Immobilization of 3-hydroxybenzoate 6-hydroxylase onto functionalized electrospun polycaprolactone ultrafine fibers: A novel heterogeneous catalyst

Article abstract of DOI:10.1016/j.reactfunctpolym.2014.05.010

3-Hydroxybenzoate 6-hydroxylase (3HB6H), an enzyme that catalyzes regiospecific para-hydroxylation of aromatic compounds, was successfully immobilized onto electrospun polycaprolactone (PCL) ultrafine fibers (424 ± 99 nm in diameter). The fibers were fabricated from 13% w/w PCL (MW ～8 × 10⁴ g/mol) dissolved in a mixed solvent of formic acid (25% v/v) and acetic acid (75% v/v) at an applied voltage of 16 kV and a fiber collection distance of 12.5 cm. Before being immobilized with 3HB6H, the surface of electrospun PCL fibers was functionalized by reacting with ethylenediamine activated with 5% v/v glutaraldehyde using aluminum sulfate as a catalyst. Effects of the immobilization process on pH tolerance and thermal stability of 3HB6H were investigated. Results indicated that 3HB6H immobilized onto PCL fibers could tolerate the changes in temperature and pH better than the free enzyme could. Therefore, the 3HB6H-immobilized PCL fibers are potentially useful as a heterogeneous catalyst under the conditions in which free 3HB6H could not endure.

Full text of DOI:10.1016/j.reactfunctpolym.2014.05.010

Accepted Manuscript
Immobilization of 3-hydroxybenzoate 6-hydroxylase onto functionalized elec-
trospun polycaprolactone ultrafine fibers: a novel heterogeneous catalyst
Tiyaporn Srisook, Thammasit Vongsetskul, Jeerus Sucharitakul, Pimchai
Chaiyen, Pramuan Tangboriboonrat
PII:
S1381-5148(14)00106-0
DOI:
http://dx.doi.org/10.1016/j.reactfunctpolym.2014.05.010
REACT 3380
Reference:
Reactive & Functional Polymers
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Revised Date:
Accepted Date:
13 January 2014
28 April 2014
23 May 2014
Please cite this article as: T. Srisook, T. Vongsetskul, J. Sucharitakul, P. Chaiyen, P. Tangboriboonrat,
Immobilization of 3-hydroxybenzoate 6-hydroxylase onto functionalized electrospun polycaprolactone ultrafine
fibers: a novel heterogeneous catalyst, Reactive & Functional Polymers (2014), doi: http://dx.doi.org/10.1016/
j.reactfunctpolym.2014.05.010
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1
Immobilization of 3-hydroxybenzoate 6-hydroxylase onto functionalized
electrospun polycaprolactone ultrafine fibers: a novel heterogeneous
catalyst
Tiyaporn Srisooka, Thammasit Vongsetskula*, Jeerus Sucharitakulb, Pimchai Chaiyenc,
Pramuan Tangboriboonrata
aDepartment of Chemistry, Faculty of Science, Mahidol University, Rama VI Road,
Ratchathewi, Bangkok 10400 Thailand
bDepartment of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Henri
Dunant Road, Patumwan, Bangkok 10330 Thailand
cDepartment of Biochemistry and Center of Excellence in Protein Structure and Function,
Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400
Thailand
*Corresponding author’s e-mail: thammasit.von@mahidol.ac.th, Tel: +66 2201-5933, Fax:
+66 2354-7151
Abstract
3-Hydroxybenzoate 6-hydroxylase (3HB6H), an enzyme that catalyzes regiospecific para-
hydroxylation of aromatic compounds, was successfully immobilized onto electrospun
polycaprolactone (PCL) ultrafine fibers (424 99 nm in diameter). The fibers were fabricated
from 13 %w/w PCL (MW ~ 8 x 104 g/mol) dissolved in a mixed solvent of formic acid
(25% v/v) and acetic acid (75% v/v) at an applied voltage of 16 kV and a fiber collection
distance of 12.5 cm. Before being immobilized with 3HB6H, the surface of electrospun PCL
fibers was functionalized by reacting with ethylenediamine activated with 5% v/v
glutaraldehyde using aluminium sulfate as a catalyst. Effects of the immobilization process
on pH tolerance and thermal stability of 3HB6H were investigated. Results indicated that
3HB6H immobilized onto PCL fibers could tolerate the changes in temperature and pH
better than the free enzyme could. Therefore, the 3HB6H-immobilized PCL fibers are
potentially useful as a heterogeneous catalyst under the conditions in which free 3HB6H
could not endure.
Keywords

