protected], Phone: +91-22-33612020, Fax: 91-22-33611020.

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Email: [email protected]

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Highlights

Microwave energy can reduce the reaction time and enhance rate of reaction.

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This review studies its use in the enzymatic synthesis of speciality esters.

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It discusses the mechanism, parameters, stability, kinetic & scalability aspects.

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Abstract: Biocatalysis exhibits distinct improvement as compared to the chemical method of synthesis with respect to simplification of operating process, improvement in end product quality and minimization of by-product. However, long duration of synthesis, slow reaction

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rate and poor conversion limit the scope of biocatalysis in the industry. Currently there is also an increased demand for the use of green technology in chemical industry. Application of microwave irradiation to the biocatalytic synthesis can address the drawbacks of biocatalysis in the industry. Microwave energy creates rampant localized super heating which enhances the reaction rate and reduces the synthesis time significantly. The present mini review article gives 1

a brief account on uses of microwave energy in enzyme processed synthesis of various speciality esters that find application in chemical industries. It briefly discusses various aspects like mechanism, instrumentation, process variables, enzyme stability, kinetics and scalability approach.

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Keywords: Speciality esters, lipases, esterification, sustainability, microwave

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1. Introduction

Microwave technology is becoming exceedingly popular in the chemical industry because it gives increased rate of conversion and yield in a short duration of time. Microwaves are

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electromagnetic radiations having frequencies in the range of 300 MHz to 300 GHz. and

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wavelengths in the range of one meter to one millimetre [1]. The common microwave

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wavelength of 12.24 cm for 2.45 GHz frequency has been mostly used for enzymatic reactions

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[2,3]. Microwave energy couples with the polar molecules in the reaction system to generate

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vibrations that are independent of the temperature of reaction. It leads to instant localized superheating due to ionic conduction [4,5]. Microwave combines two effects, i.e. non thermal

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and thermal [1]. Microwave irradiation leads to direct coupling of molecules of the reaction system which gives much better results compared to conventional heating as it leads to direct

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coupling of molecules by selective absorption [6]. Thus, some reactions which take hours to complete under conventional heating can be successful using microwave in a very short time. In the synthesis of lauric acid esters using Novozym 435, conventional heating required 24 h

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whereas, the reaction was completed in merely 8 minutes under the microwave system [7]. Also, microwave radiations improve stability profile of enzymes by delaying denaturation. Enzymes like Candida antarctica lipase B have shown improved stability under microwave system as compared to conventional heating [8, 9]. Speciality esters are chemical components that are used for their effect and performance. Their market value is high as compared to bulk 2

chemicals. They can be categorized on the basis of their application and function. Based on application they are used in agrochemicals, fragrance & flavours, personal care products, foods & nutraceuticals, textiles, polymers, dyes, lubricants, etc. Speciality esters by function include emulsifiers, viscosity builders, antioxidants, synthetic dyes, fixatives, surfactants, synthetic intermediates, pesticide synergists, etc. [10,11]. Chemical as well as enzymatic routes can be

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used for the synthesis of speciality esters. However, conventional enzymatic reactions are very slow [12,13]. One of the approaches to intensify the enzymatic reactions is through the use of

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microwave energy. Microwave heating and biocatalysis show synergistic effect with

augmentation of rate and final conversion [14]. In the enzyme catalyzed esterification of adipic

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acid with various alcohols, low microwave energy lead to an improvement in rate by a factor

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of up to 2.63 as compared to normal heating [15]. This effect was due to the higher collision

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frequency between the molecules under microwave [16,17,18]. The synergism of microwave

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energy and biocatalysis has shown good results in esterification [19] as well as transesterification reactions [20]. However, this synergistic approach in synthesis of speciality

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esters is still in a nascent stage. Therefore, it is purposeful to present a review on this niche topic. The initial part of the review is more extensive and discusses the microwave mechanism,

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working models, process variables, enzyme stability and literature survey. The latter part of the

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review discusses the kinetics and scalability aspects. 2. Mechanism of microwave reactions In conventional heating system the transport of energy into the reaction media relies on the

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thermal conductivity of the reaction medium. This may cause higher heating of the reactor than the reaction system if the thermal conductivity of the reaction medium is poor. Microwave energy on the other hand gives rise to effective internalized heating by allowing coupling of microwaves with the reaction medium [21] (Fig.1). Microwaves possess the potential to transport energy from one medium to another in a small duration of 10-9 s with every run of 3

electromagnetic energy. The relaxation time of molecules from this energy is roughly 10-5s. This suggests that the time required for the energy to advance is quicker than the time needed for the molecules to relax. This gives rise to non-equilibrium state and rapid increase in temperature that influence the reaction kinetics of the entire system [19, 22, 23]. The effect produced by microwave radiations in the chemical synthesis is a combined effect of non-

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thermal and thermal phenomenon. The thermal action consists of phenomenon like overheating, hotspots and selective heating. The non-thermal effect consists of highly polarized

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radiations. ‘Hotspot’ is a region with a high intensity of electro-magnetic fields, relative to its surroundings. Hot spots result when the materials of the reaction medium have different

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microwave absorbing properties, which lead to non-uniform temperature distribution. Hot

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spots give rise to temperature difference over several hundred degrees between neighbouring

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substances They may occur due to non-uniformity of applied field, erratic distribution of

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electromagnetic field strength, volumetric dielectric heating or variation in dielectric properties of materials of the system The non-thermal effects may have many sources such as erroneous

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temperature measurements, agitation/stirring of the reaction mixture, interactions occurring between microwave field and the organic compounds. Another cause for non-thermal effect is

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alteration in thermodynamic parameters taking place under microwave irradiation [18, 24]. Microwave radiations generate molecular rotation which stems from dipole alignment with the

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externally operating electric field (Fig.2). Microwaves strongly influence the polar molecules with high dipole moment. Lipases and substrates used in enzymatic synthesis have high dipole

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moment due to their functional groups which give them the polar nature. Polar molecules show greater tendency to absorb microwave energy as compared to the less polar molecules leading to super-molecular heating in the range of 13-26°C in excess of their boiling point. If any polar solvent has relaxation time > 65 ps, then the loss tangent (ability to absorb microwave radiation) increases with temperature which leads to the superheating of solvent above a few

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degrees of their boiling point [22]. As the temperature rises, these solvent convert more microwave energy into thermal energy. An increase in temperature causes molecules to move about more rapidly, which leads to a greater number of more energetic collisions. This occurs much faster with microwave energy, due to the high instantaneous heating of the substances above the normal bulk temperature, and is the primary factor for the observed rate

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enhancements.

