SusChemE 2015
International Conference on Sustainable Chemistry & Engineering
October 8-9, 2015, Hotel Lalit, Mumbai
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
Kirtikumar C. Badgujar and Bhalchandra M. Bhanage*
Department of Chemistry, Institute of Chemical Technology, Mumbai-400 019, India
*Corresponding author. Tel.: + 91- 22 3361 1111/2601; Fax: (+91)-22-33611020.
E-mail addresses: kirrtti@gmail.com bm.bhanage@gmail.com
1. Introduction:
The enzyme lipase {triacyl glycerol hydrolases, (E.C. 3.1.1.3)} has received special attention as a biocatalyst due to its wide
substrate range and ability to carry out various promiscuous transformations in various reaction media. However, because of
their proteomic nature, enzymes are quite sensitive, unstable, and poorly soluble in organic solvents, which limits their
direct applications as a potential biocatalyst. Besides this, application of the free/crude enzyme has several drawbacks such
as recyclability issues, lower stability to temperature, and organic reaction media. To overcome these shortcomings,
enormous efforts have been taken by various researchers to enhance the activity-stability of enzymes with the help of the
skilful immobilization technique. Among these various immobilization methodologies, the application of beads/polymer
matrixes as an immobilization carrier has received a special importance because of their easy separation (recovery) from
organic reaction media for reuse. Hence in present study, we explored a biocatalytic protocol using lipase Bukholderia
cepacia (BCL) immobilized on biodegradable Hypromellose (HY) and Poly(vinyl alcohol) (PVA) as biocatalyst
{HY:PVA:BCL}. Thus present study deals with the, (i) immobilization of lipase on HY:PVA support and (ii) its biocatalytic application with thermo-kinetic engineering analysis. Hence; we have analyzed thermodynamic parameters of
activation (Ea, ΔH⧧, ΔS⧧, and ΔG⧧) in various solvents. Besides this, various other biocatalytic parameters such as half-life
time (τ), and deactivation rate-constant (KD). In addition to this, recyclability and applicability of developed immobilization
protocol were tested for the synthesis of various commercially/industrially essential compounds.
2. Material and Methods:
Lipases Burkholderia cepacia (lipase BCL), Rhizopus oryzae (lipase ROL), and Rhizopus niveus (lipase RNL), PVA, HY,
p-nitrophenyl acetate (p-NPA), bovine serum albumin, Bradford reagent, and all other chemicals were purchased from
Sigma-Aldrich Pvt. Ltd., India. Immobilization of Lipase: Lipase was immobilized via entrapment methodology.
3. Significant Results and Discussion
In all three cases of the lipases (BCL, ROL and RNL), we found similar observation that Vmax values and substrate
affinities was found to be higher for immobilized lipase as compared to crude lipase (Table 1). These results were attributed
due to the interfacial activation phenomenon of lipase after immobilisation. In the present study, % conversion study
showed 5-6 fold improvement in the biocatalytic activity of enzyme in various solvents (polar or non-polar) after
immobilization (Table 2). This observation can be explained as; (i) scattering of lipases on immobilization support afforded
the ease of the diffusion of substrates into the active sites of enzymes. Moreover, the immobilized lipase retained maximum
activity in non-polar solvents like cyclohexane (τ = 822 h) and toluene (τ = 748 h).
SusChemE 2015
International Conference on Sustainable Chemistry & Engineering
October 8-9, 2015, Hotel Lalit, Mumbai
The τ-value decreased while KD-value increased as the polarity of the solvent increased, which might be attributed to
disturbance in micro-aqueous environment around the enzyme which losses the conformational flexibility and causes
lowering of enzyme activity in polar solvent. Thus, overall solvent study reveals the improved catalytic activity and stability
of the immobilized lipase in polar as well as non polar solvents which providing conversion of 99 % in non-polar solvents
while conversion was found ≥ 70 % in polar solvents also. Thermodynamic study (Ea, ΔH⧧, ΔS⧧, and ΔG⧧) reveals, the
feasibility of process via immobilized catalyzed reaction for the synthesis of butyrate compounds (Table 3). The protocol is
well extended to synthesize various industrially important moieties (Table 4). The immobilized-BCL reported 6-folds
superior biocatalytic activity and 8 times recyclability as compared to crude-BCL (Figure 1). The present work explains a
thermo-kinetic engineering aspect for the sustainable production of valuable compounds via biocatalytic protocols.