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Electrospinning; ultrafine fiber; enzyme immobilization; polycaprolactone; 3-
hydroxybenzoate 6-hydroxylase
1. Introduction
3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a dimer of nicotinamide adenine
dinucleotide (NADH) specific flavoprotein mono-oxygenase that contains one flavin
adenine dinucleotide (FAD) per subunit of 47 kDa [1-3]. This enzyme can catalyze the
para-hydroxylation of 3-hydroxybenzoate (3HB) at C6 to form 2,5-dihydroxybenzoate (2,5-
DHB) in Rhodococcus jostii RHA1 (Fig 1). This reaction is part of lignin biodegradation via
the gentisate pathway [1-4]. As it can use several aromatic compounds as substrates, the
enzyme has been proposed as a catalyst for performing regiospecific hydroxylation of
aromatic compounds, including phenolic compounds which are major environmental
pollutants found in wastewater from oil refineries, coking plants, coal gasification plants,
phenol resin industry and pharmaceutical processes [5].
Fig 1
Similar to other enzymes which are useful for catalyzing the reaction specifically and
efficiently, 3HB6H cannot tolerate harsh conditions such as extreme temperature and pH
[6]. In addition, it is difficult to be reused because of being homogeneous catalyst. One of
the most practicable solutions to overcome these obstacles is enzyme immobilization onto
surface of solid support via adsorption, binding, and encapsulation [7]. By binding enzyme
molecules via covalent bonds, the immobilized enzyme can be strongly attached on the fiber
surface, and, hence, can efficiently tolerate the reaction conditions.
However, the activity of enzymes immobilized on the surface of solid support normally
reduced from the free enzyme. Therefore, a suitable solid support with high surface area to
volume ratio, i.e., nanomaterials, is in demand to increase the amount of the bound enzymes.
Among several types of nanomaterials such as mesoporous silica, nanotube and
nanoparticle, the ultrafine fiber with tunable porosity and pore size to give the most efficient
immobilization is of great interest [6].

3
In general, an electrospinning technique has been used for fabrication of ultrafine fibers
because it gives the convenience to control the fiber dimension and is capable of producing
ultrafine fibers in a continuous way [8,9]. Moreover, it is simple to be setup by applying
only three major components: a high-voltage power supply, a container where a polymer
liquid (i.e., a solution or melt) is placed with a small opening to be used as a nozzle, and a
conductive fiber collection device. Under the influence of a high electric field, a partially
spherical droplet of the polymer liquid at the capillary tip is deformed into a conical one.
The increase in the electric field causes an ejection of a charged stream of the polymer
liquid, which deposits in a random fashion on the collection device in the form of a
nonwoven fabric [10].
This technique was used for preparation of enzyme-immobilized ultrafine fibers. For
example, urease was immobilized onto electrospun polyvinylpyrrolidone ultrafine fibers (7
– 86 nm) to fabricate a highly sensitive urea sensor [11], and glucose oxidase was also
attached on electrospun poly(vinyl alcohol) ultrafine fibers to produce a highly sensitive
glucose sensor [12]. Furthermore, an enzyme-immobilized solid support could be used as a
bioreactor such as lipase-immobilized poly(acrylonitrile-co-2-hydroxyethylmethacrylate)
electrospun membranes [13],
and
glucose oxidase-immobilized
poly(vinyl
alcohol)/chitosan/tea extract electrospun nanofibrous membranes [14].
In this study, 3HB6H was immobilized onto the surface-functionalized electrospun
polycaprolactone (PCL), a water-insoluble biocompatible and chemical-resistant polymer,
fibers to enhance the enzyme tolerance to changes in temperature and pH. The effects of
reaction temperature, medium pH, and reusability of the 3HB6H and 3HB6H-immobilized
PCL ultrafine fibers (3HB6H-PCL) on the 3HB6H activity were investigated comparatively
by an assay based on NADH oxidation via a spectroscopic method.
2. Experimantal
2.1 Materials
Acetic acid (≥ 99% purity) (RCI Labscan; Thailand), ethanol (≥ 99% purity) (RCI Labscan;
Thailand), ethylenediamine (EDA) (≥ 99% purity) (Sigma-Aldrich; USA), formic acid (≥
96% purity) (RCI Labscan; Thailand), glutaraldehyde (GA) (50% w/v) (Sigma-Aldrich;
USA), sodium 3-hydroxybenzoate (3HB) (Merck; USA), nicotinamide adenine dinucleotide