Enzymes under microwave irradiation may possibly behave to some extent in a different way

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and turn out to be more effective. This is due to conformational modification in enzyme under

microwave irradiation which is capable of assisting the substrate to come close to the active

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site of enzyme more easily. Microwave irradiation increases the possibility of effective

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interaction of the substrates with the biocatalyst. Also, the influence of microwave field causes

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the conformational flipping of active sites by perturbing the polar hydrogen bonding in the

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protein cluster of lipase which activates the enzymes and causes substrate binding as well as the product release from the active sites of lipase at faster rate [25].

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3. Microwave devices and working models

Domestic microwaves have a pulse irradiation system and lack a reliable system for monitoring

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the reaction temperature. The poor quality of magnetrons used in domestic microwaves

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produce inhomogeneous fields which leads to lack of safety control. Thus, due to many technical and safety issues they lost popularity and have been replaced by industrial microwaves. Commercial microwaves have an in-built magnetic stirring system, direct

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temperature control using sensors and software for proper monitoring. Industrially microwaves can be broadly classified as single-mode and multi-mode apparatus (Fig. 3). In principal both these types have a magnetron that generates electromagnetic radiations. These radiations are emitted by an antenna into the cavities were the samples are placed. In the microwave synthesis, the initial microwave power is high which increases the bulk temperature to the desired value 5

very fast. Once, this temperature is reached the microwave power decreases and eventually shuts off in order to maintain the desired bulk temperature. Cooling allows a high level of microwave power to irradiate the sample mixture directly. This improves the non-thermal microwave effects that rely on the electric field strength and simultaneously prevents over heating of the reactants by continuous removal of heat without exceeding bulk temperature.

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Generally, a cooling system comprises of a compartment which has an air intake opening. A

magnetron and transformer are placed inside the compartment along with a cooling fan, which

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forces the cooling air around the microwave into the compartment through the inlet which cools the magnetron directly. Multimode reactors contain huge cavities where the microwave field

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is spread in a much disorganized form. They are large in size and host many types of rotor

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assemblies and are used for synthesis of reactions in the batch size of 300 µL to 100 mL. In

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these reactors, microwave radiations directly proceed into the cavity of the vessel and are

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reflected towards the reaction vessel by the walls of the cavity. In multi-mode reactors, microwave radiations take up a typical wave like behaviour forming constructive and

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destructive interference patterns. This results in the formation of local ‘hot spots’ in the cavity. Because of this, multi-mode reactors are equipped with a mode stirrer which is a metal fan that

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helps to spread the microwaves throughout the cavity. Multimode microwaves have isolators which are like diodes. They protect the magnetron from reflected energy and do not allow

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microwaves to bounce back and hit the magnetron. Multimode microwave reactors for single batch scale up have a circular waveguide in which many modes of the microwave radiations

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irradiate the contents of the vessel at multiple locations for effective heating of samples in large volumes. A single 1 L reactor is placed in the closely packed cavity in the centre, which gives optimized heating for large reaction volumes. This gives relatively high field density in comparison to the regular multimode microwave ovens. In monomode reactors the microwave radiations are guided through a rectangular wave guide towards the reaction container located

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at an interval from the magnetron which creates a standing wave. Consequently, an increased microwave field density is created, causing exceedingly fast heating rates. In single mode microwaves the cavity is tuned to the frequency of the magnetron. There is uniform microwave field due to which there is no hot and cold spot formation and the microwave field is completely homogenous. Since, there is no reflected energy, isolators are not used. As single mode

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microwaves transfer 100% energy to the sample, they are smaller than multimode, and usually of a much lower power. Single mode microwave reactors allow irradiation of only one vessel

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at a time and are very simple to operate [26,27,28,29]. Use of multimode reactors have been

reported in the synthesis of methyl esters of fatty acid from soybean oil using Novozym 435

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[30] and butylgalactopyranoside catalyzed by Thermotoga naphthophila RKU-10 [31]. Mono-

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mode reactors have been employed in the synthesis of isoamyl myristate in a substrate solvent

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system [19]. They have also, been used in the enzymatic glycosidation reaction in dry media

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[32].

4. Effect of temperature, nature of solvent and power

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Factors like temperature, type of solvent and power control enzymatic microwave reactions. With a rise in temperature, microwave irradiation leads to a rise in the frequency factor which

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increases the collision of the molecules giving them more energy. The more energy they have,

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the more is the tendency of electrons of the reacting molecules to skip to higher energy state. This movement of electron increases the randomness of the system which inevitably increases the system’s entropy and reduces the activation energy leading to faster synthesis [33].

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However, at very high temperature under microwave systems there is a reduction in the rate of reaction due to denaturation of protein structure of enzyme by heat induced disruption and breakdown of weak ionic and hydrogen bonds. These bonds stabilize the typical three dimensional structure of enzyme. Microwave assisted synthesis of Geranyl cinnamate, showed a rise in the conversion from 23% to 83% and rate of reaction from 0.98 x 10-3 to 4.3 x 10-3 7

mol.l-1.min-1.g-1of enzyme with a rise in temperature from 40-70°C [34]. Similar results have been reported in the transesterification of ethyl-3-phenyl propanoate and n-butanol [35]. The solvent’s effect on the activity of enzyme is defined by its log P value. Increase in log P value of solvent shows linear increase in the lipase activity. Solvents having non- polar nature (log P> 4) show improved effect as compared to the polar solvents (log P< 2) on the functioning

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of enzyme. The non-polar solvent can permit specific absorption by reactants allowing direct

transfer of energy to take place from reactant to solvent. Also, the thin layer of water present

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outside the enzyme necessary for its activity is not disrupted by the non-polar solvents. Polar solvents display strong hydrophilic interactions with the enzyme’s water layer which affects

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its activity. In case of immobilized enzyme reactions, the reactants should be able to pass

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through the solution to the active sites of enzymes and the products formed should percolate

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into the bulk solution. In fatty acid ester synthesis, substrates display high affinity for solvents

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of non-polar origin and show greater diffusion into active sites on enzymes, which give better results. Solvent free systems have also indicated good results. In microwave supported lipase

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catalyzed synthesis of isoamyl myristate good results have been reported in both solvent free and non-polar reaction systems [19]. Acceptable outcome in substrate solvent conditions can

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be because of the presence of myristic acid which has high log P value in the surroundings of

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the enzymes. This preserves the lining of water around the enzymes. Non-polar solvents have low dielectric constants which lead to a poor solvent-microwave synergism that allows for direct heating of the reaction mixture. Polar solvents have shown better results than non-polar

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solvents in some cases possibly due to the selective heating effect of microwave. Microwave heating mechanism takes place by the dipole molecular rotation and ion conduction. Hence, microwave irradiation has a strong impact on such polar solvents, which absorb more microwave radiations. The improved effect of polar solvents can also be due to factors like their solubility, dielectric constant, polarity and conductivity [23]. 8

