Solvent stability study with thermodynamic analysis and superior biocatalytic activity of Burkholderia cepacia lipase immobilized on biocompatible hybrid matrix of poly(vinyl alcohol) and hypromellose

Article abstract of DOI:10.1021/jp5093493

In the present study, we have synthesized a biocompatible hybrid carrier of hypromellose (HY) and poly(vinyl alcohol) (PVA) for immobilization of Burkholderia cepacia lipase (BCL). The immobilized biocatalyst HY:-PVA:BCL was subjected to determination of half-life time (τ) and deactivation rate constant (K_D) in various organic solvents. Biocatalyst showed higher τ-value in a nonpolar solvent like cyclohexane (822 h) as compared to that of a polar solvent such as acetone (347 h), which signifies better compatibility of biocatalyst in the nonpolar solvents. Furthermore, the K_D-value was found to be less in cyclohexane (0.843 × 10^-3) as compared to acetone (1.997 × 10^-3), indicating better stability in the nonpolar solvents. Immobilized-BCL (35 mg) was sufficient to achieve 99% conversion of phenethyl butyrate (natural constituent of essential oils and has wide industrial applications) using phenethyl alcohol (2 mmol) and vinyl butyrate (6 mmol) at 44 °C in 3 h. The activation energy (E_a) was found to be lower for immobilized-BCL than crude-BCL, indicating better catalytic e fficiency of immobilized lipase BCL. The immobilized-BCL reported 6-fold superior biocatalytic activity and 8 times recyclability as compared to crude-BCL. Improved catalytic activity of immobilized enzyme in nonpolar media was also supported by thermodynamic activation parameters such as enthalpy (ΔH?), entropy (ΔS?) and Gibb 's free energy (ΔG?) study, which showed that phenethyl butyrate synthesis catalyzed by immobilized-BCL was feasible as compared to crude-BCL. The present work explains a thermodynamic investigation and superior biocatalytic activity for phenethyl butyrate synthesis using biocompatible immobilized HY:PVA:BCL in nonaqueous media for the first time. (Graph Presented).

Full text of DOI:10.1021/jp5093493

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Article
The Solvent Stability Study with Thermodynamic Analysis and Superior
Biocatalytic Activity of Burkholderia cepacia Lipase Immobilized on
Biocompatible Hybrid Matrix of Polyvinyl Alcohol and Hypromellose
Kirtikumar Chandulal Badgujar, and Bhalchandra M. Bhanage
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp5093493 • Publication Date (Web): 04 Dec 2014
Downloaded from http://pubs.acs.org on December 14, 2014
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The Solvent Stability Study with Thermodynamic Analysis and Superior Biocatalytic
Activity of Burkholderia cepacia Lipase Immobilized on Biocompatible Hybrid Matrix of
Polyvinyl Alcohol and Hypromellose
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*
Kirtikumar C. Badgujar and Bhalchandra M. Bhanage
Department of Chemistry, Institute of Chemical Technology, Mumbaiꢀ 400 019, India
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Corresponding author. Tel.: + 91ꢀ22 33612601/2222; Fax: +91ꢀ2224145614.
Eꢀmail address: bm.bhanage@gmail.com, bm.bhanage@ictmumbai.edu.in
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ABSTRACT
In the present study, we have synthesized biocompatible hybrid carrier of Hypromellose (HY)
and polyvinyl alcohol (PVA) for immobilization of Burkholderia cepacia lipase (BCL). The
immobilized biocatalyst HY:PVA:BCL was subjected to determination of halfꢀlife time (τ) and
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deactivation rate constant (K ) in various organic solvents. Biocatalyst showed higher τꢀvalue
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in nonꢀpolar solvent like cyclohexane (822 h) as compared to that of polar solvent such as
acetone (347 h), which signifying better compatibility of biocatalyst in the nonꢀpolar solvents.
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D
Furthermore, the K ꢀvalue was found to be lesser in cyclohexane (0.843×10 ) as compared to
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acetone (1.997×10 ) indicating better stability in the nonꢀpolar solvents. ImmobilizedꢀBCL (35
mg) was sufficient to achieve 99 % conversion of phenethyl butyrate (natural constituent of
essential oils and has wide industrial applications) using phenethyl alcohol (2 mmol) and vinyl
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butyrate (6 mmol) at 44 C in 3h. The activation energy (Ea) was found to be lower for
immobilizedꢀBCL than crudeꢀBCL, indicating better catalytic efficiency of immobilized lipase
BCL. The immobilizedꢀBCL reported sixꢀfold superior bioꢀcatalytic activity and 8 times
recyclability as compared to crudeꢀBCL. Improved catalytic activity of immobilized enzyme in
nonꢀpolar media was also supported by thermodynamic activation parameters such as enthalpy
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(
ꢁH ), entropy (ꢁS ) and Gibb’s free energy (ꢁG ) study; which showed that phenethyl
butyrate synthesis catalyzed by immobilizedꢀBCL was feasible as compared to crudeꢀBCL.
Present work explains thermodynamic investigation and superior biocatalytic activity for
phenethyl butyrate synthesis using biocompatible immobilized HY:PVA:BCL in nonꢀaqueous
media for the first time.
KEY WORDS: Thermodynamic activation study; Enzyme catalysis; Superior catalytic
activity; Solvent activityꢀstability
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1.
INTRODUCTION
The enzyme lipase {triꢀacyl glycerol hydrolases, (E.C. 3.1.1.3)} has received special attention as
biocatalyst due to wide substrate range and ability to carry out various promiscuous
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transformations in various reaction media. However due to proteomic nature, enzymes are
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quite sensitive, unstable and poorly soluble in organic solvents which limits their direct
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appliances as a potential biocatalyst. Besides this, application of the free/crude enzyme has
several drawbacks such as recyclability issue, lower stability to temperature and organic reaction
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media. To overcome these shortcomings, enormous efforts have been taken by various
researchers to enhance the activityꢀstability of enzymes with the help of skilful immobilization
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technique.