4
(NADH) (≥ 94% purity) (Cal-Biochem; Germany), PCL (≥ 99.5% purity) with MW of
80,000 g/mol (Sigma-Aldrich; USA), and tris(hydroxymethyl)aminomethane (Tris buffer)
(≥ 99% purity) (Bionoetikas; Thailand) were used as received. 3HB6H from Rhodococcus
jostii RHAl containing an N-terminal His6 tag was prepared according to the previous
protocol [2].
2.2 Fabrication of PCL ultrafine fibers
13 wt% PCL solution was prepared by dissolving PCL pellets (0.65 g) in 25% v/v formic
acid mixed with 75% v/v acetic acid (5 ml) and stirred for 3 h [15]. A high voltage power
supply (D-ES50PN-20w/QAM, Gamma High Voltage Research, USA) was used to charge
the solution loaded into a glass syringe by connecting the emitting electrode of positive
polarity to the nozzle. The grounding electrode, a fiber collection plate, was an aluminum
sheet. The applied electrical potential, the fiber collection distance, the fiber collection time,
the flow rate, and the inner diameter of needle were 16 kV, 12.5 cm, 3 h, 1 ml/h, and 0.9
mm, respectively. The morphology of PCL ultrafine fibers, sputter-coated (E – 102, Hitachi,
Japan) under vacuum, was observed by scanning electron microscope (SEM, S-2500,
Hitachi, Japan). An average diameter of the electrospun fiber mats was measured directly
from SEM at 15 kV using a Photoshop 5.0 (n ≥ 50).
2.3 Immobilization of 3HB6H onto PCL ultrafine fibers
In order to covalently immobilize 3HB6H onto the electrospun PCL ultrafine fibers, the
reactive functional groups, i.e., amine, carboxylic, and hydroxyl groups, on PCL fiber
surfaces were required. Mats of PCL fibers with an average thickness of 0.06 cm were cut
into 1 x 1 inch2 and sloughed from alumina foil before being immersed into 99% w/v EDA
solution for 2 – 5 h. After rinsing by ethanol (100 ml) and water twice, the sample was dried
at room temperature for 24 h [16,17], and, then, characterized by attenuated total
reflectance-Fourier transform infrared spectroscopy (ATR-FTIR; Equinox 55, Bruker,
Germany).
The amino groups of 3HB6H were activated by immersing the fibers into the mixture of 5%
v/v GA solution (1 ml) and aluminium sulfate (AS) (0.02 g) for 3 h [18]. Then, the fiber
mats washed with distilled water (100 ml) thrice were suspended in 150 µM 3HB6H (1 ml)
at 6º C for 48 h. When the reaction completed, the excess enzyme was removed by washing