High power causes faster movement of the molecules which in turn increases the reaction velocity. For low power input, the time taken to reach the phase transition temperature is very long. The heat generated during this period is conducted to the lower temperature locations, which leads to uniform temperature distribution. Excessive uniformity of temperature reduces heating efficiency and increases the consumption of energy. In the case of high input power,

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hot spots reach the phase transition temperature in a short time, that decreases energy consumption, but the uniformity of heat distribution in the system becomes poor. At high

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microwave powers, exceeding 50% of the microwave capacity, over 480 W the temperature becomes difficult to control. Therefore, in order to consume less energy and get more uniform

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temperature distribution, it is necessary to choose a proper input power [36]. High microwave

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power can cause drastic conformational variations in the three dimensional structure of enzyme

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[31, 37]. Experiments conducted under low microwave power below 50 W have displayed

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appropriate results under optimum parameters. Immobilized enzymes when operated under their optimum temperature give acceptable results at high microwave powers. At high

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microwave power in the range of 200-300 W, Lipozyme RM IM and Novozym 435 have displayed good results. In the synthesis of butyl galactopyranoside having surfactant properties,

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using enzyme and microwave, the effect of varying power on the reaction was investigated from 80W to 800W. The concentration of butyl galactopyranoside increased to 1.17g/L when

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the microwave power was raised to 400 W. Increasing the power over 480 W lead to a decrease in the yield due to enzyme deactivation at high power [31].

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5. Effect of microwave radiation on enzyme’s activity, stability and selectivity Microwave radiations have substantial effect on the stability, activity and selectivity of enzymes in esterification reactions, some of which are represented in Table 1. The activity of enzyme in microwave system is mostly affected by the polarity and hydration state of the reaction medium. Enzymatic activity improves as the hydrophobicity of the solvent increases. 9

Enzymes show a tendency to behave differently under microwave heating thus getting activated and energised. Enhancement in the enzyme activity have been reported in the synthesis of propylene glycol monolaurate [38] and 1-propyl acetoacetate [20] using Novozym 435 and microwave. At very high temperature, the catalytically active sites of enzymes are lost leading to its inactivation. The stability of enzymes is mostly dependent on its interactions with

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its microenvironment. Non-polar solvents under microwave improve the structural rigidity and conformation of enzymes. Thereby, improving its stability. Sometimes, even polar solvent

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under microwave lead to improved stability of enzymes. Polar solvents have strong affinity for

microwave radiations. Hence, when polar solvents are used, even a smaller power input as

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compared to non-polar solvents will be needed to create sufficient temperature conditions for

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synthesis. Under microwave heating, high polar solvents have greater ability to couple with the

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microwave energy. Thus, a power of 24 W is sufficient in butanol to maintain the temperature

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at 100°C whereas 210 W power is needed in ethyl butyrate to keep the same temperature [8]. It has been reported that Novozym 435, showed increased stability under microwave heating

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as compared to conventional heating in presence of more polar solvent like butanol. Immobilization of enzymes improves its stability and reusability in the reaction medium as it

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gets protected from harsh reaction conditions like temperature of microwave and acidity of reaction medium [25]. Reusability of enzymes strongly controls the economic feasibility of

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enzymatic processes. In the transesterification reaction between ethyl butyrate and butanol, both free and immobilized lipase from Candida antarctica was used. In both the cases enzyme

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showed better stability under microwave than conventional heating. With every run or reuse, the biocatalyst showed higher initial rate and conversion under microwave heating. After a total number of 6 consecutive batches, a factor of 2 was obtained in between the remaining activity of lipase used under the microwave and conventional systems. Even under the storage condition, when the biocatalyst was preincubated in ethyl butyrate at 100°C for 30 minutes,

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39% of its activity was retained under conventional system whereas 45% was retained under microwave system [9]. Reusability studies were performed in the synthesis of citronenyll acetate using Novozym 435 wherein, conversions were studied under both microwave and conventional hating for three consecutive cycles. It was observed that under microwave system the conversions as well as the initial rates (microwave irradiation: 0.821e-0.207(reuse);

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conventional heating: initial rate 0.631e-0.232 (reuse)) in every cycle was always higher [33]. In

microwave mediated and solvent-free enzyme catalyzed transesterification of beta-ketoesters,

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reusability of the biocatalyst was observed under both thermal and microwave conditions. It

was noticed that catalyst stability was not affected much under microwave and the

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transesterification reactions could be carried out for 10 consecutive cycles without much loss

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in activity [39].

The ability of enzymes to choose single substrate from many similar compounds is known as

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enzyme’s selectivity. Selectivity may be altered under microwave due to a change in enzyme conformation. Microwave has shown to improve the stereo selectivity of enzymes. In porcine

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lipase catalyzed transesterification of (R)-1, 2, 3, 4-tetrahydro-1-naphthol under microwave, the stereo selectivity increased by 3-9 folds as compared to ultrasonication [40]. This

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enantioselectivity may be because of the activation of participating substrate alcohol and very little participation of recovered alcohol substrate. In pharmaceuticals, most non-steroidal anti-

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inflammatory drugs like flurbiprofen, ketorolac, ibuprofen, etc. are present as a racemic mixtures, with mostly the (S) - (-) enantiomer showing the anti-inflammatory activity by

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inhibiting the COX inhibitor. The (R) - (+) enantiomer is generally inactive and shows side effects like gastrointestinal bleeding and renal impairment. Hence, the resolution of racemic mixtures of such drugs is carried out by converting the inactive enantiomer into an ester. Lipases are widely used for the kinetic resolution of racemates because they have the ability of identifying the stereogenic center, have wide substrate specificity, high regio and

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stereoselectivity. Microwave mediated enzyme catalyzed resolution of the racemic mixture (R, S)-flurbiprofen with alcohols was performed in an organic media using Novozym 435. Maximum conversion of R-flurbiprofen to its ester using microwave energy was ∼44.4% in 6 h with enantioselectivity (E) of 478, eep = 98.9% and ees= 79.07%, whereas in conventional heating the maximum conversion was 17% with enantioselectivity (E) of 126, and ees= 20.12%

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[41]. In the enzymatic resolution of RS-(±)-ketorolac using microwave energy, an increase in

initial rate of reaction up to 1.5-fold was reported. Novozym 435 catalyzed the enantioselective

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esterification of RS-(±)-ketorolac in n-octanol solvent, giving 50% conversion and >99%

enantiomeric excess in 3 h time interval at 50°C temperature and 300 rpm speed of stirring

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[42]. Similar results were observed in the enantioselective esterification of Ibuprofen in n-

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heptane solvent system catalyzed by recombinant APE 1547 (a thermophilic esterase from the

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Archaeon aeropyrum pernix K1). In conventional heating system, the activity of enzyme &

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enantioselectivity were 0.19 ± 0.05 & 37.1 ± 1.6 respectively at (60°C temperature, 150 rpm stirring speed, 60 mg enzyme dose, 8.5 pH). Microwave heating showed improvement in both

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enzyme activity (0.87 ± 0.04) & enantioselectivity (47.5 ± 2.1) at (400 W power, 60°C temperature & 150 rpm stirring speed). This enantioselectivity may be because of the activation

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of participating substrate alcohol and very little participation of recovered alcohol substrate

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[43]. In the transesterification reaction of 1-Phenylethanol, higher percentage of S-1Phenylethanol enantiomer was observed. The enantiomeric excess was 30% more in microwave system [44].

