Enhanced Biocatalytic Activity of Lipase Immobilized on

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Enhanced Biocatalytic Activity of Lipase Immobilized on Biodegradable Copolymer of Chitosan and Polyvinyl Alcohol Support for Synthesis of Propionate Ester: Kinetic Approach Kirtikumar C. Badgujar and Bhalchandra M. Bhanage* Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai-400 019, India ABSTRACT: The objective of this work was to investigate a kinetic and mechanistic analysis for the synthesis of benzyl propionate which has diverse applications. Furthermore this study illustrates the synthesis and beneficial uses of an ecofriendly support that is made up of chitosan (CHI) and polyvinyl alcohol (PVA) to immobilize lipase Pseudomonas cepacia (PCL), and it showed remarkable enhancement of biocatalytic activity over free lipase. Among various biocatalysts prepared, CHI:PVA:PCL (5:5:2) was found as a robust biocatalyst composition. The lower activation energy (Ea) of immobilized lipase (12.8 ± 0.11 kcal/ mol) than that of free lipase (16.6 ± 0.65 kcal/mol) showed better catalytic activity of immobilized lipase. Kinetic parameters were determined which showed that benzyl propionate synthesis catalyzed by immobilized PCL followed the order bi−bi mechanism with benzyl alcohol inhibition. CHI/PVA immobilized lipase biocatalyst showed enhanced (3.5 fold) catalytic activity and efficient recyclability over those of free lipase. Finally, developed protocol was applied for practical biocatalytic applications to synthesize various industrially important propionate esters. thermal, mechanical, and operational stabilities.5,7,8 To overcome theses drawbacks, several advance immobilization protocols were developed which assist in improving the catalytic activity and chemical/solvent/thermal/operational stability.5,7,9 However, there is a scope to develop an ecofriendly immobilization protocol for practical biocatalysis; hence current study reports the development of a biocompatible, eco-friendly immobilization protocol. Recently, Sheldon et al.10 and May11 proposed that natural polymers have significant importance for immobilization and biocatalysis because of their ecofriendly character; furthermore such types of polymers are used widely for the membrane reactor and bioreactor coating. A range of natural polymers such as cellulose,12 carrageenan,13 alginate,13 β-glucan,14 poly(hydroxybutyrate),15 hydroxypropyl methyl cellulose,3 etc. were used in the forms of a bead or film for various enzyme immobilizations. In the present study, we have synthesized a hybrid polymeric matrix made up of CHI and PVA which is subsequently used for lipase immobilization and practical biocatalytic applications. Chitosan16 is a linear β-1,4-linked polysaccharide material possessing a number of useful features such as good adhesion, high mechanical strength, inertness to chemical reactivity, and lack of toxicity and biodegradability, all of which favor enzyme immobilization.10,11,16 PVA is widely used for biochemical and biomedical applications endowed with excellent film forming properties such as high interfacial adhesion, flexibility, high tensile strength, nontoxic, better resistivity to organic solvent, and biocompatibility.3,10,11

1. INTRODUCTION The organic esters are broadly used in pharmaceutical, flavorfragrance, cosmetic, toiletries, foods, beverage and various home-care products.1 Conventionally these esters are synthesized by using strong mineral acids or bases at higher temperature ranging from 100 to 200 °C.2,3 These conventional practices possess several drawbacks such as, use of hazardous chemicals, lower conversions−selectivity, reaction rate, requirement of corrosion proofing costly equipment, heavy downstream processes, biproduct formation, waste minimization, and environmental hazards.1−3 Meanwhile, the extraction methods used to extract esters from the natural sources also suffers from major drawbacks like poor yield and a large solvent requirement which tends to make the process uneconomical.1−3 These conventional techniques are inadequate for the bulk production of various food-flavors and pharmaceutical and home care products; which appeals to researchers to look for the “cleaner”, “safer”, “greener”, and more “ecofriendly” alternative for synthesis of these compounds.1−3 More recently, the American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACSGCIPR) was established to encourage innovations for synthesis and development of molecules using green chemistry and technology.4 Biocatalysis is one of the most useful alternative green tools which offers several advantages such as, high selectivity, mild reaction conditions, and environmental compatibility;3 furthermore esters produced via biocatalysis are labeled as “natural” and can be used “safely” without “chemophobia”.2,3 Lipases from the hydrolases family received wide industrial attention for practical biocatalysis; these possess the great potential and broad substrate array to carry various promiscuous organic reactions at mild conditions.5−8 However, practical use of free enzyme in biocatalysis is often hampered by industrial process economics because of poor catalytic, solvent, © XXXX American Chemical Society

Special Issue: Ganapati D. Yadav Festschrift Received: March 28, 2014 Revised: June 11, 2014 Accepted: June 12, 2014

A

dx.doi.org/10.1021/ie501304e | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Industrial & Engineering Chemistry Research