5
with Tris-H2SO4 (100 ml) thrice. The resulting 3HB6H-immobilized electrospun PCL fibers
(3HB6H-PCL) were examined by ATR-FTIR, SEM, and a homemade ramé-hart contact
angle goniometer calibrated by purified water and absolute ethanol to study polarity of fiber
surface. The contact angle measurements were conducted in triplicate. The specific surface
area, total pore volume, average pore size of the pristine PCL fibers and 3HB6H-PCL were
measured using a Quantasorb JR Autosorp-1 gas sorption system (Quantachrome, Boynton
Beach, FL) by the BET technique. All the samples were degassed at 300°C before
measurement.
2.4 Enzyme activity assay
Enzyme activity of free 3HB6H, i.e., absorbance per second (A/s), was measured by a
continuous assay based on NADH oxidation. Absorbance values of the mixtures were
recorded using a UV-Vis spectrophotometer at 360 nm (ε360 = 4.26 mM-1cm-1) (Agilent
8453, Agilent Technologies, USA). Assay reactions in triplicate were performed in 0.1 M
Tris-H2SO4 buffer (pH 8) at 25 ⁰C in a reaction medium (1 ml) containing 0.2 mM NADH
and 1 mM 3HB. The apparent catalytic constant (kcat(app)) of 3HB6H was calculated from the
plot of initial rates of NADH oxidation versus the 3HB6H concentrations (0.01, 0.05, 0.1,
0.2, 0.4, 0.6 and 1.2 µM). One unit of activity is defined as 1 µmole NADH consumed/min.
The enzyme activity of 3HB6H-PCL (1 inch2) was evaluated by the aforementioned method
in triplicate. Since the immobilized enzyme has a much greater Km value for NADH than
3HB6H in solution, the reaction was monitored at 390 nm (ε390 = 1.23 mM-1cm-1) to avoid
reaching the detection limit of a spectrophotometer. To determine an apparent Michaelis-
Menten constant (Km(app)) of 3-hydroxybenzoate (3HB), the 3HB concentrations of 20, 40,
80, 160, 320, and 640 uM were used in the assays that contained 1 mM NADH. To measure
the Km(app) of NADH, the NADH concentrations of 0.1, 0.2, 0.4, 0.8, and 1 mM in the assays
that contained 10 mM 3HB were used. The kcat(app) of 3HB6H-PCL was measured from the
assay that contained 1 mM NADH and 10 mM 3HB.
The amount of immobilized 3HB6H on the PCL fibers was determined by measuring the
absorbance of the 3HB6H solutions before and after the enzyme immobilization at 452 nm
(ε452 = 11 mM-1cm-1 [2]). The difference in the absorbance of enzyme solutions before and

6
after the 3HB6H immobilization was equivalent to the amount of 3HB6H attached on the
PCL fibers. These measurements were conducted in triplicate.
2.5 Effect of reusability, temperature and pH on enzyme activity
The effect of reusability of 3HB6H-PCL (10 times) on their enzyme activity was assessed
by the method mentioned in Section 2.4. The results of each measurement were compared
with the starting enzyme activity. To study the effect of temperature on the enzyme activity,
both 3HB6H-PCL and free 3HB6H (0.05 µM) were incubated at 37 and 50 °C. 3HB6H-PCL
(30 mg) were washed by 0.1 M Tris-H2SO4 at pH 8.0 (100 ml) thrice and were immersed in
the reaction medium (5 ml). The assay reaction was performed at 25 °C. Absorbance of the
reaction was measured at 360 nm every 30 s to monitor the initial rate of NADH oxidation.
Samples were collected as 1 ml portion for the measurement and were added back into bulk
solutions after absorbance reading. All the experiments were run in triplicate.
The effect of pH (5.5, 6, 7, 8, 9 and 10) on the enzymatic activity was investigated by either
immersing 3HB6H-PCL (30 mg) or adding 3HB6H in the reaction medium containing 0.1
M ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), 52 mM Tris and 52 mM
o
ethanolamine at 6 C for 15 days. The enzyme activities of 3HB6H-PCL and free 3HB6H
were measured in triplicate by the similar process, and the results were compared to each
other under the same conditions.
3. Results and discussion
3.1 ATR-FTIR of surface functionalized PCL ultrafine fibers
The relationship between time of fiber immersion and the amount of amide groups on the
surface of fibers treated with EDA solution was investigated by ATR-FTIR spectroscopy.
The spectra of both untreated and EDA-treated PCL fibers at various immersion times (2-5
h) are shown in Fig 2.
Fig 2
All the spectra show the characteristic peaks of PCL chains at 2950 cm-1 (CH2 stretching),
1725 cm-1 (C=O stretching), and 1300-1000 cm-1 (C-O stretching of PCL chains). After