Table 1 Determination of kinetic parameters of immobilized and crude (free) enzyme
No
1
2
3
4
5
6
7
Lipase
Vmax
Km
(µmol/min/mg)
(mM)
Burkholderia cepacia lipase (BCL)
Crude BCL
69.93
5.24
Immobilized HY:PVA:BCL
77.51
3.48
Rhizopus oryzae lipase (ROL)
Crude ROL
33.78
6.90
Immobilized HY:PVA:ROL
35.97
4.99
Rhizopus niveus lipase (RNL)
Crude RNL
28.40
8.77
Immobilized HY:PVA:RNL
30.76
7.84
Control support HY:PVA
000
000
Catalytic
efficiency
Efficiency
factor (%)
13.33
22.17
100
166.32
4.84
7.20
100
148.54
3.23
3.92
000
100
121.18
000
Scheme: Application of immobilized protocol for synthesis of various butyrate compounds
Table 2 Solvent activity-stability study: % conversion, half life time and thermal deactivation rate constant determination
% conversiona
Solvent
a
Improved activity
τ
KD×10-3
Crude
Immobilized
(folds)
(h)
(h-1)
1) Cyclohexane
19
99
5.20
822
0.843
2) Toluene
17
99
5.86
748
0.926
3) DIPE
15
89
5.93
594
1.166
4) MTBE
12
76
6.33
388
1.786
5) 1,4 Dioxane
12
74
6.16
409
1.694
6) Acetone
11
71
6.45
347
1.997
Reaction condition: Phenethyl alcohol, 2 mmol; butyrate door, 6 mmol; solvent toluene upto 3 cm3; biocatalyst: immobilized
lipase loading 35 mg or crude lipase 7 mg; temperature 44 oC; orbital rotation speed, 175 rpm, time 3h; Conversion based on
GC analysis; DIPE-Di-isopropyl ether; MTBE- Methyl tert-butyl ether.
SusChemE 2015
International Conference on Sustainable Chemistry & Engineering
October 8-9, 2015, Hotel Lalit, Mumbai
Table 3 Thermodynamic parameters for the immobilized lipase and crude lipase in toluene a
a
R2
ΔH≠
51.23
0.995
48.6
69.72
0.976
67.1
Lipaseb
Ea
1) Immobilized lipase HY:PVA:BCL
2) Crude lipase BCL
c
ΔS≠
ΔG≠
0.994
-164.21
99.26
0.972
-122.66
104.93
d
R2
Thermodynamic activation parameters were calculated in between the temperature range of 26-44 oC; bsolvent used for
reaction media is toluene; unit of Ea (KJ/mol); unit of ΔH≠: (KJ/mol); unit of ΔS≠: (J/mol· K); unit of ΔG≠: (KJ/mol); cR2 for
Ea values; dR2 for ΔH≠ values.
Table 4 Application of the developed protocol for the synthesis of various butyrate esters from corresponding alcohols a
a
% Conversion obtained by biocatalyst c
Immobilized lipase
Crude lipase
Initial
reaction
rateb
Time
(h)
1
0.00521
3.5
99
12
2
0.00588
3.5
99
14
3
0.00600
3
99
13
4
0.00487
4.5
99
11
5
0.00377
6
70
10
6
0.00613
3
99
18
7
0.00598
3
99
14
8
0.00603
3
99
17
9
0.00504
4.5
99
13
10
0.00596
4
99
15
Entry
Butyrate ester
Reaction condition: Alcohol:butyrate donor, 2:6 mmol; immobilized HY:PVA:BCL (2:2:1) - 35 mg; temperature 44 oC;, time
3h; bInitial rate (mmol·mL·min-1); cconversion based on GC analysis.
SusChemE 2015
International Conference on Sustainable Chemistry & Engineering
October 8-9, 2015, Hotel Lalit, Mumbai
Figure 1: Recyclability study: Phenethyl alcohol, 2 mmol; vinyl butyrate, 6 mmol; solvent toluene upto 3 cm3; immobilized
HY:PVA:BCL (2:2:1)- 35 mg or crude lipase BCL- 7 mg; temperature 44 oC; orbital rotation speed, 175 rpm, time 3h.
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 KD for non-polar solvent which indicating better stability of
lipase in non-polar solvents than polar solvent. 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 butyrate compounds which providing excellent (99 %) conversion of the product when reaction was
catalyzed by HY:PVA:BCL. Immobilized lipase showed six-fold higher catalytic activity than the crude-BCL. Furthermore,
developed immobilized biocatalyst is well stable in various polar as well as non-polar solvents which providing conversion >
70 % in non-polar solvents also. Thermodynamic activation parameters (ΔH ≠, ΔG≠, ΔS≠) also showed that reaction favoured
when catalyzed by immobilized-BCL. Furthermore, Ea, ΔH≠, ΔG≠ and ΔS≠ were also deduced for the various polar and nonpolar solvents which indicating the better feasibility of reaction in non-polar 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 novel and robust biocatalyst preparation, application and thermodynamic
investigation in various organic media.
References:
[1] K.C. Badgujar and B.M. Bhanage. J. Phys. Chem. B. 118, 2014, 14808-14819, 2014
[2] K.C. Badgujar and B.M. Bhanage. Process Biochem. 49, 2014, 1304-1313, 2014.
[3] P. M. Kosaka et. al. Langmuir, , 23, 2007, 12167-12173