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6. An overview of microwave assisted enzymatic synthesis Modern microwave technology gives way to safe synthesis of esters at low temperature conditions favourable to enzymes. Few papers in the literature have reported the use of microwave to synthesize speciality esters which are discussed below and also summarized in Table 2. Novozym 435 (Candida antarctica lipase B immobilized using an acrylic resin 12

support) has shown good results under microwave as well as conventional heating. Other enzymes like Subtilisn (cross linked enzyme agglomerate); Bacillus stearothermophilus and free enzyme such as Porcine pancreatic lipase have also shown good results under microwave. Just like immobilization, even cross linking of enzymes by agglomeration stabilizes the enzyme with preservation of enzymatic activity [48]. In the esterification reaction of dodecanoic acid

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with different sugar derivatives such as α-D-glucopyranoside and α, α- trehalose to form esters with surfactant properties, enhanced conversion and selectivity were observed under

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microwave heating. Microwave energy gave conversion over 95% while, conventional heating only reached up to 50% in 5 h [32]. Esterification reaction of lauric acid with different alcohols

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from C1- C5 under both heating methods was carried out in hexane solvent using Candida

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antarctica lipase B and Burkholderia cepacia. The conversion was dependent on the carbon

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chain length. The duration of reaction was reduced from 24 h in conventional method to 8 min

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in microwave heating. Microwave heating gave a conversion of 64% using Novozym 435 [7]. Propylene glycol monolaurate finds application as emulsifier in the cosmetics. Its synthesis

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under microwave with the help of lipase Novozym 435 has been reported in the literature. The solvent 1, 4-dioaxane showed maximum conversion at 60°C. The pre-exponential factor under

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microwave irradiation at 60°C improved by 2.84 times as compared to conventional heating [38]. Enzymatic transesterification of ethyl cinnamate with citronellol was carried out in

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organic media under microwave to form a perfumery ester. An improvement in enzyme activity and conversion was observed due to increased collision between substrate and enzyme under

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microwave. N-caprylic acid finds wide application in perfumery and dyes industry. Enzymatic esterification of n-caprylic acid with C2 - C10 n-alcohol was carried out under microwave as well as conventional heating methods. Microwave reaction definitely showed an improvement in terms of conversion and reduction in reaction time. The rate of reaction was dependant on the alcohol chain length [49]. In transesterification reaction of citronellol with vinyl ester in

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toluene, microwave showed conversion of ~90% in 120 min and conventional method gave a conversion of 80%. Thus, microwave irradiation was instrumental in intensifying the process and improving the conversion [18]. The reaction synthesis of isoamyl myristate, a flavour and fragrance ester has been studied in a substrate solvent system using microwave irradiation. The reported conversion was 96% in 1 h under following conditions: 60°C, 400 rpm and 0.38%

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[19]. Ascorbyl palmitate having antioxidant properties has also been reported to be synthesized

under microwave irradiation. Ascorbyl palmitate synthesis showed a conversion of 71 % in 1

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h reaction time, 70°C temperature condition, 1:3 substrate molar ratio and 15 % w/w of enzyme

dose using microwave technology. Under the same conditions but without using microwave

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heat it showed a conversion of only 40% in 24h [50]. In one experimental work, fatty acids

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(C4:0–C18:1) were reacted with ascorbic acid under hexane and lipases system (Bacillus

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stearothermophilus SB-1 and Burkholderia cepacia RGP-1). This synthesis reaction attained

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equilibrium in 30 seconds at 1.35 kW microwave power [51]. In the microwave assisted transesterification reaction between methyl esters of fatty acids and ascorbic acid in a solvent

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free medium, maximum conversion of 70-80% was obtained in just 35-45 s [51]. A selective herbicide, 4-chloro-2-methylphenoxyacetic acid ester was synthesized under microwave

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irradiation using Novozym 435 in 1, 4- dioxane solvent. The initial rate of reaction was increased by 2-fold under microwave assistance and conversion was increased from 26% to

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83% under 6 h reaction time [52]. Adipic acid esters are used as freeze- resistant plasticizers used in cold resisting agricultural plastic film, freezing food, packaging films etc. [53]. Low

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energy microwave mediated enzymatic synthesis of adipic acid esters resulted in a rise by a factor of up to 2.63 as compared to conventional route. No change in the activation energies of both the heating modes was noticed. At 60°C using Novozym 435, microwave gave conversion of 78% and mechanical stirring of 73% in h [15]. Enzyme catalyzed synthesis of starch esters used as food and pharmaceutical additives was performed under microwave in a solvent free

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system. Lipase from Staphylococcus aureus (SAL3) immobilized by CaCO3 was used to augment the reaction between starch and oleic acid. Highest conversion of 76% was obtained at a starch/oleic acid molar ratio of 0.18 and 44°C in 4 h incubation time. Modification of starch into starch ester greatly improves its water resistance and thermo plasticity [54]. Microwave intensification of synthesis of medium chain triglycerides, used in nutraceuticals and

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pharmaceuticals has been reported showing conversion of 97.8% in 20 min. Conventional

synthesis reported a conversion of 40% in 180 min [55]. β-keto esters behave as synthons or

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starting materials for the synthesis of many drugs in pharmaceuticals. The transesterification

of methyl acetoacetate, a β-keto ester with alcohols of different chain lengths was studied under

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microwave irradiation as well as conventionally. Conventional heating showed conversion of

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57% and microwave gave conversion of 74% in 60 min. It was noted that Novozym 435 gave

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the best results. Increase in the chain length of alcohol showed good results under microwave.

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Initial activity was enhanced by 2.2-4.6 under microwave system [20]. Alkyl benzoate esters used in the flavour and pharmaceutical industry were synthesized under microwave radiations.