7
chemical modification, the small peaks at 1630 (C=O stretch of amide groups) and 1570 cm-
1
(N-H deformation of amide groups) prove the success of introducing the amide groups
onto the fiber surface. With increasing the reaction time from 0 to 4 h, the amount of amino
groups on the fiber surface increased, and the amide peaks of 5 h-treated PCL fibers were as
high as those of 4 h-treated ones. Therefore, the chosen time to functionalize the PCL fibers
was 4 h.
3.2 Analysis of 3HB6H-PCL
The morphology and fiber diameters of the untreated and EDA-treated PCL ultrafine fibers
were observed under SEM and the micrographs of PCL fibers, EDA-treated PCL fibers, and
3HB6H-PCL are shown in Fig 3(a)-(c).
Fig 3
In Fig 3(a), the smooth and uniform ultrafine fibers with an average diameter of 191 45 nm
were observed. After the EDA-functionalization, PCL fibers with higher average diameter
of 424 99 nm and rougher surface were obtained as shown in Fig 3(b). The rough and
wrinkled surface was probably due to the PCL chain scissions and the shrinkage of PCL
fibers. Fig 3(c) reveals that the 3HB6H-PCL with diameter of 424 112 nm were intact
during the 3HB6H immobilization process. To confirm that both EDA functionalization and
3HB6H immobilization onto PCL increased the polarity to PCL fibers, the contact angle of
the untreated PCL fibers, EDA-treated PCL fibers, and 3HB6H-PCL was studied and the
images of the ramé-hart contact angle goniometer are displayed in Fig 4.
Fig 4
Due to the existence of amino groups, the EDA and GA-treated PCL fibers (Fig 4(b)) were
much more hydrophilic than the PCL counterparts (Fig 4(a)). Similarly, the highest
hydrophilicity of 3HB6H-PCL (Fig 4(c)) was caused from several polar functional groups
such as carboxylic, amine, and amide groups on their surface. Both the EDA and GA-treated
PCL fibers and 3HB6H-PCL were then analyzed by ATR-FTIR, and the spectra are shown
in Fig 5.

8
Fig 5
In the spectra (a) and (b), the peaks at 2900 cm-1 (CH2 stretching), 1750cm-1 (C=O
stretching), and 1200 cm-1 (C-O stretching of PCL fibers) were observed. Small peaks at
1500-1600 cm-1 attributing to amide groups were also noticed. Differently, the spectrum
5(b) shows the broad overlapped peak at 3000-3500 cm-1 corresponds to carboxylic and
amine groups of 3HB6H. Hence, the results confirmed that the 3HB6H was successfully
immobilized onto the EDA-treated PCL fibers.
The BET results revealed that the specific surface area, total pore volume, and average pore
size of the pristine PCL ultrafine fibers and 3HB6H-PCL were 74.5 m2/g, 1.22 x 10-1 cm3/g
and 65.3 Å for the pristine PCL fibers, and were 17.7 m2/g, 4.84 x 10-2 cm3/g and 110 Å,
respectively for 3HB6H-PCL. The decrease in the specific surface area and total pore
volume with the increased average pore size after aminolysis and enzyme immobilization
was probably resulted from the chain scission of PCL chains and the shrinkage of PCL mats.
This probably led to the increase in the fiber diameter.
The amount of immobilized 3HB6H molecules on 3HB6H-PCL was determined by the
method mentioned in Section 2.4. The results revealed that the amount of 3HB6H on the
PCL fibers was ~0.199 µmol/inch2. The kcat(app) of the free enzyme was assessed from the
plot of the initial rate of NADH oxidation (µM/s) versus the free 3HB6H concentration
(µM) as shown in Fig 6.
Fig 6
Fig 6 reveals that the kcat(app) of the free 3HB6H was 21 s-1. The kinetic parameters obtained
from the experiments described in Section 2.4, i.e. kcat, Km(app) of 3HB, and Km(app) of
NADH, were used to represent the different activities of the free and immobilized 3HB6H
as shown in Table 1.
Table 1
The parameters shown in Table 1 indicate that the Km(app) of 3HB and NADH of the
immobilized enzyme was ~3 and ~52 times greater than that of the free enzyme,

