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(HPMC) were used for enzyme immobilization in the form of film,
[15] and Chang and Juang [17] utilized CH for the chymotrypsin and
of Candida rugosa lipase. Dong et al. [19] applied the organoben-
tonite for immobilization of the porcine pancreatic lipase. Hilal et al.
eral techniques such as structure modification, enzyme and genetic
engineering were developed to improve the enzyme stability and
activity [1,3–7]. Furthermore, various immobilization protocols
were developed [3–7,10–21] which showed the improvement in
the pH, thermal and operational stability of enzyme. Besides these
immobilization protocols, considering limitations of the traditional
supports in the enzyme immobilization, many efforts have been
made to develop an ideal ecofriendly immobilization support to
prepare the robust biocatalyst.
In present protocol a polymeric film was prepared by using
ternary blend of PLA, PVA, and CH which is biodegradable. The
prepared film has good biocompatibility and stability as compared
to the binary blend [22]. PLA is a biodegradable, biocompatible,
ecofriendly biopolymer produced from starch like materials [23].
PVA is an excellent compatibilizing polymer for preparing mis-
emulsification, high tensile strength, non-toxic nature, biodegrad-
able, stabilization of blend and essentially resistant to organic
solvent which makes them more ideal for lipase immobilization
lack of toxicity and its biodegradable nature [10–14]. In addi-
tion to this, free hydroxyl and amino groups in CH is expected
to offer the higher degree of immobilization. Recently, Grande
and Carvalho [22] synthesized a ternary blend film of PLA-PVA-CH
and the outcome invites the researcher for application in enzyme
immobilization.
In continuation to our research interest in amidation reactions
[27–31] and bearing in mind the goal of non-toxicity, low cost,
recovery, reusability of the catalyst, high substrate specificity and
development of green protocol. Certain enzymes such as amidase
and protease, etc. are reported for the amide bond formation reac-
tion. However, they have a major drawback as they hydrolyze the
amide bond readily which lowers the yield and activity in the
organic solvent [32]. Lipase has numerous benefits in comparison
with other enzymes such as greater tolerance to organic solvents,
high stability, easy viability, wide substrate array [25,32]. Taking
the advantage of this, we were interested in developing amida-
tional transesterification reactions. Acetamides are the biologically
active molecules and are widely used in biomedical applications,
enzyme inhibitor and herbicides along with the pharmacy inter-
mediate [31]. To best of our knowledge acylation of amine via
immobilized lipase on biocompatible and biodegradable ternary
blended thin film has not yet reported. Considering this issue, we
have studied the synthesis of acetamides using vinyl acetate as
an acyl donor with lipase CCL immobilized on a ternary blend
biopolymer (PLA PVA CH) as a biocatalyst. Also a mechanistic
pathway was elucidated for the lipase catalyzed acetamide synthe-
sis reactions. Furthermore we characterize the immobilized lipase,
examine kinetic-thermodynamic parameters, leaching, activity,
stability and recyclability.
2. Experimental
2.1. Materials
Mucor javanicus lipase (AmanoM, lipase MJL, white powder, ≥10,000 U/g),
was kindly gifted by Amano Enzymes (Nagoya, Japan) while Candida cylindracea
lipase (lipase CCL, yellow-brown powder, ≥2000 U/g), C. rugosa lipase (lipase CRL,
light brown powder, ≥2000 U/g), PVA (Mw. 9000–10,000), CH (Brookfield viscos-
ity > 200.0 cps) and p-NPP were purchased from Sigma–Aldrich Ltd., India. Bovine
serum albumin (BSA) and Folin-Ciocalteu reagent was purchased from Hi Media
Ltd., India. PLA was synthesized in the lab by the Marques et al. procedure [33].
Other all chemicals of analytical grade were purchased from Hi media (I) Pvt. Ltd.
and S.D. Fine Chemicals Ltd.
Formation of the C N bond has great importance in the nat-
the life beginning bio-molecules such as amino acids, DNA, RNA,
proteins, micro cycles, glycopeptides, amino glycosides, and alka-
loids as well as in the synthesis of many active pharmaceutical
intermediates (API) [24]. However its formation is a challenging
task in organic synthesis. The American Chemical Society Green
ACS GCIPR concluded that C N bond formation was one of the
most exploiting key steps for production of many pharmaceutical
intermediates [25]. More recently in a survey of pharmaceuti-
cal industries, Carey et al. [26] found that 15 (12%) of acylation
reactions while 84 (66%) N-acylation reactions were involved in
tance of N-acylation reactions [26]. In multistep synthesis of a
drug molecule, acylation is a basic elementary step for protec-
tion of reactive amine which blocks nucleophilic as well as basic
character of amines and thus avoids the interference in multi-
chemicals such as the acid/thionyl/oxalyl chloride or by using
azabenzotriazole, carbonyldiimidazole, dicyclohexylcarbodiimide
[24,25]. Each of these above methods has several drawbacks such
as production of toxic and harmful side products, shock sensitivity,
poor atom efficiency, non-ecofriendly methodology [24–26].
2.2. Immobilization of lipase
Initially ternary blend of polymer was prepared with reported procedure of
Grande and Carvalho [22] with slight modification followed by the immobiliza-
tion of lipase. CH (50 mg) was dissolved in 1% acetic acid solution while PVA
(300 mg) was dissolved in deionized water (2%, w/w solution) separately. The PLA
(50 mg) was dissolved in chloroform (2%, w/w solution). Each solution was stirred
for 30 min at 1200 rpm separately and was filtered by Whatman paper filter. Then
CH and PVA solution was mixed in a beaker with PLA solution and stirred for 4 h
at 1500–1600 rpm. After 4 h, the crude extract lipase (100 mg; twice the amount
of CH) dissolved in 2 mL deionized water; was added to ternary blend and moder-
ately stirred for 1 h at 150–180 rpm. Finally this immobilized enzyme into ternary
blend was carefully poured in Teflon Petri-dish, followed by drying at 40–45 ◦C for
40–48 h. A thin film of immobilized lipase polymer formed was cut off into small
pieces of 2–3 mm2 and stored in plastic container at 4–8 ◦C until use. Theoretically,
100 mg of crude extract CCL was loaded on the 400 mg of support. The practical
protein content was calculated by Folin and Lowry assay. The prepared immobi-
lized biocatalyst was characterized by various techniques such as thermal analysis,
% water content, surface morphology study, I.R. and further exploited for N-acylation
reactions (Fig. 1).
2.2.1. Immobilization of lipase using cross linking agent glutaraldehyde
Process for the immobilization of lipase using cross linking agent glutaraldehyde
was same as described above in Section 2.2. The only minor change was made as
after addition of crude extract lipase; the 0.5% of glutaraldehyde solution (25%) was
added to ternary blend and moderately stirred for 1 h at 150–180 rpm. Rest of the
immobilization process followed was same as indicated in the above Section 2.2.
2.3. Lipase activity assay
Lipase activity of crude extract and immobilized lipase was determined in trip-
licate by hydrolysis of p-nitrophenyl palmitate (p-NPP) at 410 nm wavelength using