Among these various immobilization methodologies; the application of beads/
polymer matrices as an immobilization carrier has received a special importance because of their
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easy separation (recovery) from organic reaction media to reuse.
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Recently, Cantone et al. and May proposed that, use of natural polymeric material is an
attractive way and receiving increasing importance for enzyme immobilization due to extended
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application in membrane bioreactor designing.
Additionally, biodegradable polymer for
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immobilization fulfils the ecoꢀfriendly and greener aspect for biocatalysis.
For this
purpose, various natural (i) carbohydrates polymers such as cellulose, starch, dextran, agarose,
βꢀglucan and chitin or (ii) proteins such as albumin and gelatine or (iii) biocompatible polymers
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such as poly vinyl alcohol, poly hydroxyl butyrate and poly lactic acid preferably were used.
In recent year, cellulose and their derivatives are extensively used in biochemical and medical
sciences, mainly because of potential application and properties like biocompatibility and
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biodegradability.
HY is cellulosic water soluble polysaccharide composed of hundreds
of βꢀ1ꢀ4 linked glucose unit which possesses excellent film forming properties, flexibility,
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texture, biodegradability and stability. Furthermore, the presence of free OHꢀgroups in HY
provided enhanced immobilization effect. PVA is used widely as biopolymer in biomedical
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sciences and tissue engineering because of excellent properties such as, flexibility,
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biocompatibility nonꢀtoxicity and adhesiveness. Herein, we make an attempt to immobilize the
lipase BCL on a biodegradable HYꢀPVA blended biocompatible carrier; which will contribute
an individual advantages in a single film. In present study, an entrapment method was used to
immobilize the lipase BCL since, entrapment of the enzyme within support matrix was carried
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out at the time of support HYꢀPVA matrix formation. The entrapment immobilization
methodology has several benefits such as (i) it is a simple and nonꢀtedious method which is
usually free from the use of crossꢀlinking chemical reagent and multistep immobilization
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process; (ii) entrapment methodology provide protection to enzymes from direct exposure of
the outer environment (chemical/ solvent/ temperature) which tends to enhance stability and
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activity of biocatalyst in reaction media;
(iii) moreover entrapment reduces the direct
leakage of the enzyme from the support matrix which tends to improve the recyclability of the
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biocatalyst. Thus, it is likely that the lipases not only entrapped within the polymer matrix, but
also form an additional multipoint interaction within the matrix through Hꢀbonding, ionic, and
hydrophobic interactions which tend to produce an efficient immobilization and consequently a
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stable robust biocatalyst.
The figure 1 showing diagrammatic illustration of immobilization
mechanism via entrapment methodology. Thus, in present study immobilization of lipase was
carried out into matrix of HY:PVA at the time of matrix formation in water as a greener media;
this developed HY:PVA:BCL biocatalyst was then applied for practical biocatalytic applications
and thermodynamic investigation to synthesize phenethyl butyrate.
Please insert here Figure 1
Phenethyl butyrate is a colourless liquid having pleasant fruity smell; it is an essential
constituent of Pelargonium graveolens, Narcissus sp. Fragaria vesca, Michelia champaca,
passiflora edulis, etc. It has fresh fruity jasmine like smell and can be used as a foodꢀflavouring
agent for apricot, banana, bayberry, cherry, berry, jasmine, kiwi, pineapple, rose, vanilla,
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strawberry, etc. In addition to this, Phenethyl butyrate is broadly used in decorative cosmetics,
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bodyꢀlotions, faceꢀcreams, fragrances, shampoos, soaps, showerꢀgels and other toiletries.
Phenethyl butyrate was included in the list of grantedꢀA substances by the Council of Europe for
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safe use in foodstuffs; moreover its estimated production is metric of tons per year. These
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valuable esters are commercially obtained by conventional chemical synthesis at higher
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temperature using concentrated acid/base or by natural extraction from the plant resources.
However, both these methods having several drawbacks and discourages production of these
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esters which are extensively utilized in houseꢀhold and food products. In recent time, the
American Chemical Society Green Chemistry Institute pharmaceutical round table (ACSꢀ
GCIPR) was set up to encourage innovations for synthesis of valuable molecules using
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sustainable green chemistry and technology.
If theses esters are obtained through biological
routes (e.g., microbial bioꢀconversion of natural precursors or bioꢀcatalysis), then the resulting
esters can be labelled as “Natural”; furthermore, chemoꢀphobia attitude of the customer is also
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avoided for the houseꢀhold products which tagged them as "Green Product".
The worldwide flavour and fragrance in food market was worth to USD 22 billions in 2011; and
figure is projected to exceed USD 30 billions upto 2017, with a compounded annual growth rate
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CAGR) of 5.6 %. Furthermore, the overall global market for the cosmetics and toiletries was
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contributed at about 200 billion €. Thus, bearing in mind the goal of wide scope and
applications of these compounds, we explored an enzymatic synthesis of phenethyl butyrate
using biodegradable HY:PVA:BCL as biocatalyst, which tag it as “Clean, Green and Natural
product”. Till date, (i) HY:PVA immobilization support for lipase BCL and (ii) its study for
biocatalytic application with thermodynamic analysis has not been reported in scientific
literature and hence finds a great scope. Thus, in the present article; we have analyzed
≠
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thermodynamic parameters of activation (ꢁH , ꢁS and ꢁG ) in various solvents for phenethyl
butyrate synthesis. Besides this, various reaction parameters for phenethyl butyrate synthesis,
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, D
activation energy (Ea), half life time (τ) deactivation rateꢀconstant (K ) for various solvents
were determined. In addition to this, recyclability and applicability of developed immobilization
protocol were tested for synthesis of various commercially/ industrially essential butyrate esters.
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2.