9
respectively. In addition, kcat(app) of 3HB6H-PCL, i.e. the enzyme activity of the immobilized
enzyme, was ~1,180 times less than that of the free enzyme.
3.3 Effect of reusability, temperature and pH on the enzyme activity
The reusability of 3HB6H-PCL, an important index for being a good heterogeneous catalyst,
is illustrated by the graph of the residual activity of 3HB6H-PCL versus the reuse time as
shown in Fig 7.
Fig 7
Fig 7 shows that the activity of 3HB6H-PCL decreased approximately 4% per each reused
time. The loss in 3HB6H activity related to the inactivation of enzyme possibly was caused
by the denaturation of protein [6]. However, after three-repeated uses, the activity of the
3HB6H-PCL slightly decreased and was almost constant at > 60% of the original 3HB6H
activity. It was because the 3HB6H, a protein, molecules were bound to the PCL fibers
making their conformation relatively stable to the free enzyme in the aqueous solutions.
Thus, the immobilization of enzyme on PCL fibers allowed the effective reuse of 3HB6H.
The effects of incubating temperature and incubating time on the %residual activity of both
3HB6H and 3HB6H-PCL were studied. The %residual activities plotted with incubation
time at 37 and 50 oC are shown in Fig 8 (a) and (b), respectively.
Fig 8
Fig 8 (above) revealed that the residual activities of 3HB6H (light gray) and 3HB6H-PCL
(dark gray) at 37oC decreased with increasing incubation time. After 3 h of incubation, the
residual activities of 3HB6H-PCL were higher than 50%, which were more than those of
3HB6H because of the covalent bonds between 3HB6H and PCL leading to the high
conformational stability of the enzyme at high temperature. With increasing temperature,
the residual activities of free enzyme abruptly decreased from 100 to 5% within 15 min,
whereas those of 3HB6H-PCL were ~20% under the same reaction time. This indicated that
almost all of the 3HB6H molecules were denatured [6]. Therefore, the enzyme
immobilization process improved the durability of the enzyme at 50oC.

10
The effects of pH (5.5-10) of the reaction mixture on the activities of 3HB6H and 3HB6H-
PCL incubated at room temperature for 15 days were then examined. The data are presented
in Fig. 9.
Fig 9
In Fig. 9, the maximum activities of free and immobilized enzymes were obtained at pH 7
and 8, respectively. At pH 5.5- 8, the residual activity of free enzyme was clearly less than
that of the immobilized one. It was explained that the free enzyme molecules could be
denatured easier [6]. However, at pH 9 and 10, the enzyme activity of 3HB6H was higher
than that of the immobilized enzyme. It was probably because PCL, a polyester, could be
degraded in the alkaline conditions via the hydrolysis reaction. Then, the 3HB6H molecules,
bound to the surface of the fibers by more than one amine groups of the enzyme, were
probably destroyed during the hydrolysis of PCL. In addition, with increasing the storage
time, the enzyme activity decreased.
4. Conclusions
3HB6H was successfully immobilized onto electrospun PCL fibers with diameters of
424 99 nm. First, the PCL fibers were prepared from 13 %w/w PCL (MW ~ 8 x 104 g/mol)
at 16 kV and 12.5 cm. Then, they were treated by EDA for 4 h, GA, and 3HB6H,
respectively. The success of 3HB6H immobilization on the PCL fibers was confirmed by
ATR-FTIR spectra. The study on the kinetics of NADH degradation revealed that the
amount of loaded enzyme on PCL ultrafine fibers was 131 27 µg enzyme/g fibers.
Moreover, 3HB6H-PCL could be reused at least 10 times with >60% of the original 3HB6H
activity at pH 8 and 25 °C, whereas 3HB6H could not be reused. Furthermore, 3HB6H-PCL
tolerated the changes in temperature (37 and 50° C), pH (5.5 – 8.0), and storage time (15
days) better than the 3HB6H did. Hence, the 3HB6H-PCL is potentially used as a
heterogeneous catalyst under the conditions in which free 3HB6H cannot tolerate.
ACKNOWLEDGEMENTS
Research grants from The Thailand Research Fund (TRF)/Mahidol University
(TRG5680070) to T.V., from TRF/Commission on Higher Education to P.T.
(RTA5480007), from Thailand Ministry of Science and Technology to T.V., from Mahidol