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crude lipase, and this composition was denoted as CHI:PVA:lipase (200:200:80) meaning CHI:PVA:PCL (5:5:2). 2.3. Protein Content and Lipase Activity Assay. The protein content was measured by the Bradford assay in set of triplicate using bovine serum albumin (BSA) as a standard reference to construct the calibration curve.15 At first, the initial (free) amount of lipase protein (PCL/ROL/MJL) used to load on the support was determined. After immobilization, the CHI/ PVA lipase matrix was removed and the Petri dish was rinsed to determine the nonimmobilized amount of protein. Thus, the amount of protein immobilized on the CHI/PVA support is the difference between the initial amount of protein used to load on the support and the nonimmobilized amount of protein found after washing of Petri dishes.15 Lipase activity of free and CH/PVA immobilized lipase was studied in triplicate through spectrophotometrically at 410 nm by hydrolysis of p-nitrophenyl acetate (p- NPA) with minor modification in reported procedure by Pencreacha and Baratti.21 In standard assay condition, the reaction mixture consists of 2 mg of free lipase (or equivalent quantity of the immobilized lipase) in 1 mL of iso-octane. The reaction was initiated by the addition of 1 mL of 15 mM, p-NPA dissolved in isopropanol solution as a substrate and incubated at 37 °C for 1 min. After a minute, 200 μL of reaction mixture was taken out and added to 800 μL of deionized water to extract p-nitro phenol in aqueous phase. Finally, 200 μL of potassium phosphate buffer solution (50 mM, pH 7.2) was added to above solution so that, p-nitro phenol extracted in the aqueous phase gives pale yellow color which was used to measure absorbance. The one unit (U) lipase activity was defined as micromoles of p-nitro phenol released per minute by hydrolysis of p-nitro phenyl acetate (p-PNA) by one milligram of lipase under given standard assay conditions at 37 °C. Furthermore, specific activity, percent protein immobilization, and percent lipase activity yield was determined by the following equations

Thus, the binary hybrid material of CHI and PVA is an attractive support for immobilization which incorporates the individual polymer properties in a single hybrid polymer.3,10,16 Furthermore, the method of preparation is very simple and the presence of free −OH and −NH2 groups offers a higher degree of immobilization; which makes a CHI:PVA support more attractive and ideal for lipase immobilization.5,9,10,16 This hybrid biocatalyst was then applied for synthesis of benzyl propionate which is recognized as a safe compound by the European and U.S. Food and Drug Administration.17 Benzyl propionate is a colorless to slightly pale yellow liquid with a fruity smell and is widely used as a fragrance ingredient in antiperspirant, decorative cosmetics, shower gel, shampoos, body lotions, toiletries, and noncosmetic products such as household cleaners and detergents.18 Naturally it occurs in the Michelia champaca, Prunus species, Tanacetum parthenium and Melon while used as a flavoring agent for almond, apple, banana, coconut, grape, cherry, strawberry, plum, etc.18,19 In 2008, the global estimated use of benzyl propionate ester was nearly 100 t/y.18,20 Moreover, the universal business for cosmetics and toiletries was estimated almost around 200 billion €; while for flavors-fragrances and beverages it was estimated nearly 20.3 billion USD.1 Thus, considering the extensive scope and importance of esters, we make an attempt to explore kinetic modeling for benzyl propionate synthesis using CHI:PVA based immobilized lipase as a biocatalyst, which has great scope in the scientific literature. In the present study, the influence of various reaction parameters for benzyl propionate synthesis was studied in detail. Furthermore, initial reaction rate, activation energy, various kinetic parameters {Vmax, Ki(BA), Km(BA), Km(VP)}, and possible mechanisms were proposed. In addition to this, operational and shelf life stability and the applicability of developed protocol were tested to synthesize the various industrially important propionate esters.

% protein immobilization

2. MATERIAL AND METHODS 2.1. Enzymes and Chemicals. Lipase PCL (Pseudomonas cepacia, activity ≥23 000 U/g) and lipase MJL (Mucor javanicus, activity ≥10 000 U/g) were gifted by Amano Enzymes (Japan). Lipase ROL (Rhizopus oryzae, activity ≥30 000 U/g) was purchased from the Fluka India Pvt. Ltd. CHI (Brookfield viscosity >200), PVA (Mw 9000−10 000), vinyl propionate (VP), and p-nitro phenyl acetate (p-PNA) were purchased from Sigma Aldrich Pvt. Ltd., India. Benzyl alcohol (BA), bovine serum albumin, and all other solvents/chemicals were bought from Hi-Media Pvt. Ltd., India. 2.2. Immobilization of the Lipase. Immobilization of lipase on the CHI/PVA matrix was carried out very simply in water as a green solvent at room temperature (25−30 °C). PVA (200 mg) was dissolved in distilled water (10−15 mL) while CHI (200 mg) was dissolved in distilled water (1% w/w acetic acid solution) in a separate beaker and stirred at 1200 rpm for 1 h. Each solution was filtered to remove undissolved particles. Finally, PVA solution was slowly added into the CHI solution and stirred vigorously for 4 h at 1500 rpm. After 4 h, native lipase (80 mg) dissolved in distilled water (2−3 mL) was added to the CHI/PVA blend and stirred gently at 160 rpm for 1 h. The immobilized blend was then carefully poured in a Teflon dish and allowed to dry at 44−45 °C for 48 h. A thin film of CHI:PVA:lipase was formed; which was then cut off into small pieces of 2−3 mm2 size and stored at 8−10 °C. Thus, theoretically 400 mg (0.4 g) support was loaded by 80 mg of