.1
MATERIALS AND METHODS
Enzymes and chemicals
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Lipase Burkholderia cepacia (lipase BCL, ≥ 30,000 U/g), Rhizopus oryzae (lipase ROL, ≥
0,000 U/g) and Rhizopus niveus (lipase RNL, ≥ 1500 U/g), PVA, HY, pꢀnitrophenyl acetate (pꢀ
3
NPA), Bovine serum albumin, Bradford reagent and all other chemicals were purchased from
SigmaꢀAldrich Pvt. Ltd, India.
2.2
Immobilization of lipase
Preparation of hybrid support matrix film of HY and PVA was carried out in water as a greener
immobilization media and subsequently lipase BCL was immobilized at the time of HY:PVA
matrix formation. The HY (0.2 g) and PVA (0.2 g) was dissolved in 20 mL distilled water in
separate beaker. Both the solutions were stirred for 60 minutes at 1200 rpm; later on both
solutions were mixed together and allowed to stir for 3 h at 1300ꢀ1400 rpm. After 3 h, the native
lipase (0.1 g) was dissolved in 2ꢀ4 mL of distilled water which was added to above
immobilization matrix solution. It was then moderately stirred for 120 rpm for 1 h. Finally,
immobilized matrix (HY:PVA:BCL) was carefully poured in a Teflon dish and allowed to dry at
35ꢀ40°C for 40 h. A thin film of immobilized lipase BCL was formed which was then cut into
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small pieces of 2ꢀ4 mm size and stored at 3ꢀ5°C.
2.3
Determination of lipase activity and protein content of protein binding yield
The lipase activity of crude and various HY:PVA immobilized lipase was studied in triplicate
by spectrophotometrically at 405 nm by hydrolysis of pꢀnitro phenyl acetate (pꢀNPA) with
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slight modification in reported procedure by Pencreac’h and Baratti . In the standard assay
condition, reaction mixture consists of 2 mg of crude lipase (or equivalent quantity of the
HY:PVA:immobilized lipase) in 1 mL of nꢀheptane solvent. The reaction was started by
addition of 1 mL of 12 mM, pꢀNPA dissolved in 2ꢀpropanol solution as an enzyme substrate
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and incubated at 40 C for 10 minutes. Later on, 300 ꢂl of reaction mixture was taken out and
added to 700 ꢂl of deionised water to extract pꢀnitro phenol (pꢀNP) in aqueous phase. Finally,
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00 ꢂl of potassium phosphate buffer solution of pH 8.0 was added to above solution so that,
extracted pꢀNP give pale yellow colour which was used to measure the absorbance at 405 nm.
The lipase activity was described as µmole of pꢀNP released per minute by per mg of the
lipase under the prescribed standard assay condition.
The protein content of the crude lipase or protein binding yield for the immobilized lipase
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was determined by the Bradford method at 595 nm. A detailed procedure is as follow: At
first, initial protein content was determined from crude/free lipase which was used to
immobilize into support. Finally after complete removal of immobilized lipase matrix from
petriꢀdish; the petriꢀdish was rinsed and subjected for determination of unꢀimmobilized
amount of protein. Therefore, the amount of protein immobilized on PVA:HY support was
the difference between the initial (crude) protein content used to load and unꢀimmobilized
amount of protein found after washing petri dishes. BSA was used to construct the internal
standard calibration curve; subsequently, % lipase activity, % protein binding yield, specific
activity and % activity yield was determined.
2.4
Determination of kinetic parameters (Km, Vmax) and % water content
The maximum reaction velocity (Vmax) and MichaelisꢀMenten constant (Km) for the crude
lipase and immobilized lipase (BCL/ ROL/ RNL) was determined by plotting the Lineweaver–
Burk plot using pꢀNPA hydrolytic assay reaction in the range of substrate concentration 2ꢀ26
mM. The procedure used is same as indicated in above section 2.3. The % water content of
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various crude, immobilized lipases (BCL, ROL, RNL) and control support was determined by
using the Karl Fischer method (784 KFP Titrino).
2.5
Determination of halfꢀlife time (τ) and deactivation rate constant (K
lipase in various organic solvents
D
) for immobilized
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Influence of various organic solvents on catalytic activity and stability was determined for
HY:PVA:BCL (2:2:1) immobilized lipase. The immobilized lipase HY:PVA:BCL (2:2:1) was
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incubated in different nonꢀpolar and polar organic solvents for 96 hours at 44 C. After
incubation, the solvent was removed by filtration and lipase activity was determined for
immobilized lipase BCL as above indicated procedure (section 2.3) to investigate the halfꢀlife
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time (τ) and deactivation rate constant (K ) using following formula.
N / N = exp(−K / T ).........(1)
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N / N = exp(− ln 2 /τT ).....(2)
0
Where, N is the actual activity of lipase sample after incubation at given time T, N
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is the actual
activity of fresh (unincubated) lipase sample, τ is the halfꢀlife time and K is the deactivation
D
rate constant. The above equations are derived from the firstꢀorder kinetics; since it is well
known fact that enzyme follows the first order decay kinetics.
2.6
Experimental setup and analysis
Phenethyl butyrate synthesis involves addition of phenethyl alcohol and vinyl butyrate in a 10
mL glass reaction vessel of 1.6 cm i.d. with a glass lid. The reaction mixture was diluted by
toluene to make volume of 3 mL. Immobilized lipase BCL (35 mg) was added to initiate the
reaction; later on reaction vessel was kept in orbital shaker at given temperature and agitation
speed. Reaction mass sample 10 ꢂl was withdrawn periodically and analyzed by using the Perkin
Elmer, Clarusꢀ400 gas chromatograph equipped with the flame ionizing detector (FID) and
capillary column. The detector and injector temperature was 280 and 80 °C respectively.
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2.7
Initial reaction rate determination
The initial reaction rate was deduced by the concentration versus time profile graph for the
initial 25 % conversion; these curves were plotted by using the polynomial graph and
corresponding derivative was equated to zero to obtain the initial reaction rate for the
phenethyl butyrate synthesis (Scheme 1).