11
University Faculty of Science to T.V., from TRF (RSA5580050) to J.S., and from TRF
(RTA5680001) to P.C. are gratefully acknowledged.
References
[1] S. Montersino, W.J.H. van Berkel, Functional annotation and characterization of 3-
hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1, Biochim. Biophys. Acta
1824 (2012) 433-442.
[2] J. Sucharitakul, T. Wongnate, S. Montersino, W.J.H. van Berkel, P. Chaiyen, Reduction
kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1, Biochemistry
51 (2012) 4309-4321.
[3] J. Sucharitakul, C. Tongsook, D. Pakotiprapha, W.J. van Berkel, P. Chaiyen, The
reaction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodoccocus jostii RHA1
provide an understanding of the para-hydroxylation enzyme catalytic cycle, J. Biol. Chem.
288(49) (2013) 35222-35236.
[4] C.N. Jones, R.A. Cooper, Catabolism of 3-hydroxybenzoate by the gentisate pathway in
Klebsiella pneumoniae M5a1, Microbiology 154 (1990) 489-495.
[5] L. Zídková, J. Szőköl, L. Rucká, M. Pátek, J. Nešvera, Biodegradation of phenol using
recombinant plasmid-carrying Rhodococcus erythropolis strains, Int. Biodeterior.
Biodegrad. 84 (2013) 179-184.
[6] Z.G. Wang, L.S. Wan, Z.M. Lui, X.J. Huang, Z.K. Xu, Enzyme immobilization on
electrospun polymer nanofibers: an overview, J. Mol. Catal. B-Enzym. 56 (2009) 189-195.
[7] W. Tischer, F. Wedekind, Immobilized enzymes: methods and applications, Chemistry,
200 (1999) 95-123.
[8] S. Ramakrishna, K. Fujihara, W. Teo, T. Lim, Z. Ma, An introduction to electrospinning
and nanofiber, World Scientific Publishing, Singapore, 2005.
[9] N. Bhardwaj, S.C. Kuundu, Electrospinning: a fascinating fiber fabrication technique,
Biotechnol. Adv. 28 (2010) 325-347.
[10] T. Vongsetskul, P. Kongjumnean, P. Sunintaboon, R. Rangkupan, P. Tangboriboonrat,
Electrospun composite fibers of polyvinylpyrrolidone with embedded poly(methyl
methacrylate)-polyethyleneimine core-shell particle, Polym. Bull. 69 (2012) 1115-1123.
[11] K. Sawicka, P. Gouma, S. Simon, Electrospun biocomposite nanofibers for urea
biosensing, Sensor. Actuat. B-Chem. 108 (2005) 585-588.