= (initial amount of protein − nonimmobilized amount of protein)/initial amount of protein

% lipase activity =

immobilized lipase activity initial (free) lipase activity

specific activity of free lipase free lipase activity = free amount of protein content specific activity of immobilized lipase immobilized lipase activity = immobilized amount of protein content

2.4. Experimental Set and Analysis. Benzyl propionate synthesis involves the addition of benzyl alcohol (2 mmol) and vinyl propionate (5 mmol) in 8 mL glass reaction vessel of 2 cm i.d. with a glass lid. The reaction mixture was diluted by isooctane to make up 3 mL. Later on, 54 mg of immobilized lipase CHI:PVA:PCL biocatalyst was added to initiate the reaction. The reaction was placed at 50 °C in an orbital shaker with an agitation speed of 160 rpm (Scheme 1). Reaction mixture samples of 10 μL were taken out at regular intervals and analyzed by using the Perkin-Elmer, Clarus- 400 gas chromatoB



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support and aggregation of enzyme is no longer possible after immobilization,7,15 while free enzyme may suffer from agglomeration and mass transfer diffusion in iso-octane solvent.15 Thus in all, the specific activity was also found to be higher for the immobilized lipase than free lipase follows series of lipase PCL > ROL > MJL. This also confirms that (i) immobilization on CHI/PVA support dramatically improves enzyme activity due to interfacial activation and (ii) CHI/PVA support is proficient for the immobilization of enzyme.15,21 3.2. Screening of Lipases from Various Sources. Initially lipase catalytic activity of various free and immobilized (PCL, ROL, MJL) lipases was tested for benzyl propionate synthesis under similar set of reaction conditions to find out best suitable biocatalyst (Scheme 1, Figure 1). It was observed

Scheme 1. Benzyl Propionate Synthesis Catalyzed by CHI/ PVA Immobilized Lipase

graph (GC) equipped with flame ionizing detector and capillary column. The oven temperature of gas chromatography was kept at 70 °C for 3 min with a rise of 10 °C min−1 up to 220 °C for 30 min. The products formed are confirmed by gas chromatography−mass spectroscopy (GC-MS, Shimadzu QP2010) analysis. 2.5. Recyclability of Immobilized Lipase. Immobilized lipase was subjected to determine the repetitive use for benzyl propionate synthesis. After completion of 2.5 h, the reaction mixture was filtered through the Whatman filter paper and the biocatalyst was rinsed three times with iso-octane solvent to remove traces of product or reactant if any. Later on, it was dried at 40 °C for 12−14 h and stored in plastic container at room temperature (25 °C) until it was used for the next recycle. 2.6. Shelf Life Stability Study. Various immobilized lipase batches were stored in airtight plastic containers at room temperature (25 °C). After regular time interval, the residual lipase activity was determined by lipase activity assay (as per section 2.3). The residual lipase activity was determined by comparison with the fresh run during each time which was considered as a control (100% activity). 2.7. Initial Rate Determination. The initial reaction rate was determined using a concentration vs time profile plot for the initial 25% conversion; these curves were plotted by using the polynomial graph and the corresponding derivative was equated to zero to obtain the initial reaction rate for the benzyl propionate synthesis.

Figure 1. Screening of lipases from various sources for synthesis of benzyl propionate (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg (or equivalent free lipase); temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

that free lipases MJL, ROL, and PCL gave 3, 8, and 28% conversion; while the immobilized lipase {CHI:PVA:lipase (5:5:2)} MJL, ROL, and PCL gave 11, 19, and 99% conversion for benzyl propionate synthesis respectively. Free lipases provided lower conversion may be attributed due to aggregation in nonaqueous media which was prevented after immobilization and showed significant improvement in catalytic activity up to 3.5 times (Figure 1).7 Thus in present study, CHI/PVA immobilized lipase PCL was found to be very effective and versatile biocatalyst in nonaqueous media while lipase ROL and MJL are good potential biocatalyst but here show lesser catalytic activity as compared to CHI/PVA immobilized PCL. 3.3. Influence of Lipase Loading on Support. The influence of the lipase PCL loading (in the range of 20−140 mg) on CHI:PVA polymer support (400 mg) was studied for

3. RESULTS AND DISCUSSION 3.1. Protein Content and Lipase Activity Assay. A protein content and lipase activity assay was performed to determine the immobilization of the protein on the support and to check enzyme catalytic efficiency after immobilization.15,21 The protein content for the free as well as immobilized lipase followed the sequence as lipase MJL > PCL > ROL; while lipase activity for the free as well as immobilized lipase followed the sequence as lipase PCL > ROL > MJL (Table 1). The percent protein immobilization was found to be more than 90% for all three immobilized lipases which showed that protein was successfully immobilized on the support.15 Furthermore, lipase activity was found be improved after immobilization of lipase (Table 1). This fact can be explained as follows; enzymes are well dispersed on the CHI/PVA