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Please insert here Schemeꢀ1
3.
RESULT AND DISCUSSION
3.1
Lipase activity and protein content
The lipase hydrolytic activity assay for crude and various immobilized HY:PVA lipase followed
the sequence: lipase BCL > ROL > RNL (Table ST1: please see supplementary information for
ST1); similar trend was also found for the protein content of crude lipases. All three immobilized
lipases, showed higher activity than crude lipase; these results demonstrated improvement in the
catalytic activity which was attributed to interfacial activation after immobilization of lipase on
5ꢀ8, 24ꢀ27
the support matrix.
Moreover, this fact can be explained as lipase was well scattered on
the HY:PVA support and aggregation of lipase is no longer possible after immobilization; while
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crude lipase agglomerated and suffered from the mass transfer diffusion.
The protein loading
was found to be greater than 90 % in each case which concluding that protein was immobilized
on the support. Herein, the lipase BCL showed the highest lipase activity and % protein binding
yield (Table ST1). The improved % lipase activity and higher % protein binding yield showed
6,7,17
that HY:PVA polymer support was proficient for lipase immobilization.
Activity yield for
immobilized lipase followed the order of lipase BCL (161 %) > RNL (140 %) > ROL (136 %).
Activity yield is ratio of specific activity of immobilized lipase to crude lipase which indicating
25
the better retention of lipase activity after immobilization on HY:PVA support. Hung et al. got
highest activity yield of 91 % when lipase was immobilized on chitosan support. Thus lipase
activity assay and protein content study demonstrated that HY:PVA support was proficient for
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the lipase immobilization, while interfacial activation was the basic cause for improvement in
4
ꢀ9,26,27
lipase activity after immobilization on HY:PVA matrix.
3.2. Determination of the Km and Vmax for the free and immobilized lipases
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In all three cases of the lipases (BCL, ROL and RNL), we found similar observation that
Vmax values was found to be higher for immobilized lipase as compared to crude lipase,
while Km value was found to be lower for the immobilized lipase as compared to crude lipase
(
Table 1; entries 1ꢀ7) . The Km value for immobilized lipase BCL was found to be lower
3.48 mM) than crude lipase BCL (5.24 mM), while the Vmax value for immobilized lipase
(
BCL was found to be higher (77.51) than crude lipase BCL (69.93) (Table 1; entries 1,2).
Similar observation was obtained for lipases ROL and RNL. The increase of Vmax values of
the immobilized lipases could be explained by several hypotheses such as (i) immobilized
lipase scattered into immobilization matrix could easily contact with the substrates molecules
6
,11,12
which enhances the catalytic rate;
(ii) immobilization matrix could benefit the “open
state” conformation of lipase and offered easy access of substrates to active sites of
7
,18,19
enzyme;
(iii) the interfacial activation causes change in enzyme conformation after
6,17
immobilization which improve the biocatalytic activity of the lipases; (iv) free enzyme
aggregation is avoided after immobilization which facilitates easy massꢀtransfer of the
12,27
substrates to active catalytic sites.
Thus in present study, the improvement of the Vmax
value may be due to the more efficient conformation of immobilized lipases as compared to
11ꢀ17
crude/free lipase.
The results of the Km value demonstrated higher affinity of the immobilized lipase to the
substrate since, Km value of immobilized lipases were lower than that of crude lipase (Table
11,12
1).
As mentioned earlier, the immobilization facilitates the dispersion of enzyme into
support matrix, thus the higher substrate concentration was found around the immobilized
enzyme. Consequently, the immobilized lipase apparent affinity for the substrate was
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1,12,17ꢀ19
positively enhanced.
As a result of Km and Vmax values, the immobilized lipases
possessing the higher catalytic efficiency (Vmax/Km) as compared to crude lipase (Table 1).
Thus in all, the conformational orientation might be changed from “close form” to an “open
6
,7,15
form” after the immobilization of lipase into support.
The improvement of the kinetic
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parameters of immobilized lipases (BCL, ROL and RNL) signifying that, the immobilized
lipase could be used efficiently for organic synthesis reactions instead of crude lipase.
Please insert here Tableꢀ1
3.3
D
Determination of the Halfꢀlife time (τ) and thermal deactivation rate constant (K )
Biocatalysis in nonꢀconventional media is a challenging task as enzymes are sensitive to the
organic media hence, it is important to study the influence of various organic solvents on
28
immobilized lipase activity. Enzymes follows firstꢀorder kinetics from which the halfꢀlife time
D
(τ) and thermal deactivation rate constant (K ) were studied for various tested organic solvents
28
(
Table 2). Higher τ and lesser K
D
value demonstrated better activityꢀstability of the lipase for
1
7,28
given organic solvent.
In the present study, immobilized lipase retained maximum activity in
nonꢀpolar solvents like cyclohexane (τ = 822 h) and toluene (τ = 748 h). The τꢀvalue decreased
while K ꢀvalue increased as the polarity of the solvent increased, which might be attributed to
disturbance in microꢀaqueous environment around the enzyme which losses the conformational
D
5
ꢀ8
28
flexibility and causes lowering of enzyme activity in polar solvent. Recently Silva et al.
immobilized lipase BCL on silicaꢀPVA matrix which having the halfꢀlife time of 478 h.
Furthermore % conversion study showed 5ꢀ6 fold improvement in the biocatalytic activity of
enzyme in various solvents (polar or nonꢀpolar) after immobilization (Table 2). This observation
can be explained as; (i) formation of crude enzyme aggregates was avoided after immobilization
of lipase on the polymeric support (ii) scattering of lipases on immobilization support afforded
6
ꢀ9
the ease of the diffusion of substrates into the active sites of enzymes. Thus overall solvent
study reveals the improved catalytic activity and stability of the immobilized lipase in polar as
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well as nonꢀpolar solvents which providing conversion of 99 % in nonꢀpolar solvents while
6
ꢀ8
conversion was found ≥ 70 % in polar solvents also.