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Many alcohols such as n-butanol, n-hexanol, n-pentanol, benzyl alcohol and enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM were screened for the process. Microwave

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system showed improvement in the initial rate of reaction up to 6.5 folds. Novozym 435 gave the best results. Conversion of ~31% under conventional scheme and ~97% under microwave

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system was observed in 6 h [56]. Pharmaceutical intermediate, Butyl dipheyl methyl mercapto acetate is used in the synthesis of Modafinil, which is a CNS stimulating drug. Enzymatic

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esterification of dipheyl methyl mercapto acetate with n-butanol under toluene solvent was studied. There was an increase in conversion from 11% (conventional) to 18% (microwave) and enzyme activity from 0.0013 mol L-1h-1 (conventional) to 0.0021 mol L-1h-1 (microwave) in 10 h time at 60°C [57]. Microwave heating effects were investigated in the synthesis of xylitol mono esters, which are used as drugs for treatment of malignant tumours. Esterification

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of xylitol with many fatty acids was performed in a system free of solvent using Penicillium Camembertii lipase. Highest conversion of 39.82% was achieved in 9 h under mechanical agitation 63.10% in 6 h under microwave [58]. Free fatty acids extracted from blackcurrent oil contain γ-linolenic acid. It is important to enrich γ-linolenic acid for it to be used as a dietary supplement. The enrichment performed by carrying out esterification of free fatty acids from

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blackcurrent oil with n-butanol using microwave at 30°C using Lipozyme gave the best results.

In 2 h, microwave heating gave 49.1% and conventional heating gave 43.8% conversion [59].

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Microwave mediated lipase catalyzed synthesis of glycerol monolaurate, an antiviral agent was

performed using Novozym 435. Good conversion was observed in just 30 min under

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microwave and it was 1.5 times higher than the conventional system [60]. Ethyl lactate used as

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a solvent and degressar was synthesized under microwave using phophonium ionic liquid

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system. The reaction duration was reduced from 24 h to 7 h under microwave system [61].

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From the discussion of case studies above it is evident that microwave energy is instrumental in giving better outcome in terms of conversion in comparison to conventional heating.

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The study of application of microwave energy can be extended from enzymatic synthesis of speciality esters to biofuels, although this topic is vast and can form a separate review

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altogether. Microwave energy has been utilized substantially in the biocatalytic synthesis of biofuels from oils and algae. Fatty acid methyl esters have been synthesized from soybean oil

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and methanol at a temperature of 40°C in presence of tert-amyl alcohol solvent. Under microwave, 94% conversion was observed in 12 h, whereas conventional method took 24 h to

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achieve the same conversion [30]. The biocatalytic synthesis of biodiesel by transesterification of palm oil and short chain alcohol like ethanol was carried out using Pseudomonas fluorescens immobilized lipase under a microwave heating system. A conversion of 97.56 % was observed in 12-h reaction, this was six fold times higher than the conversion observed in a mechanical stirring system [63]. A unique hybrid method was developed for synthesis of biofuels in which

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enzymatic hydrolysis was combined with acid catalyzed esterification. In the enzymatic hydrolysis, three phase partitioning (TPP) and microwave drying were used to improve the activity and stability of enzymes. The treated enzyme was further used for hydrolysis of waste cooking oil at 40°C in a bi-phasic media. It was observed that the enzyme activity was improved by 1.6 times when using microwave method as compared to conventional technique

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[64]. Thus, microwave energy can significantly improve the conversions in a short time as compared to conventional heating.

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7. Enzyme and reaction kinetics

The enzyme kinetic study measures the reaction rate and investigates the effect of varying

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process parameters on the reaction. A detailed study of an enzyme’s kinetics reveals the

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catalytic mechanism of action of enzyme, its controlling factors and inhibitors. Majority of

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enzymatic reactions are bi-substrate in nature and follow a sequential or ordered mechanism.

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Microwave energy greatly influences the kinetics of an enzymatic reaction by improving the initial rate, Vmax. Kinetic studies of some ester synthesis have been reported in Table 3. These

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esters have been synthesized using Novozym 435 Candida antarctica lipase B (CALB) immobilized on a macro porous polyacrylic resin beads. A detailed kinetic rate study of

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citronellyl acetate was performed. The Lineweaver Burk’s plot of inverse rate of reaction vs. inverse concentration of citronellol showed parallel lines at low concentrations for both

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microwave and conventional methods. The slopes of the lines were unaffected by concentration of a fixed substrate. This indicated ping-pong dissociation mechanism. Higher Vmax value

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under microwave study (0.0368 kmol/m3·s·g) as compared to conventional study (0.0268 kmol/m3·s·g) clearly depicts rate enhancement. The kinetic constants for conventional synthesis were, KmA = 0.705 kmol/(m3.g-enz), KmB = 1.67 kmol/(m3.g-enz), KiA = 0.502 kmol/(m3.g-enz), KiB = 1.602 kmol/(m3.g-enz) and for microwave synthesis were, KmA = 0.422 kmol/(m3.g-enz), KmB = 1.25 kmol/(m3.g-enz), KiA = 0.150 kmol/(m3.g-enz), KiB = 0.307

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kmol/(m3.g-enz). The study of kinetic modelling under microwave irradiation showed an increase in the turnover number of the lipase. Michaelis constant values were reduced due to an increase in affinity of citronellol and vinyl acetate towards enzyme’s active sites. Also, higher collisions between molecules, lead to lower values of dissociation constants under microwave heating [33]. The kinetic studies of microwave assisted synthesis of isoamyl

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myristate, indicate a dissociation mechanism like ping-pong where one product is liberated

before the second substrate is added to the complex. Substrate inhibition was indicated at high

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doses of isoamyl alcohol with the following kinetic parameters; Vmax = 7.05 mol L-1 min-1, KmA = 1.45 mol/L, KmB = 1.07 mol/L & Ki = 1.23 mol/L [19]. Ternary complex mechanistic path

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with inhibition by geraniol at higher concentrations was suggested in the microwave mediated rate study of Geranyl cinnamate. Geraniol formed a dead-end inhibitory complex with the

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enzyme at high doses. Evaluated kinetic constants are as follows: Vmax (mol L-1) 0.0051, KmA

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(mol L-1) 0.244, KmB (mol L-1) 0.325, KiA 0.0015, and KiB 0.0327 [34]. Ping-pong bi-bi dissociation mechanism with inhibition by both the substrates is observed in the kinetic study

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of 4-chloro-2-phenoxyacetic acid esters synthesis using microwave energy. The initial rate graph showed parallel lines with slopes of the line not affected at lower concentration of

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substrates [52]. A ping–pong bi–bi model with inhibition by n-butanol at very high concentration was optimum for kinetics of synthesis reaction of n-butyl dipheyl methyl

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mercapto acetate [57]. The various kinetic variables were evaluated. The model was used to simulate the rate of reaction data which was similar to the experimental values. Similarly, in

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the transesterification reaction of ethyl-3-phenylpropanoate and n-butanol using microwave, the initial rate study and progress data curve indicated that the enzyme obeys ping-pong bi-bi mechanistic path with alcohol inhibition [35]. In the transesterification reaction of methyl acetoacetate with alcohols, the SSE value for ping-pong bi-bi model was the lowest in both conventional and microwave systems. Hence, ping- pong bi-bi model showed the best fit in

18

both systems. The kinetic constants showed an improvement in the microwave method. There is a significant improvement in the reaction rate (Microwave heating, Vmax = 0.6322 mol L1

min-1 and Conventional heating, Vmax = 0.5761 mol L-1min-1) due to the synergism between

microwave and enzyme catalysis wherein the enzyme receives better activation. The Michaelis Menten’s constant value (Microwave heating, KmA= 7.20, KmB = 0.1061 and Conventional

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heating, KmA= 9.11, KmB = 1.096) showed a drop in the microwave method indicating improved affinity of substrates to the enzymes [20]. From the available data it is evident that most

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microwave assisted enzymatic reactions synthesizing esters follow ping-pong dissociation mechanism. The evidence of sequential mechanisms in microwave promoted ester synthesis

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using lipase is present but is rare.