Table 1. Protein Content, Lipase Activity, and Specific Activity Study for Various Free and Immobilized Lipasesa protein loadedb

lipase activityc

specific activityd

sample

free or initial

immobilized

% protein immobilization

free or initial

immobilized

% lipase activity

free or initial

immobilized

1. MJL 2. PCL 3. ROL

41.67 ± 1.14 33.89 ± 0.29 27.45 ± 0.82

37.68 ± 0.60 31.39 ± 0.68 24.97 ± 1.03

90.43 ± 3.80 92.61 ± 1.23 90.94 ± 1.74

7.28 ± 0.37 37.68 ± 1.34 14.78 ± 0.58

8.367 ± 0.46 53.99 ± 0.23 19.03 ± 0.44

114.93 ± 4.07 143.29 ± 4.43 128.70 ± 2.31

0.174 ± 0.005 1.110 ± 0.04 0.538 ± 0.011

0.222 ± 0.01 1.720 ± 0.044 0.762 ± 0.019

Values are given as mean value ± standard deviation. bProtein loaded: μg/mg of support. cLipase activity: U/mg of support. dSpecific activity: U/ μg. a

C



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synthesis. However in the present study, it showed lower initial reaction rate and conversion (8%); this may be because acid acts as a potent inhibitor for enzyme activity.24 Alternatively, ester synthesis can be carried out using various esters such as methyl propionate or vinyl propionate as a propanoyl donor (Figure 3). When methyl propionate used as a donor, then it liberates the methyl alcohol as a side product which competes with benzyl alcohol for nucleophilic attack on carbonyl ester and inhibits the reaction rate to yield only 17% conversion.24 Moreover, the polar nature of the methyl alcohol disturbs the presence of the microaqueous system in the enzyme structure and causes a decrease in the enzyme activity and subsequent reaction rate.4,24 However to overcome these difficulties; vinyl propionate was used as a activated propanoyl donor which provided significant conversion of 99% in 2.5 h (Figure 3). When vinyl propionate is used as a propanoyl donor, then it liberates the vinyl alcohol as a side product which immediately tautomerized into the acetaldehyde and did not compete with benzyl alcohol for nucleophilic attack. 3.5. Influence of Rotatory Shaker Rotation. This is a micro-aqueous solid−liquid system in which CHI/PVA supported biocatalyst is in the solid phase while both reactants are in the liquid phase. Hence, it is essential to check the influence of mass transfer (Figure 4). It was observed that initial

the benzyl propionate synthesis to obtain robust biocatalyst composition (Figure 2). It was observed that as lipase PCL

Figure 2. Influence of lipase loading on support for synthesis of benzyl propionate (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

loading increases from 20 to 80 mg on CHI:PVA (200:200); the corresponding benzyl propionate conversion also increases. The lower catalytic activity at lower lipase loadings is believed to be due to the availability of the lesser catalytic sites to carry out the transformation; similar type of results were observed to Bosley and Peilow22 when lipase was immobilized on porous polypropylene support. Thus, the maximum conversion (99%) of the benzyl propionate was obtained when 80 mg lipase was loaded on 400 mg of CHI:PVA (200:200) immobilization support in 2.5 h. However further increase in the lipase loading from 80 to 140 mg showed decrease in the catalytic activity (Figure 2). The lower catalytic activity at higher lipase loadings is believed to be due to crowding of lipase on support which creates difficulty in diffusion of the substrate to the active site of enzyme.23 3.4. Influence of Propanoyl Donor. Various propanoyl donors were screened to obtain good conversion of propionate ester (Figure 3). It was observed that propanoic acid has been used conventionally as a good donor for propionate ester

Figure 4. Influence of rotatory shaker speed (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 70−190 rpm; time, 2.5 h).

reaction rate and percent conversion increases as rotation speed increases from 70 to 160 rpm while increase in speed from 160 to 170 rpm does not affect the initial reaction rate and conversion. A similar type of result was also found by Yadav and Trivedi25 for n-octyl acetate synthesis catalyzed by novozyme 435. However, further increases in reps per minute from 170 to 190 showed slight decrease in initial rate and conversion. It was observed that beyond 170 rpm, little polymer supported biocatalyst was thrown outside of the liquid phase reaction media and did not contribute to catalyzing the reaction which leads to decrease in the conversion (Figure 4). Thus optimum rotation speed was kept at 160 rpm, to carry further all kinetic experiments. 3.6. Influence of Reaction Media. Organic solvent for enzymatic reaction should be chosen in such a way that it should offer good solubility of substrate in reaction media without affecting the catalytic power of enzyme.7,9,10 Thus, in

Figure 3. Influence of propanoyl donor for synthesis of benzyl propionate (reaction conditions: benzyl alcohol, 2 mmol; propanoyl donor, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time 2.5 h). D



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relationship with the biocatalyst amount ranging from 12 to 54 mg (Figure 6A). The highest conversion (99%) was obtained when 54 mg of biocatalyst was used to carry out the transformation. Further, increase in the catalyst amount from 54 to 60 mg showed marginal increase in reaction rate while no effect was observed for the percent conversion which indicates the presence of more number of active catalytic sites than actual required.23,26 Figure 6A showed linear increment of initial rate and percent conversion with the amount of biocatalyst, which signify that reaction is kinetically controlled.27,28 Hence, 54 mg was found to be optimize biocatalyst amount and utilized further to carry all kinetics experiments. The sensitivity of initial reaction rate to the amount of biocatalyst is given by a powerlaw model equation (Figure 6B).28

the present study various hydrophilic and hydrophobic solvents were screened and results showed that, hydrophobic solvent such as iso-octane has good compatibility with the enzymes and providing excellent conversion of 99% in 2.5 h (Figure 5). This