Please insert here Tableꢀ2
.4 Screening of lipases (crude or immobilized)
3
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Various three crude and immobilized lipases namely BCL, ROL and RNL with various support
compositions were screened for phenethyl butyrate synthesis to determine the catalytic
efficiency under analogous reaction condition (Figure 2A). It was found that crude BCL, ROL
and RNL gave the 17, 5 and 4 % conversion while immobilized BCL, ROL and RNL gave 99,
22 and 16 % conversion of the phenethyl butyrate synthesis respectively. This was accounted as,
crude lipase forms aggregates in nonꢀaqueous media which causes complexity in diffusion of
6
,17
substrates to access the catalytic sites.
However, catalytic sites are easily accessible which
causes easy diffusion of substrates after immobilization of lipase on HY:PVA based support due
4
ꢀ10
to phenomenon of interfacial activation.
Moreover, screening study reveals the big difference in conversion especially for the
immobilized lipases ROL and RNL as compared to BCL. Since immobilized lipase BCL
providing superior biocatalytic activity as compared to the crude/free lipase. However similar
extent of superior biocatalytic activity was not observed for immobilized lipase ROL and
RNL as compared to the respective crude lipase. This large variation was attributed to the
2
9
presence of % water content (Table 1). Wehtje and Adlercreutz observed that, bacterial
lipases showed maximum catalytic activity at higher % water content while fungal lipases
showed maximum catalytic activity at moderate or lower % water content. In present study
also, it was observed that after immobilization the % water content was found to be higher as
compared to respective crude lipase (Table 1; entries 1ꢀ7). Hence, immobilized bacterial
lipase BCL showed superior (sixꢀfold) biocatalytic activity, while immobilized fungal lipases
ROL and RNL showed improved (fourꢀfold) biocatalytic activity.
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Similar observations were reported by Valivety et al. and Ma et al. wherein they found
optimal catalytic activity for the fungal lipases such as RNL and ROL at lower % water
6
content. In recent review, Adlercreutz proposed that % water content causes large variation
6
in the lipase biocatalytic activity. Moreover, in literature review study Adlercreutz found that
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lipase BCL showed maximal catalytic activity at higher % water content while fungal lipases
of Candida, Rhizomucor, Rhizopus species showed optimal activity at lower % water
29ꢀ32
content.
However, in present case of the ROL and RNL, when the timeꢀspan increases to 12ꢀ14 hours
then immobilized lipase leads to provide conversion > 90 % while that of crude/free lipase
provided only upto 20ꢀ25 % conversion. Thus, screening study showed that immobilized
lipase HY:PVA:BCL (2:2:1) acted as robust biocatalyst among various composition which
showing almost 6ꢀfold higher catalytic activity than crude lipase and lead to achieve the
highest conversion (99 %) in 3h.
Please insert here Figureꢀ2
3.5 Effect of lipase loading on carrier
Different amounts of the lipases BCL were loaded (immobilized) on fixed amount of support to
obtain the robust biocatalyst with the maximum catalytic activity (Figure 2B). The effect of
amount of lipase BCL loading from 60ꢀ150 mg on 400 mg of HYꢀPVA polymer supports were
studied for the phenethyl butyrate synthesis under the similar reaction conditions. It was found
that (i) as the lipase loading increased from 60 to 100 mg the corresponding phenethyl butyrate
conversion also increased; this was attributed to increase of catalytic sites with higher lipase
33
loading; (ii) Further increase in the lipase loading from 100 to 120 mg showed almost similar
% conversion; this might be due to cause that excess catalytic sites did not take part in the
6
,33
reaction;
(iii) However further, increase in lipase loading from 120 to 150 mg showed
decrease in % conversion which might be attributed to difficulty in substrate diffusion to access
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the active sites of enzyme molecules because of heavy lipase loading on HY:PVA support.
33
Similar type of lowering of yield was accounted by Salis et al. for over lipase loading on
support. Thus, maximum conversion (99 %) of the phenethyl butyrate was obtained in 3 h when
lipase loading was the 100 mg (Figure 2B); this composition was indicated as the PVA:HY:BCL
(2:2:1).
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3.6 Effect of butanoyl donor
Conventionally butanoic acid used as butanoyl donor for butyric ester synthesis; however acid
works as a potent inhibitor and ceases enzyme activity which leads to provide lower %
conversion (11 %) and initial reaction rate (Figure 3A). Hence, butyrate ester synthesis can be
performed by reaction of alcohols with alkyl butyrate or vinyl butyrate esters. When alkyl
butyrate (methyl butyrate or ethyl butyrate) was used as butyrate donor then it liberates alcohol
(methanol or ethanol) as a byꢀproduct which compete with phenethyl alcohol for nucleophilic
attack and inhibits the rate of desired reaction. Furthermore, these byꢀproducts (alcohols) are
polar in nature which may tend to distort the microꢀaqueous system around the enzyme and
6
,33
22
causes lowering of reaction rate. Similar alcoholic inhibition was reported by the Pelt et al.
for amide synthesis. However, these difficulties can be overcome when activated butyrate donor
1
,17,22
such as vinyl butyrate was used.
Vinyl butyrate is an activated butyrate donor speices,
liberating vinyl alcohol as a byꢀproduct which tautomerizes into acetaldehyde and do not take
34
part in reaction. Herein, vinyl butyrate provided highest initial reaction rate and conversion (99
%) for phenethyl butyrate synthesis in 3 h and used for further study.
Please insert here Figureꢀ3
3.7 Effect of solvent
The choice of organic solvent can have significant effect on the enzyme performance and hence
8
,28
it is mandatory to screen the suitable solvent;
furthermore reaction solvent helps for
17,35
dissolution of the substrate and ease of the substrate diffusion process in the reaction media.