The energy required for the reactants to overcome the state of transition and get converted into

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products is defined as activation energy ‘Ea’. When the reaction rate increases, the activation

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energy requirement to overcome the barriers of energy reduces that leads to faster product formation through enzyme-substrate transition state. Under microwave irradiated systems, Ea

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is much lower than that in conventional systems indicating faster rate of reaction. In biocatalytic synthesis of Isoamyl butyrate under solvent free system, it was observed that the

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highest activation energy was 7 kJ/mol which was much lower than 16 kJ/mol in conventional system. The lesser value of enthalpy (ΔH) under microwave (5.15 kJ/mol) vs. conventional

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method (13.75 kJ/mol) at 313 K indicated quick formation of the enzyme-substrate complex and that lesser amount of energy is required for the elongation and compression of the chemical

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bond present in the molecules to reach transition state. The change in entropy (ΔS) showing negative values under microwave suggested spontaneity of the reaction. Microwaves increase collisions of molecules which increase the systems entropy [25]. Similar results were obtained in microwave assisted transesterification of ethyl-3-phenylpropanoate with n-butnol using Novozym 435. A drop in activation energy from 5.3 kcal/mol to 4.6 kcal/mol from

19

conventional to microwave mode of heating indicated fastening of reaction rate [35]. Biocatalytic esterification of n-alcohol with n-caprylic acid in a solvent free system lead to a drop in activation energy from 29.76 kJ/mol to 23.29 kJ/mol while switching systems from conventional to microwave heating [49]. In enzymatic synthesis of citronellyl acetate, the frequency factor was raised from 0.319 (m3/mol) s-1 in conventional system to 1.7 ×10-3

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(m3/mol) s-1 in microwave system. Microwave heating enhanced collision between molecules

which enhanced entropy of the system leading to an increase in frequency factor. The activation

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energy for both the systems [Microwave: 8.5 kcal/mol; Conventional: 8.2 kcal/mol] were comparable indicating no change in mechanism due to the mode of heating [33]. Similarly in

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enzymatic synthesis of isoamyl myristate [19] and n-butyl dipheyl methyl mercapto acetate

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[57], the energy of activation in microwave as well as conventional heating were almost the same, indicating that the increase in rate of reaction was due to increased frequency factor.

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8. Challenges, scale – up & techno-economics

Most microwave-assisted reactions reported in the literature have been carried out on a less

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than 1 g batch size i.e. typically 1–5 mL of reaction volume. For this process to become completely accepted as a large scale industrial technology there is still a need to develop

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strategies that can eventually provide products routinely on a multi-kilogram scale. Thus, development of large reactors is required to fully commercialize microwave operations.

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However, there are a few challenges in scaling-up of microwave reactions like limited penetration depth of microwaves, temperature control and design of reactor. An important

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aspect in processing large volumes under pressure in a microwave field is the safety aspect, as any malfunction or rupture of a large pressurized reaction vessel may cause industrial hazard. In microwave reactions using enzymes, the stability of enzyme is also a major issue which needs to be considered while scaling up. Also, esterification reactions are equilibrium reactions which require removal of water. Hence, scaling-up of enzymatic esterification reactions for

20

synthesis of fine chemicals using microwave technology can be quite challenging. Two techniques for microwave synthesis on a large scale (>100 mL volume) can be considered. Large scale microwave synthesis can be performed using batch-type multimode or monomode reactors processing less than 1000 mL volume or continuous-flow techniques (multi- and monomode) to overcome the problems associated with the scale-up of microwave reactors. In

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the batch scale-up, a single large reaction vessel can be employed instead of multiple small

reaction vessels which solves the problem of continuous charging and discharging. With

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today's commercially available single-mode microwave cavities, large volumes of 50 mL can be processed under sealed-vessel. There are different vessel types available for this kind of

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upscale in a linear fashion from 0.05 to 50 mL. In single large sealed vessels, heterogeneous

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reactions can be carried out in presence of solvents and pressure. Despite this versatility, the

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limiting factor is the vapour pressure generated by a superheated solvent due to safety concerns.

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Thus, additional safety features need to be incorporated in this kind of set-up. In large sized vessels poor penetration depth of microwaves can create an issue. If the reaction system does

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not comprise of toxic reagents and superheated solvents, the reaction can be carried out in open vessels. Under open-vessel conditions, higher volumes (>1000 mL) can be processed. In the

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case of equilibrium reactions like esterification open-vessels are advantageous for driving reactions to completion when the by-product is a volatile component. Reflux- condensers can

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also be used in this kind of set-up for removal of water formed during esterification. Scaling up of batch scale reactors can have many limitations. The preferable option for processing more

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than 1000 mL reaction volumes under sealed-vessel conditions is a continuous-flow method. In such reactor systems, a microwave-transparent coil is placed in the cavity of a single or multimode microwave reactor through which the reaction mixture is passed. The previously optimized reaction time under batch microwave conditions now needs to be related to a residence time at a specific flow rate. The single-mode continuous flow microwave reactors

21

present today in the market allow processing of small volumes. Large volumes can be processed in continuous-flow reactors placed in multimode microwave ovens. Continuous flow micro- reactors increase the selectivity and heat transfer of the enzymatic reactions. They also help in maintaining constant product quality over the period of time. A combination of continuous flow micro- reactor with microwave allows rapid heating and cooling of the

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reaction system. Presence of solids, viscous liquids, waxy substances, heterogeneous catalysts, etc. can be challenging for continuous processing as this may lead to plugging and obstruction

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of reactors. Since, enzymatic esterification reactions involve the use of solid lipase and substrates, single-mode stop–flow reactors can be used which were recently developed. In these

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types of reactors, peristaltic pumps that are capable of pumping slurries and solid reagents are

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employed to fill a batch type reaction vessel with the reaction mixture. The product mixture is

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pumped out of the system after processing under microwave and the system is then ready to

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receive the next batch for operation. In order to process volumes of less than 1000 mL a batch process can be suitable. Kilogram quantity of product can be achieved by carrying out

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sequential runs in batch mode. Continuous flow protocol can be considered when larger quantities of a specific product have to be prepared on a regular basis [65, 66, 67, 68, 69]. Scale