V = kE1[E]kE2

(1)

Where, V is the initial rate of reaction, [E] is the amount of biocatalyst, kE1 is the kinetic constant, and kE2 is the order of reaction; eq 1 can be rearranged as follows in eq 2a, Figure 5. Influence of reaction media (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

ln(V ) = kE2 ln[E] + ln(kE1)

(2a)

y = 0.9804x − 9.168

(2b)

Equation 2b was obtained by plotting a graph (Figure 6B), thus, by comparing eqs 2a and 2b, we can deduce a relationship of initial rate for a given amount of biocatalyst as

was attributed due to better uphold ability of microaqueous system in hydrophobic solvents which maintain the conformational mobility of enzyme molecules to perform catalytic activity.7,25 This microaqueous system around the enzyme may distort in the hydrophilic solvent which leads to yield poor conversion in tetrahydrofuran (75%), acetone (69%), methyl tert-butyl ether (62%), and methylene dichloride (56%). The present study indicates that prepared CHI/PVA biocatalyst adapted to show the moderate catalytic activity in various hydrophilic solvents also (Figure 5); finally, iso-octane was selected as a solvent of choice and used for further biocatalytic experiments.25 3.7. Influence of Biocatalyst Amount. The amount of biocatalyst is a crucial parameter by industrial economical point of view;23 therefore the effect of immobilized biocatalyst amount was studied in the range of 12−60 mg. It was observed that initial reaction rate and percent conversion showed a linear

V = 0.1043 × 10−3[E]0.98

(3)

The above model was valid within the range of 12−54 mg of biocatalyst loading at the given reaction conditions, while the model showed that the immobilized lipase catalyzed reaction is first order (order of reaction is 0.98 ≈ 1).28 3.8. Influence of the Molar Quantity of Reactant. In a kinetic modeling study, it is imperative thing to find out the influence of any substrate which may causes possible inhibition to the enzyme activity.28 Hence, various experiments were performed to determine the effect of substrate molar quantities on enzyme activity.29 Initially molar quantity of benzyl alcohol was kept constant (2 mmol) while the molar quantity of vinyl propionate was varied from 1 to 6 mmol; under similar reaction conditions. Experimentally it was found that initial reaction rate and conversion increased significantly with increase in the molar quantity of vinyl propionate from 1 to 5 mmol (Figure

Figure 6. (A) Influence of biocatalyst amount on initial rates and percent conversion. (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 12−60 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h). (B) Determination of relation of initial rate versus biocatalyst amount. E



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acetate synthesis catalyzed by C. rugosa lipase immobilized on a polyvinylidene fluoride membrane. 3.9. Influence of Temperature. It is well reported in the literature that immobilization may improve the thermal stability of enzyme;5 but still, it is obligatory to find the optimal temperature as enzymes are easily susceptible to thermal denaturation.9,10,26,31 Furthermore, temperature is a crucial key parameter as it assists to solubilize reactants in reaction media and boost the molecular-collision interface;26−31 hence, a number of experiments were performed to find out the optimal temperature under similar reaction conditions. It was observed that the initial reaction rate and conversion gradually increases with increase in temperature from 30 to 50 °C (Figure 9). This

7); while further increases in molar quantity from 5 to 6 mmol showed insignificant improvement in the conversion. Thus,

Figure 7. Influence of molar quantity of vinyl propionate (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 1−6 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

vinyl propionate worked as an activated propanoyl donor species, signifying no inhibitory action within studied range (Figure 7). A similar type of effect was seen by Kuo et al.29 for use of vinyl acetate during phenylethyl acetate synthesis. In another set of experiments the molar quantity of vinyl propionate was kept constant (5 mmol) while the molar quantity of benzyl alcohol was varied from 1 to 5 mmol, under otherwise similar reaction conditions. Here, it was observed that initial rate and conversion increased moderately when the molar quantity of benzyl alcohol was changed from 1 to 2 mmol (Figure 8); further increase in the benzyl alcohol molar

Figure 9. Influence of temperature (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 30−55 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

was due to reason that as temperature increases the molecular collision interaction increases which causes decrease in the energy barrier between reacting molecule and enzyme− substrate complex formation; this decrease in energy barrier causes improvement in reaction rate and conversion.28 However, further increase in the temperature from 50 to 55 °C showed increase in the initial reaction rate while percent conversion (99%) was found to be constant (Figure 9). Thus, to keep the minimum possible temperature, we selected 50 °C as optimal temperature and used it for all further experiments. The Arrhenius plot was made to calculate the apparent activation energy (Ea) on the basis of ln(initial rates) versus reciprocal of temperature in the range of 30−50 °C.27,31,32 The Ea for benzyl propionate synthesis catalyzed by the free and immobilized lipase was found to be 16.6 ± 0.65 and 12.8 ± 0.11 kcal/mol which lies in between the range of enzymatic reactions (Figure 10).28,31 The higher Ea of free lipase was attributed due to difficulty in mass transfer, as free lipase leads to agglomerate in the organic media; while lower Ea was observed with immobilized enzymes usually indicates easy mass transfer (diffusion) of substrate, since the catalytic sites are easily accessible to substrate after immobilization of enzyme on support matrix. Thus, the lower Ea value of immobilized lipase indicates better catalytic efficiency and lesser energy requirement as compared to free lipase for corresponding benzyl propionate synthesis.27,28,32,33 3.10. Influence of Reaction Time and Comparison of Immobilized Lipase to Free Lipase. Immobilized lipase