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In present study, six various (polar and nonꢀpolar) solvents were studied and results were
demonstrated that, nonꢀpolar solvents possess excellent compatibility with the enzyme
molecules; while polar solvents provided moderate conversion (≥ 70 %) in 3 h (Table 1). This
was due to reason that essential microꢀaqueous environment around the enzyme coat was
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5
ꢀ9
remains active in nonꢀpolar solvent; while microꢀaqueous coat may distorted in polar solvents
27,28
and looses the enzyme conformational flexibility.
Interestingly immobilized biocatalyst
provided good activityꢀstability and better conversion (> 70 %) in polar solvents also;
furthermore immobilized lipase showed five to seven fold superior catalytic activity than crude
lipase for each solvent (Table 1). Thus nonꢀpolar solvent cyclohexane and toluene was the best
suitable solvent giving 99 % conversion of the corresponding product; while toluene was used
further for all experiments.
3.8
Effect of mass transfer
Present system is the microꢀaqueous heterogeneous solidꢀliquid system where catalyst is in solid
phase while both reactants are in liquid phase; hence it is essential to check effect of mass
17,35,36
transfer (Figure 3B).
In present system it was found that initial reaction rate and %
conversion was increased with increase in agitation speed from 50 to 175 rpm; further raise in
the agitation speed from 175 to 200 rpm did not show any significant improvement in the initial
reaction rate and % conversion. These results signifying that, as soon as the mass transfer barrier
achieved; then there was no influence of agitation speed on mass transfer for corresponding
5ꢀ8,17,35
biotransformation;
rpm.
hence further kinetic experiments were carried out at the speed of 175
3.9 Effect of biocatalyst amount
Effect of HY:PVA:BCL biocatalyst amount ranging from 5 to 40 mg was studied to find out
appropriate quantity of biocatalyst; since biocatalyst amount is crucial factor to govern the
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industrial economics. It was observed that initial reaction rate and (%) conversion of phenethyl
butyrate increases linearly with increase in amount of biocatalyst from 5 to 35 mg; since more
the amount of biocatalyst, more will be catalytic sites available to access the substrate which
3
3
subsequently increases the % conversion rate (Figure 4). However, increase in biocatalyst
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amount to 40 mg (data point not shown in figure 4) showed slight increase of initial reaction rate
but no significant improvement was observed in % conversion; which indicating the presence of
17,33
more catalytic sites at 40 mg biocatalyst amount.
The overall conversion was found to be 99
%
with 35 mg of HY:PVA:BCL (2:2:1) biocatalyst amount; hence other all experiments were
performed with use of 35 mg biocatalyst. Additionally, the figure 4 showed linear relationship of
biocatalyst amount versus initial reaction rate; which indicating the kinetically controlled
35,36
synthesis of phenethyl butyrate.
Please insert here Figureꢀ4
.10 Effect of molar quantity of phenethyl alcohol and vinyl butyrate
3
Several experiments were performed to determine the possible inhibition of any substrate on
enzyme activity by changing mole ratio of both substrates systematically. The mole ratio of
phenethyl alcohol was kept constant (2 mmol) while the mole ratio of vinyl butyrate was varied
from 1 to 6.5 mmol; under the similar reaction condition (Figure 5A). It was observed that initial
reaction rate increases as mole ratio of vinyl butyrate increases from 1 to 6.5 mmol; however %
conversion was found to be constant when mole ratio changes from 6 to 6.5 mmol. Thus, vinyl
2
2,35,36
butyrate acted as activated butanoyl donor, which liberated vinyl alcohol as a side product.
This vinyl alcohol is no longer available for the nucleophilic attack as it immediately
o
17,22,35
35
tautomerized to low boiling acetaldehyde (21 C).
Yadav and Trivedi as well as Kuo et
36
al. obtained similar type of observation in their study for novozyme catalyzed reaction.
In another set of experiment, concentration of vinyl butyrate (6 mmol) was kept constant while
concentration of phenethyl alcohol was varied from the 1 to 4 mmol (Figure 5B). It was found
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that, there was gradual increase in % conversion and initial reaction rate with increase in mole
ratio of phenethyl alcohol from 1 to 2 mmol. However further increase in mole ratio from 2 to 4
mmol showed continuous decrease in % conversion and initial reaction rate; this may be
endorsed by deadꢀend inhibition complex formation at higher alcohol (phenethyl alcohol)
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1
7,35,36
concentration.
Similar type of activity inhibition at higher concentration of alcohol was
35,36
reported previously in literature.
butyrate was 2:6.
Thus optimized molar quantities of phenethyl alcohol:vinyl
Please insert here Figureꢀ5
3
.11 Effect of temperature
Temperature plays an important role (i) to enhance reaction rate (ii) to enhance the catalytic
interactions (iii) to lower the viscosity of reaction medium (iv) to improve the solubility of
1
7,20,35
substrate in the reaction media and (v) to improve for enzyme activity.
Hence, it was
mandatory to check the optimal temperature for enzymatic reactions as enzymes are highly
4ꢀ9
susceptible to higher temperatures. The influence of temperature was studied in the range of
o
2
5ꢀ44 C (Figure 6A). It was observed that % conversion as well as initial reaction rate rose up
o
linearly with increase in temperature from 25ꢀ44 C; and it gave 99 % conversion of the
o
corresponding butyrate ester in 3 h at 44 C. This was attributed as raise in temperature increases
the molecular collision interaction of reactants which leads to decrease in the energy barrier
between reacting molecule and enzymeꢀsubstrate complex formation and thus subsequently
3
7ꢀ40
improves the reaction rate.
Herein, we kept minimum possible temperature to attain the
o
greener aspect hence, 44 C was the lowest optimum temperature used for further experiments.