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- up of microwave reactor systems for synthesis of fine chemical esters has been very rare or is in its nascent stage. A commercially available continuous flow reactor operated as a batch

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loop system has been reported to be used in the enzyme triggered synthesis of cosmetic esters in the industry. Esterification of with vinyl acetate and (S) - Pyroglutamic acid with n-decanol

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resulting in the formation of cosmetic esters have been successfully scaled up in the industry using the laurydone process [70]. Energy efficiency is an important aspect while considering microwave assisted synthesis. Energy efficiency of microwave energy from electrical energy is around 50-65% which indicates that around 35-50% electrical energy is not utilized. The energy efficiency of

22

microwave irradiated reactions depend of various factors like volume of sample, nature of solvent, penetration depth of microwave and dissipation power of microwave device. Poor efficiencies can result when high power of microwave is used for very small reaction volume. Actual microwave power needs to be considered for all energy calculations. Energy return on investment for a microwave assisted transesterification synthesis was studied. This reaction

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operated at 300 W power and 300 rpm speed gave a yield of 81% in 9 minutes. The energy

study of this system showed that production of 1 kg of biofuel needed only 0.46 kWh electricity

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whereas 2.13 kWh electricity can be generated through 1 kg of biofuel [71]. Similarly, in the

one-step method of synthesis of FAME (fatty acid methyl esters) from Rhodotorula glutinis by

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combining lipid extraction with acid-catalyzed transesterification in a microwave reactor it was

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observed that energy required for production of biofuel was 20% less than the energy produced

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by the biofuel in 20 minutes reaction time [72]. A transesterification reaction using vegetable

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oil and methanol in 1:6 molar ratio of oil/alcohol was carried out using microwave. Energy consumption calculations revealed that the continuous-flow microwave at a flow rate of 7.2

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L/min feedstock consumed 26 kJ/L energy, 4.6 L batch type microwave utilized 90.1 kJ/L energy and conventional technique consumed 94.3 kJ/L of energy. This showed that

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microwave flow technique for the transesterification was the most energy-efficient as

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compared to conventional and batch type microwave techniques [66]. 9. Conclusion

Consumer demands for eco-friendly products have led to the development and progress of

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biocatalysis in the industry. However, long duration of biocatalysis has always been the major downside of these processes. Microwave and enzymatic synthesis of speciality esters has proved to be a fruitful and synergistic combination in many cases reported in the literature. Microwave has improved the stability and activity of the enzyme in many studies. Selection of right parameters like solvent system, power, temperature, enzyme and substrates for optimum

23

results is very crucial and challenging at the same time. Scale-up of microwave assisted esterification and transesterification reactions has limitations like poor microwave penetration power, temperature control, enzyme sensitivity, etc. Hence, microwave systems still need to be explored in the chemical industry and have great potential for future work and research

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globally.

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63. P.C. Da Rós, L. Freitas, V.H. Perez, H.F. de Castro, Enzymatic synthesis of biodiesel from palm oil assisted by microwave irradiation, Bioprocess Biosyst. Eng. 36 (2013) 443-51. 64. N, Saifuddin, A.Z. Raziah , H.N. Farah, Production of biodiesel from high acid value waste cooking oil using an optimized lipase enzyme/acid-catalyzed hybrid process, E-J. Chem. 6

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(2009) 485-495. 65. M. D. Bowman, J. L. Holcomb, C. M. Kormos, N. E. Leadbeater, V. A. Williams,

SC R

Approaches for Scale-Up of Microwave-Promoted Reactions, Org. Process Res. Dev. 12 (2008) 41–57.

66. T.M Barnard, N.E. Leadbeater, L.M. Stencel, M.B. Boucher, Continuous-Flow Preparation

N

U

of Biodiesel Using Microwave Heating, Energy & Fuels 21 (2007) 1777-1781.

A

67. K. M. Amore, N. E. Leadbeater, Microwave-Promoted Esterification Reactions:

M

Optimization and Scale-Up, Macromol. Rapid Commun. 28 (2007) 473–477. 68. A. Stadler, B. H. Yousefi, D. Dallinger, P. Walla, E. Van der Eycken, N. Kaval, C. O.

ED

Kappe, Scalability of Microwave-Assisted Organic Synthesis. From Single-Mode to

PT

Multimode Parallel Batch Reactors Org. Proc. Res. Dev., (2003), 7 (5), pp 707–716. 69. M. A. Surati, S. Jauhari, K. R. Desai, A brief review: Microwave assisted organic reaction,

CC E

Archives of App. Sci. Res. 4 (2012) 645-661. 70. H. C. Mark, L. Dressen, H.P. Bastiaan, V. D. Kruijs, J. Meuldijk, J. Vekemans, L. A.

A

Hulshof, Flow Processing of Microwave-Assisted (Heterogeneous) Organic Reactions, Org. Process Res. Dev. 14 (2010) 351–361. 71. F. Motasemi, F.N. Anin, A review on microwave-assisted production of biodiesel. Renew. Sust. Energ. Rev. 16 (2012) 4719–4733.

32

72. C. J. Chuck, D. L. Hing, R. Deana, L. A. Sargeant, R. J. Scott, R.W. Jenkins, Simultaneous microwave extraction and synthesis of fatty acid methyl ester from the oleaginous yeast

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Rhodotorula glutinis, Energy, 69 (2014) 446-454.

33

List of Figures: Fig. 1: Conventional and Microwave heating mechanisms Fig. 2: Mechanism of microwave assisted enzymatic synthesis Fig. 3: Schematic diagram of industrial microwave: mono mode (A), multi - mode for

A

CC E

PT

ED

M

A

N

U

SC R

IP T

parallel synthesis (B) and multi - mode for single batch scale-up synthesis (C)

34

IP T SC R U N A M ED

A

CC E

PT

Fig. 1: Conventional and Microwave heating mechanisms

35

IP T SC R U N A M ED

A

CC E

PT

Fig. 2: Mechanism of microwave assisted enzymatic synthesis

36

IP T SC R U N A M ED

Fig. 3: Schematic diagram of industrial microwave: mono mode (A), multi mode for

A

CC E

PT

parallel synthesis (B) and multi mode for single batch scale-up synthesis (C)

37

List of Tables: Table 1: Effect of microwave on activity, stability & selectivity of enzymes Table 2: Effect of microwave vs. convention heating in the synthesis of esters Table 3: Kinetic constants for microwave assisted & Novozym 435 (Candida antarctica lipase