Figure 8. Influence of molar quantity of benzyl alcohol (reaction conditions: benzyl alcohol, 1−5 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

quantity from 2 to 5 mmol showed steady decrease in the reaction rate and conversion. The decrease in percent conversion and initial reaction rate was attributed to the inhibitory effect of the benzyl alcohol which may form a deadend inhibition complex at higher concentration;28,29 furthermore the polar nature of alcohol disturbs the enzyme microaqueous system (at higher concentration) which causes inhibition of enzyme activity. A similar type of alcohol inhibition was observed to Kuo et al.29 for 2-phenylethyl F



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decrease of initial rate at higher concentration of benzyl alcohol which signifys that benzyl alcohol acts as an inhibitor;25,28,29,31 this fact was also supported by study of influence of the molar quantity of benzyl alcohol (Figure 8). Now, by considering the initial rate, here we scrutinized model which exactly suitable for given experimental data. Clenden notation was used to show the steps involves in the ping pong bi−bi model with inhibition of benzyl alcohol as follows:28

Figure 10. Arrhenius plot for free and immobilized PCL lipase (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 30−50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

E + VP ⇄ E−VP

(4)

E−VP ⇄ E* + VA

(5)

E* + BA ⇄ E*−BA

(6)

E* − BA ⇄ E + BP

(7)

E + BA ⇄ E−BA

(8)

The equation obtained for above mechanism is

provided 99% conversion in 2.5 h while after prolonged reaction time (4 h), it was found that conversion of the desired product was not altered showing that reaction was performed in forward direction.3,28 However free enzyme provided only 28% conversion of desired product in 2.5 h; while percent conversion is linearly increases with the time and gave 38% conversion after 4 h (data not shown). This observation showed enhanced catalytic activity of the CHI/PVA immobilized lipase (3.5 fold) as compared to free lipase. 3.11. Kinetic Modeling and Determination of Reaction Mechanism. Kinetic modeling and mechanistic study of a reaction are very important aspects for the reactor designing and to scale up the process.28 Lipase catalysis involving two substrates reaction basically follows order bi−bi or a ping pong bi−bi mechanism.28,29 However, in the literature various forms of order bi−bi or ping pong bi−bi models have been proposed involving inhibition of one or both substrates.24−32,34 In the present kinetic modeling study, various kinetic parameters for benzyl propionate synthesis were determined using the initial rate data. The Lineweaver−Burk graph was constructed by plotting the reciprocal of initial rates at different concentrations of benzyl alcohol (Figure 11). The Lineweaver−Burk plot showed

V=

Vmax[BA][VP] K m(BA)[VP](1 + [VP]/K i(BA)) + K m(VP)[BA] + [BA][VP] (9)

The steps involves in the order bi−bi model with inhibition of benzyl alcohol as follows:28 E + VP ⇄ E−VP

(10)

E−VP + BA ⇄ E−VP−BA

(11)

E−VA−BP ⇄ E−VA + BP

(12)

E−VA ⇄ E + VA

(13)

E + BA ⇄ E−BA

(14)

The equation obtained for above order bi−bi mechanism is V=

Vmax[BA][VP] K i(BA)K m(VP) + K m(BA)[VP] + K m(VP)[BA] + [BA][VP] (15)

Where, E = enzyme, VP = vinyl propionate, BA = benzyl alcohol, BP = benzyl propionate, VA = vinyl alcohol, E* = activated enzyme, V = initial rate of reaction; Vmax = maximum rate of reaction; [BA] = initial concentration of benzyl alcohol; [VP] = initial concentration of the vinyl propionate; Km(BA) and Km(VP) = Michaelis−Menten constant of benzyl alcohol and vinyl propionate; Ki(BA) = inhibitory constant of benzyl alcohol. The kinetic parameters indicated in Table 2 were scrutinized by the nonlinear regression analysis using software package Table 2. Various Kinetic and Statistical Parameter for the Benzyl Propionate Synthesis Catalyzed by CHI/PVA Immobilized Lipase PCL entry 1 2 3 4

Figure 11. Lineweaver−Burk plot (reaction conditions: benzyl alcohol, 2−5 mmol; vinyl propionate, 1−5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

5 6 7 G

kinetic/statistical parameters

value (order bi−bi model)

Various Kinetic Parameters Vmax 0.03368 (mmol mL−1 min−1) 1.07446 Km(BA) (mmol mL−1) Km(VP) (mmol mL−1) 0.67467 Ki(BA) 5.16464 Various Statistical Parameters R2 0.999807 rmsd 8.58 × 10−6 SSE 1.88 × 10−9

values (ping pong bi− bi model) 0.016284 −0.22365 2.661757 160.5203 0.3715439 0.004836 5.84 × 10−6



