K. Dutta, V.V. Dasu / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 150–156
151
shift towards ester. This reversible reaction was previously shown
to follow a Ping–Pong Bi Bi mechanism [14,17].
26.5 kDa) was added to 2.5 ml of isooctane containing 0.25 M fatty
acid and 0.25 M alcohol. All reagents were previously dried over
4 A molecular sieves. The tubes were kept in a horizontal shaker at
˚
Cutinase was previously isolated and purified from Burkholde-
ria cepacia NRRL B 2320. In this investigation, we have studied the
B. cepacia cutinase catalyzed synthesis of alkyl esters in isooctane
solvents under various conditions. Here, we have also studied the
kinetics for the synthesis of butyl butyrate from butyric acid and
butanol in isooctane.
37 ◦C. At regular intervals, 300 l of the mixture (for butyric acid) or
tube and centrifuged at 10,000 rpm for 1 min to remove the sus-
pended enzyme particles. The progress of reaction was determined
by measuring the decreasing profile of acids by titration with 0.01 N
NaOH using phenolphthalein as indicator and also with Lowry and
Tinsley method [18]. According to Lowry and Tinsley method 700 l
(for butyric acid) or 900 l (for all other acids) isooctane was added
to the samples. Cupric acetate aqueous solution (0.2 ml) containing
pyridine (5%, w/v, pH 6.0) was then added into the tube and the
After centrifugation at 2500 rpm for 5 min, the upper organic phase
was measured by a UV/visible spectrophotometer (Cary 100, Var-
ian) at 715 nm. The formation of ester was also confirmed by gas
chromatography. Water percentage in the reaction mixture was
determined by Karl Fisher titration method [2,4]. All reactions and
analysis were performed in duplicate.
2. Materials and methods
Butanol, pentanol, hexanol, octanol, butyric acid, valeric acid,
caproic acid and octanoic acid purchased from sigma. Isooctane
˚
(Merck) was dried over 4 A molecular sieve. Tris (hydroxymethyl)
amino methane (Tris) was also purchased from Sigma. The cutinase
enzyme used for reaction was isolated from B. cepacia NRRL B 2320
(previously known as Pseudomonas cepacia).
2.1. Production and purification of enzyme
The following medium was used for the production of cuti-
nase (g l−1): beef extract, 4.0; peptone, 17.8; urea, 5.0; KH2PO4,
3.0; KCl, 0.64; MgSO4·7H2O, 5.55 and cutin 10.1. Initial pH of the
medium was adjusted at 7.0. A 2% of inoculum from the seed culture
was added to 200 ml of the production medium in 1000 ml Erlen-
meyer flasks. The flasks were then incubated in a shaking incubator
at 28 ◦C and 180 rpm. After 96 h of fermentation, cells were cen-
trifuged at 8000 rpm for 10 min at 4 1 ◦C to obtain the cell free
broth containing extracellular cutinase. All purification steps were
carried out at 0–4 ◦C unless otherwise indicated. All chromato-
graphic runs were monitored for protein at 280 nm. The enzyme
was purified in four steps. The crude enzyme was precipitated with
finely powdered ammonium sulphate at 80% saturation. The pre-
cipitate was collected by centrifugation at 8000 rpm for 30 min and
dissolved in minimal amount of 20 mM Tris–HCl buffer (pH 8.0) and
dialyzed against the same buffer for 24 h. Dialyzed ammonium sul-
phate fraction was applied to a 2 cm × 50 cm CM-650 TOYOPEARL®
column (Tosho Corporation, Tokyo, Japan) and eluted using linear
gradient of NaCl (0–500 mM). After ion exchange chromatography
two steps of gel filtration chromatography was used (Sephadex
G100 and Sephacryl S-300 (Sigma)) to get purified homogenous
cutinase enzyme. This purified enzyme was used for further
study.
2.3.1. GC analysis of esters
Synthesis of fatty acid ester was analyzed by gas chromatograph
(Varian 390). The diluted aliquots of the reaction mixture were
injected into CPSIL 8CB column and compounds were detected by
FID. The injector and detector temperature were set at 250 ◦C. The
program (temperature and time) and retention time (tR) used for
different esters are given below.
Butyl butyrate: 150 ◦C for (0.5 min) −15 ◦C/min-250 ◦C (10 min);
•
•
•
•
•
•
•
•
•
•
•
•
•
tR 2.42 min
Butyl valerate: 150 ◦C for (0.5 min) −15 ◦C/min-250 ◦C (10 min);
tR 3.14 min
Butyl hexanoate: 150 ◦C for (0.5 min) −15 ◦C/min-250 ◦C
(10 min); tR 4.38 min
Butyl octanoate: 150 ◦C for (0.5 min) −30 ◦C/min-250 ◦C (10 min);
tR 6.65 min
Butyl decanoate: 150 ◦C for (0.5 min) −30 ◦C/min-270 ◦C
(10 min); tR 15.5 min
Butyl Palmitate: 150 ◦C for (0.5 min) −30 ◦C/min-300 ◦C (10 min);
tR 20.3 min
Ethyl butyrate: 100 ◦C for (0.5 min) −15 ◦C/min-150 ◦C (10 min);
tR 1.36 min
Ethyl valerate: 100 ◦C for (0.5 min) −15 ◦C/min-150 ◦C (10 min);
2.2. Cutinase assay
tR 2.55 min
Ethyl hexanoate: 50 ◦C for (0.5 min) −30 ◦C/min-160 ◦C (10 min);
The cutinase hydrolytic activity was measured by following the
hydrolysis of p-nitrophenyl butyrate (p-NPB) (Sigma) as substrate.
An aliquot of (0.02 ml) culture supernatant was added to 0.98 ml of
reaction mixture, which was prepared by adding 1 ml 23 mM pNPB
in tetrahydrofuran to 40 ml of 50 mM potassium phosphate buffer
containing 11.5 mM sodium deoxycholate. The reaction was moni-
tored for 15 min at 37 ◦C and absorbance of released p-nitrophenol
was measured at 410 nm. One enzyme unit is defined as the amount
of enzyme required to release 1 M of p-nitrophenol per min under
assay conditions. The cutinase production from B. cepacia NRRL B
2320 was confirmed previously (data not shown) using cutinase
specific substrate, p-nitrophenyl (16 methyl sulphone ester) hex-
adecanoate (p-NMSH).
tR 2.15 min
Pentyl butyrate: 150 ◦C for (0.5 min) −30 ◦C/min-250 ◦C (10 min);
tR 3.58 min
Hexyl butyrate: 150 ◦C for (0.5 min) −30 ◦C/min-250 ◦C (10 min);
tR 4.18 min
Octyl Butyrate: 150 ◦C for (0.5 min) −30 ◦C/min-250 ◦C (10 min);
tR 5.43
Decyl butyrate: 100 ◦C for (0.5 min) −30 ◦C/min-150 ◦C (10 min);
tR 2.20 min
2.4. Kinetic study for butyl butyrate synthesis
Originally Michaelis–Menten equation was derived for kinetics
of single substrate reaction. However, a reaction involving two sub-
strates may also thought to obey the Michaelis–Menten kinetics, if
the reaction rate depends on the concentration of both the sub-
strates, so that if one substrate concentration varied while other
maintaining constant, the reaction behaves like a single substrate
reaction obeying Michaelis–Menten kinetics.
2.3. Enzymatic synthesis of alkyl esters
In the standard protocol, ester synthesis was carried out in
screw-capped test tubes as bioreactors. Unless otherwise speci-
fied, 0.1 mM of enzyme in lyophilized powder form (calculated on
the basis of molecular mass of enzyme as obtained from SDS PAGE