12
[12] G. Ren, X. Xu, Q. Lui, J. Cheng, X. Yuan, L. Wu, Y. Wan, Electrospun poly(vinyl
alcohol)/glucose oxidase biocomposite membranes for biosensor applications, React. Funct.
Polym. 66 (2006) 1559-1564.
[13] X.J. Huang, A.G. Yu, Z.K. Xu, Covalent immobilization of lipase from Candida rugosa
onto poly (acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for
potential bioreactor application, Bioresource. Technol. 99 (2008) 5459-5465.
[14] L. Ge, Y.S. Zhao, T. Mo, J.R. Li, P. Lin, Immobilization of glucose oxidase in
electrospun nanofibrous membranes for food preservation, Food. Control. 26 (2012) 188-
193.
[15] T. Srisook, P. Chaiyen, J. Sucharitakul, T. Vongsetskul, Fabrication of 3-
hydroxybenzoate 6-hydroxylase immobilized ultrafine fibers by electrospinning technique,
Proceedings of pure and applied chemistry international conference 2013. Chonburi,
Thailand, (2013) 553-556.
[16] Y. Zhu, C. Gao, X. Liu, J. Shen, Surface modification of polycaprolactone membrane
via aminolysis and biomacromolecule immobilization for promoting cytocompatibility of
human endothelial cells, Biomacromolecules 3 (2002) 1312-1319.
[17] G. Irenan, B. Jolanta, Z. Karolina, Chemical modification of poly (ethylene
terephthalate) and immobilization of the selected enzymes on the modified film, Appl. Surf.
Sci. 255 (2009) 8293-8298.
[18] Y. Wang, Y.L. Hsieh, Aldehyde functionalized cellulose support for hydrogels, J. Appl.
Polym. Sci. 118 (2009) 2489-2495.

Figure
1
3HB6H
3-hydroxybenzoate
2,5-dihydroxybenzoate
Figure 1 Reaction of 3-hydroxybenzoate with 3HB6H

2
e)
d)
c)
b)
a)
4000
3500
3000
2500
2000
1500
1000
Figure 2 ATR-FTIR spectra of PCL ultrafine fibers treated with EDA solution for a) 0, b) 2,
c) 3, d) 4, and e) 5 h of immersion time

3
Figure 3 SEM images of a) PCL fibers, b) EDA-treated PCL fibers, and c) 3HB6H-PCL

4
a) 123⁰±3⁰
b) 27⁰±4⁰
c) 0⁰
Figure 4 Images of the ramé-hart contact angle goniometer on a) PCL fibers, b) EDA-
treated PCL fibers, and c) 3HB6H-PCL

5
b)
c)
a)
4000
3500
3000
2500
2000
1500
1000
Wavenumber (cm-1)
Figure 5 ATR-FTIR spectra of (a) EDA-treated PCL fibers, (b) EDA and GA-treated PCL
fibers, and (c) 3HB6H-PCL

6
30
25
20
15
10
5
y = 21.389x
R² = 0.9964
0
0
0.2
0.4
0.6
0.8
1
1.2
[3HB6H] (µM)
Figure 6 The rate of NADH oxidation (M/s) vs. the 3HB6H concentrations (0.01, 0.05, 0.1,
0.2, 0.4, 0.6, and 1.2 µM)

7
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
Reuse time (Times)
Figure 7 Residual activity (%) of 3HB6H-PCL as a function of the reuse time (times) at 25
°C and pH 8

8
(a)
100
80
60
40
20
0
0
15
30
45
60
120
180
Incubation time (min)
(b)
100
80
60
40
20
0
0
15
30
45
60
120
Incubation time (min)
Figure 8 Residual activity (%) and incubation time of free 3HB6H (light gray) and 3HB6H-
PCL (dark gray) at 37oC (a) and 50oC (b)

9
120
100
80
60
40
20
0
5.5
6
7
8
9
10
pHs
Figure 9 Effect of pH on the enzyme activity of 3HB6H (light gray) and 3HB6H-PCL (dark
gray) incubated for 15 days

Table
Types of Enzyme
3HB6H
Km(app) of 3HB (µM) Km(app) of NADH (µM)
kcat(app) (s-1)
21
463 [1]
14245
484 [1]
3HB6H-PCL
~2,500
1.78 × 10-2
Table 1 Steady-state kinetic parameters for 3HB6H and 3HB6H-PCL