Article

polymath 6.0 version; the Levenberg−Marquardt (LM) method (function of polymath) was used for nonlinear regression analysis. The kinetic parameter values from Table 2 showed that the order bi−bi mechanism was well fitted and stable for the benzyl propionate synthesis while the bi−bi ping pong model gave some negative parameters which are unrealistic and hence ruled out. According to the order bi−bi model, the value of the Michaelis−Menten constant was found to be lower for vinyl propionate than benzyl alcohol (Km(VP) < Km(BA)) which signifies higher affinity of the vinyl propionate toward the immobilized lipase for formation of the propanoyl−enzyme complex.28 Ki for the benzyl alcohol was found to be 5.16; a similar type of Ki value was found by Yadav and Pawar31 for the enantioselective resolution. Furthermore, statistical parameters showed a very small sum of squared error (SSE) and root means square deviation (rmsd) for order bi−bi mechanism which indicates a good fit of the order bi−bi model (Table 2). The parity plot (graph not shown) of the experimental versus theoretical rate gave an excellent correlation coefficient (R2 = 0.99) which demonstrated that the proposed model is valid for the given system. According to the order bi−bi mechanism, initially vinyl propionate bound to immobilized lipases (because of higher affinity of vinyl propionate)28 to form a vinyl propanoyl− enzyme complex (E−VP). Later on benzyl alcohol combined with the E−VP complex to form a ternary complex (E−VP− BA) which isomerized into new ternary complex (E−VA−BP). This isomerized ternary complex was broken down into desired ester benzyl propionate (BP); while the binary complex (E-VA) of enzyme subsequently releases enzyme (E) and vinyl alcohol (VA). This vinyl alcohol (VA) was tautomerizes to low-boilingpoint acetaldehyde (20.2 °C), which cannot acts as a further substrate for enzyme and thus shifting the equilibrium toward desired ester formation.35 However at higher concentration of benzyl alcohol, a binary inhibition complex (E−BA) was formed in between the enzyme and benzyl alcohol instead of enzyme and vinyl propionate which reduces the enzyme activity and subsequent rate of reaction.28 3.12. Influence of Catalyst Recyclability. The major benefit of immobilization is the recyclability which is an essential factor in making the process economically and practically feasible;5,8,10 hence, immobilized lipase was subjected for the recyclability study under similar reaction conditions. The catalyst recyclability was studied up to the four cycles, which provided a conversion of 72% at the end of four cycles in 2.5 h. The decrease in conversion may be caused by inactivation of enzyme by continuous exposure to the alcoholic substrate in each cycle and somewhat due to leaching of the enzyme from the support.5,7,28,29 In comparison to the immobilized lipase, the fresh free lipase provided only 28% conversion without any reuse which showed the effectiveness of the immobilization on polymer support (Figure 12). 3.13. Shelf Life Stability. Shelf life stability at a given temperature is one of the key issues for biological and industrial application. The activity assay showed that there was marginal decrease in the lipase activity up to 4% in a period of four months when it was stored at 25 °C; which indicated remarkable shelf life stability of the immobilized lipase within the studied period of 4 months. Thus, the shelf life stability study also confirms that enzyme and immobilization support interaction is capable to maintain the stability and confirmation of the enzyme proteomic structure (Table 3).36,37

Figure 12. Recyclability of immobilized lipase PCL (reaction conditions: benzyl alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg/ free lipase loading 10 mg; temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h).

Table 3. Shelf Life Stability Study of the Immobilized Lipase When Stored in an Air Tight Plastic Container at 25 °C entry

time interval (months)

% residual lipase activity

1 2 3 4 5

fresh 1 2 3 4

100 99.1 ± 0.24 98.41 ± 0.45 97.83 ± 0.92 95.94 ± 1.02

Experiments were performed in triplicate, and values were given as mean value ± standard deviation.

3.14. Influence of Substrate Moieties. The practical feasibility of the developed protocol was tested for synthesis of numerous propionate esters using immobilized lipase PCL as a biocatalyst (Figure 13). The conversion of short chain alcohol such as butyl and isobutyl alcohol was hampered little due to the polar nature of alcohol which provided moderate conversion of 83 and 74% for the corresponding product respectively in 2.5 h.24,25,32 Phenethyl and furfuryl alcohol showed similar reactivity like benzyl alcohol to give 99% conversion in 2.5 h. Citronellol provided moderate conversion of 86% in 2.5 h; this may be attributed due to long hydrophobic tail of citronellol which resided in the vicinity of enzyme due to hydrophobic−hydrophobic interaction and tends to obstruct the enzyme activity.27 Cyclohexyl alcohol is a secondary alcohol which showing lower reactivity because of the steric hindrance at the α-carbon which leads to provide poor conversion (30%) of desired product in 2.5 h.38 In a similar way, steric hindrance and acidic nature of phenol greatly hamper the enzyme activity to give lesser conversion (18%) of phenyl propionate 2.5 h.38