Please insert here Figureꢀ6
3.12 Reaction profile with respect to time
Figure 6B showed the profile of the product formation by crude and immobilized lipase with
respect to time. The highest conversion (99 %) was achieved in 180 minutes when reaction
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was catalyzed by immobilized lipase BCL; while crude lipase BCL was providing 17 and 30
%
conversion in 180 and 270 minutes respectively (Figure 6B). Thus, the profile of the
product formation with respect to time showed superior biocatalytic activity of the
immobilized lipase (HY:PVA:BCL) as compared to the corresponding crude lipase BCL. It
was observed that, % conversion of the desired product was continuously increases with
respect to time up to 180 minutes, signifying that reaction was performed in the forward
direction. Moreover, after prolonged reaction time (270 minutes) the % conversion of the
desired product was not altered; which indicating the facts such as (i) reaction was not revert
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7,18,22
into backꢀward direction and (ii) final product formed was a stable one.
It is noteworthy
to mention that lipase was solely responsible to catalyze the reaction and to form the desired
product, since, there was no product formation (no % conversion) was observed in control
17,18
reaction (reaction without lipase BCL).
3.13 Catalyst recyclability
3
ꢀ7
One of the important feature of immobilization is recyclability of the biocatalyst; hence
immobilized lipase (PVA:HY:BCL) was subjected to check recyclability for phenethyl butyrate
synthesis in 3h (Figure 7). After 3h; reaction mixture was filtered and immobilized lipase was
washed with nꢀheptane (1.5 mL × 3 times) to remove the stick of any reactant /product. Later on,
immobilized lipase was dried for 10 h at 35ꢀ40°C and stored at room temperature (25 °C) until
used for next cycle. The marginal decrease in conversion was observed after two reuses
providing 94 % conversion while conversion was decreased further upto 74 % after eight
recycles in 3 h (Figure 7). This decrease in conversion was attributed to deactivation of lipase in
2,35,36
each cycle by continuous exposure to alcoholic substrate
However, % conversion of the
phenethyl butyrate synthesis can be improved and achieved upto 99 % in studied eight recycle
by increasing reaction time span (from 3h to 5h), as enzyme activity was decreased in each
recycle hence required more time span to achieve 99 % conversion (study not shown).
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Please insert here Figureꢀ7
3
.14 Thermodynamic study for immobilized and crude lipase in toluene
The temperature causes acceleration of reaction rate is generally consistent with the Arrhenius or
37ꢀ39
37
Eyring theory.
In general the lower energy barrier, the faster is reaction rate. The energy of
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activation was calculated by the Arrhenius equation (3); while other thermodynamic parameters
were calculated by the Eyring equation (4).
k = Aexp(Ea / RT)....................(3)
≠
≠
kh = Tk
B
exp(ꢀS / R)exp(−ꢀH / RT ).....(4)
≠
≠
Where, k, A, E
a
B
, T, k , h, R, ꢁS and ꢁH are reaction rate constant, Arrhenius collision factor,
energy of activation, absolute temperature, Boltzmann constant, Planck’s constant real gas
constant, entropy and enthalpy of activation respectively.
Activation energy (Ea) was calculated by the conventional Arrhenius plot in the temperature
range of 299 to 317 K (Table 3). The Ea for crude and immobilized lipase was found to be 69.72
and 51.23 KJ/mol respectively; the higher Ea value for the crude lipase BCL indicated the (i)
higher energy requirement (ii) lower catalytic efficiency and (iii) lesser reaction rate as
4
,35,36
compared to HY:PVA immobilized lipase BCL for the corresponding system.
Recently,
40
Xin et al. reported 65.05 KJ/mol Ea of lipase catalyzed ethyl ferulate synthesis. The decrease in
≠
enthalpy of activation (ꢁH ꢀvalue) for immobilized lipase was attributed to easier formation of
≠
enzymeꢀsubstrate complex; however ꢁH ꢀvalue increased when reaction was catalyzed by crude
lipase which indicates complexity of the reaction due to the presence of diffusion restrictions of
41,42
substrate as crude enzyme was agglomerated in the organic solvents.
≠
The feasibility and ease of reaction is best accounted by ꢁG ꢀvalue; lower is the free energy
37ꢀ39
≠
change then more feasible is the reaction.
Thus lower ꢁG ꢀvalue was found for immobilized
37,39
enzyme which indicates the feasibility of reaction as compared to crude lipase BCL.
The
≠
ꢁ
S ꢀvalue represent the difference in the extent of local disorder between transition state and
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ground state.
The entropy change is the measure of extent of disorder; large decrease in
≠
ꢁ
S ꢀvalue for immobilized lipase showed lesser disorder of reaction which concluded that,
39
immobilized lipase favours the reaction in much better way than the crude lipase. Thus after
immobilization of lipase BCL on PVA:HY support, the lipase confirmation causes easy
exposure of substrate to active sites of enzyme and increases the feasibility of reaction.
Please insert here Tableꢀ3
0
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.15 Thermodynamic study of immobilized enzyme in various solvents
It is likely that thermodynamic studies can provide valuable information about the reaction
≠
feasibility at various temperatures. It was observed that a ꢁH value was increases as solvent
polarity increases (Table 4). The lower ꢁH value of immobilized lipase was found in nonꢀpolar
solvent which indicated ease of formation of enzymeꢀsubstrate complex because of more flexible
43
≠
enzyme conformation. Higher the ꢁH value, higher is the amounts of stretching or squeezing
≠
of chemical bonds to achieve the transition state/activated complex; this higher ꢁH value was
observed in polar solvent which may attributed to disturbance in microꢀaqueous environment
41,43
around enzyme and subsequent disturbance in structure of enzyme in polar solvent.
The
≠
37ꢀ39
positive value of ꢁH showed that reaction is endothermic in nature.
≠
Lower ꢁG value was found for the nonꢀpolar solvent which suggested that, the conversion of
activated enzymeꢀsubstrate complex into products was more spontaneous and feasible in the
37,41,45
≠
nonꢀpolar solvent.
Entropy (ꢁS ) is measure of extent of disorder, according to the second
39,42,45
law of thermodynamics, lower entropy value indicate more ease of reaction.
. In present
≠
study ꢁS was found to be lower for nonꢀpolar solvents which indicate more favourable
environment for the reaction; while extent of disorder (entropy) increases as the polarity of the
solvent increases (Table 4).