A

CC E

PT

ED

M

A

N

U

SC R

IP T

B) catalyzed esterification reactions following ping-pong bi-bi model

38

I N U SC R

Table 1: Effect of microwave on activity, stability & selectivity of enzymes

Product

M

PT

Butyl butyrate

ED

1-propyl acetoacetate

A

Propylene glycol monolaurate

CC E

Butyl butyrate

Citronenyll acetate

A

(R)-1,2,3,4-tetrahydro-1-naphthylacylate

S- Flurbiprofen

S- Keterolac

S-Ibuprofen

Reaction condition

Activity

Novozym 435 (Candida antarctica lipase B), organic solvent, 30°C-70°C Novozym 435, toluene, 50°C

Higher

Novozym 435 (Candida antarctica lipase B, substrate solvent (ethyl butyrate and butanol butanol), 100°C

No effect

Free Candida antarctica lipase B, organic

Stability

Selectivity

Reference

-

[38]

No effect

[20]

Higher

-

[8]

No effect

Increased

-

[9]

Novozym 435 (Candida antarctica lipase B), Toluene, 50°C Porcine pancreatic lipase (PPL), benzene, 35°Ccreatic lipase (PPL) lipase tic lipase

Higher

Higher

-

[33]

Higher

-

Higher

[40]

Novozym 435 (Candida antarctica lipase B), organic solvent, 50°C Novozym 435 (Candida antarctica lipase B), n-octanol, 50°C

-

-

Higher

[41]

Higher

-

Higher

[42]

Recombinant APE 1547

Higher

-

Higher

[43]

-

Higher

medium, 100°C

39

I N U SC R ( Archaeon aeropyrum pernix K1), n-heptane, 60°C

S-1-Phenylethanol

Supported solvent free 70°C-80°C

M

A

Sucrose ester of fatty acids

ED

Butyl butyrate

Higher

No effect

Higher

[44]

Porcine pancreatic lipase, B. stearothermophilus SB1 and B. cepacia RGP-10 lipases,

Higher

-

No effect

[45]

Higher

-

-

[46]

Higher

-

-

[47]

organic solvent, 80°C Cutinase, substrate solvents (ethyl butyrate and butanol) , 50°C α-chymotrypsin, organic medium,25°C60°C

A

CC E

PT

N-acetyl-L-phenylalanine ethyl ester

lipase, system,

40

I N U SC R

Table 2: Effect of microwave vs. convention heating in the synthesis of some esters Microw ave heating

Solve nt

Tempera ture

A

Enzyme

M

Application Industry

α-D-glucopyranosides ester Personal care, food, feed additive

Novoz ym 435

Lauric acid esters

Surfactants, Cosmetic

ED

Ester

Citronellyl Acetate

Fragrance, Perfumery

R-1-phenyl ethyl aceate

PT

Tim e

Conversi on

Ref.

-

5h

95%

5h

50%

[32]

60°C90°C

8 min

64%

24h

-

[7]

60°C

120 min

80%

120 min

60 min

96%

60 min

Toluen e

Fragrance, Fixative,

Novoz ym 435

Solve nt free

Anti-oxidants

Novoz ym 435

Tert amyl alcohol

60°C

1h

71%

24h

40%

[50]

Novoz ym 435

nhexane

60°C

2h

48.78%

2h

42.85%

[62]

A

Ascorbyl palmitate

Subst rate solve nt

Conversi on

Novoz ym 435

CC E

Isoamyl myristate Modifier

Novoz ym 435

-

Tim e

Conventional heating

Chiral building block

60°C

90%

56%

f f f f f

[33]

[19]

41

I N U SC R

Novoz ym 435

1, 4dioxan e

60°C

6h

83%

6h

26%

[52]

Adipic Acid Esters acidulant

Gelling agent,

Novoz ym 435

1,4dioxan e

60°C

3h

78%

3h

73%

[15]

Foods,

Novoz ym 435

Solvent free

90°C

20 min

97.8%

150 min

40%

[54]

Chemical intermediate

Novoz ym 435

Toluen e

50°C

60 min

74%

60 min

57%

[20]

n-hexyl benzoate fragrance

Food flavour &

Hexane

60°C

6h

97%

6h

31%

[56]

n-butyl dipheyl intermediate methyl mercapto acetate

Pharmaceutical

Novoz ym 435

Toluen e

60°C

10h

18%

10h

11%

[57]

xylitol fatty acids ester Surfactant

Emulsifier,

Penicil lium Camem bertii

Solvent free

4560°C

6h

63.10%

9h

39.82%

[58]

Solublizer, lubricant,

Lipozy me

Isoocta ne

30°C

2h

49.1%

2h

43.8%

[59]

Glycerol Emulsifier, surfactant, preservative monolaurate n-butyl-3-phenylpropanoate Flavour, Foods

Novoz m 435

Emulsi on system nhexane

60° C

30 min

125.9 mol/L

30 min

84 mol/L

[60]

70°C

2h

~96%

2h

~91%

[35]

A

CC E

Novoz ym 435

PT

1-propyl acetoacetate for drugs

M

triglycerides

ED

Medium chain Nutraceuticals

A

4-chloroHerbicide, Agrochemical 2-methylphenoxyacetic acid ester

butyl γ –linolenate fragrance

Novoz ym 435

42

I Chiral building block

Novoz ym 435

nhexane

65– 85°C

3.5h

17.07 mol /L

24h

17.84 mol/L

[31]

60°C

2h

48.78%

2h

42.85%

[62]

A

CC E

PT

ED

R-1-phenyl ethyl aceate

Organi c solvent

N U SC R

synergist

Therm otoga naphth ophila RKU10 (TH 1577)

A

Surfactant,

M

Butyl galactopyranoside emulsifier, pesticide

43

I N U SC R

Table 3: Kinetic constants for microwave assisted & Novozym 435 (Candida antarctica lipase B) catalyzed esterification reactions following ping-pong bi-bi model

A

4-chloro-2phenoxyaceti c acid esters n-butyl dipheyl methyl mercapto acetate

M

m ol/ L

Ki mol /L

1.25

0.1 5

0. 30

-

[33]

1.07

-

-

1.23

[19]

0. 03

-

7.05

CC E

Geranyl cinnamate

B

0.42

0.06

Isoamyl alcohol

Isoamyl myristate

KmA mol/L

KiA mol /L

PT

Citronellol Citronellol acetate

Ki

KmB mol/ L

ED

Vmax ( mol L-1 min1 )

Inhibiting substrate

Ester

Reference

A

Kinetic constants

1.45

[34]

Geraniol

0.00 51

0.24

0.32

0.0 015

4-chloro-2phenoxyacetic acid and nbutanol

0.00 41

0.01

0.09

0.2 0

0. 35

-

[52]

10.1 2

0.49

1.01

-

-

1.0

[57]

n-butanol

44

I 2.7 x 10-4

0.18

-

-

0.63

7.20

0.10

-

-

N U SC R

No substrate inhibition

3.7x 10-6

1.1 x 10-4

[35]

-

[20]

A

CC E

PT

ED

M

Methyl acetoacetate esters

n-butanol

A

Ethyl-3phenylpropa noate ester

45