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CONCLUSION In conclusion, lipase PCL was successfully immobilized on the CHI/PVA based ecofriendly support. Lipase activity and protein content analysis showed that CHI/PVA support was efficient for the immobilization. This immobilized biocatalyst was then applied for the practical biocatalytic application to perform synthesis of benzyl propionate. The biocatalytic application evaluates the enhancement of immobilized enzyme catalytic activity (3.5-fold) over that of free lipase. The influence of reaction parameters such as propanoyl donor, H



Article

Figure 13. Influence of various alcoholic substrate moieties for synthesis of propionate esters (reaction conditions: alcohol, 2 mmol; vinyl propionate, 5 mmol; solvent iso-octane up to 3 cm3; immobilized biocatalyst 54 mg, temperature 50 °C; rotatory shaker speed, 160 rpm; time, 2.5 h). lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide. J. Biotechnol. 2011, 156, 48−51. (4) Pelt, S. V.; Teeuwen, R. L. M.; Janssen, M. H. A.; Sheldon, R. A.; Dunn, P. J.; Howard, R. M. Pseudomonas stutzeri lipase: a useful biocatalyst for aminolysis reaction. Green Chem. 2011, 13, 1791−1798. (5) Hanefeld, U.; Gardossi, L.; Magner, E. Understanding enzyme immobilisation. Chem. Soc. Rev. 2009, 38, 453−468. (6) Saibi, W.; Abdeljalil, S.; Masmoudi, K.; Gargouri, A. Biocatalysts: Beautiful creatures. Biochem. Biophys. Res. Commun. 2012, 426, 289− 293. (7) Adlercreutz, P. Immobilisation and application of lipases in organic media. Chem. Soc. Rev. 2013, 42, 6406−6436. (8) Sharma, R.; Chisti, Y.; Banerjee, U. C. Production, purification, characterization and application of lipases. Biotech Adv. 2001, 19, 627− 962. (9) Matero, C.; Palomo, J. M.; Fernandez-Lorente, G.; FernandezLafuente, R.; Guisan, J. M. Improvement of enzyme activity, stability and selectivity via immobilization technique. Enzyme Microb. Technol. 2007, 40, 1451−1463. (10) Sheldon, R.; Pelt, S. V. Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 2013, 42, 6223−6235. (11) May, S. W. New applications for biocatalysts. Curr. Opin. Biotechnol. 1997, 8, 181−186. (12) Pronk, W.; Boswinkel, G.; Riet, K. V. Parameters influencing hydrolysis kinetics of lipase in a hydrophilic membrane bioreactor. Enzyme Microb. Technol. 1992, 14, 214−220. (13) Elnashar, M. M. M.; Mostafa, H.; Morsy, N. A.; Awad, G. E. A. Biocatalysts: isolation, identification, and immobilization of thermally stable lipase onto three novel biopolymeric supports. Ind. Eng. Chem. Res. 2013, 52, 14760−14767. (14) Vaidya, B. K.; Singhal, R. S. Use of insoluble yeast β-glucan as a support for immobilization of Candida rugosa lipase. Colloids Surf. B. 2008, 61, 101−105. (15) Mendes, A. A.; Oliveira, P. C.; Vélez, A. M.; Giordano, R. C.; Giordano, R. L. C.; de Castro, H. F. Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis. Int. J. Biol. Macromol. 2012, 50, 503−511. (16) Macquarrie, D. J.; Hardy, J. J. E. Applications of Functionalized Chitosan in Catalysis. Ind. Eng. Chem. Res. 2005, 44, 8499−8520. (17) Food Additives Permitted for Direct Addition to Food for Human Consumption. Code of Federal Regulations, Part 172, Title 21, Volume 3, Chapter I; 21CFR172.515, 2013. (18) McGinty, D.; Letizia, C. S.; Api, A. M. Fragrance material review on benzyl propionate. Food Chem. Toxicol. 2012, 50, S486−S490. (19) http://www.thegoodscentscompany.com/data/rw1001772.html (accessed Mar 24, 2014).

substrate concentration, catalyst loading, reaction media, temperature, rotatory shaker speed, and reusability were studied systematically. In addition to this, a power law model was deduced to show the correlation between biocatalyst amount and initial rate; in addition to this, it showed that the reaction is first order. Furthermore, the activation energy (Ea) for free as well as immobilized lipase was determined, showing the better catalytic efficiency of the immobilized lipase. Various reaction kinetic parameters were scrutinized using nonlinear regression analysis for bi−bi ping pong and order bi−bi mechanisms. A kinetic model indicated that reaction followed the order bi−bi mechanism with inhibition by the benzyl alcohol; furthermore, there was an excellent agreement between the experimental and theoretical data which indicated the validity of proposed model. The CHI/PVA immobilization protocol is recyclable and showed excellent shelf life stability. The developed CHI/PVA immobilization protocol showed ease of use for the wide substrate array to synthesize various industrially important propionate esters.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: + 91- 22 3361 2601/2222. Fax: +91- 22 2414 5614. Email address: [email protected], bm.bhanage@ ictmumbai.edu.in. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.C.B. is greatly thankful for the CSIR (Council of Scientific and Industrial Research, India) and DBT (Department of Biotechnology, India) for providing a fellowship and research grant (BT/PR4192/PID/6/623/2011).

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REFERENCES

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Enhanced Biocatalytic Activity of Lipase Immobilized on

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