Please insert here Tableꢀ4
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3.16 Application of immobilized protocol for synthesis of various compounds
The practical viability of developed immobilization protocol was tested by synthesis of various
butyrate compounds using PVA:HY:BCL immobilized lipase as a biocatalyst (Scheme 2). Initial
reaction rate for short chain alcohol like ethyl/ butyl alcohol was little fraught due to polar nature
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3
5
of alcohol which provided yield of 99 % in 3.5 h (Table 5; entries 1,2). The aliphatic branched
alcohol provided excellent conversion of 99 % for corresponding butyrate product in 3 h (Table
5
; entry 3). nꢀOctyl alcohol showed poorer initial reaction rate and yielded 99 % of octyl
butyrate in 4.5 h because of the long hydrophobic chain which causes strong hydrophobicꢀ
hydrophobic interaction with the enzyme structure and tends to decrease the enzyme activity
4
6,47
(Table 5; entry 4).
Cyclohexyl alcohol is a secondary alcohol which providing sluggish
conversion of 70 % of the corresponding butyrate in 6 h, because of the steric hindrance at the
neighbouring αꢀcarbon (Table 5; entry 5). However, industrially important alcohols such as
benzyl alcohol and cinnamyl alcohol provided excellent conversion (99 %) of corresponding
butyrate ester in 3 h as like phenethyl butyrate (Table 5; entries 6,7). The 2ꢀmethyl benzyl
alcohol provided lower initial reaction rate hence 99 % conversion was achieved in 4.5 h; this
was attributed to presence of sterically bulky methyl group at ortho position (Table 5; entry
3
5,48
9).
Surprisingly, heterocyclic aromatic alcohols such as 3ꢀpyridine methanol provided
excellent yield of 99 % in 4h (Table 5; entry 10). In all studied substrates, (i) immobilized lipase
provided 99 % conversion of desired product (except cyclohexanol) and (ii) immobilized lipase
provided 6 to 9 fold higher conversion of the corresponding butyrate ester than crude lipase in
specified time; which showing superior biocatalytic activity of HY:PVA immobilized lipase
BCL. Thus, developed protocol is well extended for the practical biocatalytic applications to
synthesize variety of industrially important butyrate compounds by greener enzymatic route.
Products are well characterized by available physical characterization techniques such as GCꢀ
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MS analysis, HꢀNMR and CꢀNMR. (Please see supplementary information for product
characterization)
Please insert here Schemeꢀ2
Please insert here Tableꢀ5
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CONCLUSION
Lipase BCL was successfully immobilized on a biodegradable carrier made up of HY/PVA
using water as a medium for immobilization. Immobilized lipase showed higher τ and lower K
D
for nonꢀpolar solvents which indicating better stability of lipase in nonꢀpolar solvents than polar
solvents. Lipase activity assay and protein content study showed that HY/PVA immobilization
support is proficient for enzyme immobilization. Various reaction conditions and parameters
were studied in details for synthesis of phenethyl butyrate which providing excellent (99 %)
conversion of the product when reaction was catalyzed by HY:PVA:BCL. Immobilized lipase
showed sixꢀfold higher catalytic activity than the crudeꢀBCL. Furthermore, developed
immobilized biocatalyst is well stable in various polar as well as nonꢀpolar solvents which
providing conversion > 70 % in nonꢀpolar solvents also. Thermodynamic activation parameters
≠
≠
≠
(
ꢁH , ꢁG , ꢁS ) also showed that reaction favoured when catalyzed by immobilizedꢀBCL.
≠
≠
≠
Furthermore, Ea, ꢁH , ꢁG and ꢁS were also deduced for the various polar and nonꢀpolar
solvents which indicating the better feasibility of reaction in nonꢀpolar solvents. Finally, this
biocatalyst was used to synthesize various industrially important butyrate esters. The biocatalyst
PVA:HY:BCL was efficiently reused up to 8 recycles. The present protocol demonstrated the
robust biocatalyst preparation, application and thermodynamic investigation in various organic
media.
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ACKNOWLEDGEMENT
Author Kirtikumar Badgujar is grateful to Department of BiotechnologyꢀDBT, India
(
BT/PR4192/PID/6/623/2011) and Council of Scientific & Industrial ResearchꢀCSIR, India
09/991(0015)/2011ꢀEMRꢀI) for providing research funds.
(
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SUPPORTING INFORMATION
Lipase activity assay, protein content study and characterization of the product are provided in
supplementary information. Supporting information is available free of charge via the Internet at
http://pubs.acs.org
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0
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Scheme 1 Immobilized lipase catalyzed synthesis of phenethyl butyrate.
Scheme 2 Application of immobilized protocol for synthesis of various butyrate compounds
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Tables
Table 1 Determination of kinetic parameters and water content
No Lipase
V
max
K
m
Catalytic Efficiency Water content
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(µmol/min/mg) (mM) efficiency
(%)
(% w/w)
Burkholderia cepacia lipase (BCL)
1
2
Crude BCL
Immobilized HY:PVA:BCL
69.93
5.24
13.33
100
166.32
1.34
9.98
77.51
3.48
22.17
Rhizopus oryzae lipase (ROL)
3
4
Crude ROL
Immobilized HY:PVA:ROL
33.78
6.90
4.84
100
148.54
0.94
8.97
35.97
4.99
7.20
Rhizopus niveus lipase (RNL)
5
6
7
Crude RNL
Immobilized HY:PVA:RNL
Control support HY:PVA
28.40
30.76
000
8.77
7.84
000
3.23
3.92
000
100
121.18
000
0.81
8.26
7.94
Catalytic efficiency is ratio of maximum velocity (Vmax) to MichaelisꢀMenten constant (Km)
Efficiency (%) = (Catalytic efficiency of immobilized lipase / catalytic efficiency of crude
lipase) ×100
Values presented are average values of triplicate.
29
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