Development of novel support for penicillin acylase and its application in 6-aminopenicillanic acid production

Article abstract of DOI:10.1016/j.mcat.2019.110484

There is an ever-increasing demand for the β-lactam bulk intermediate 6-aminopenicillanic acid (6-APA) that has wide applications in the synthesis of newer generations of semisynthetic penicillins. It is commercially synthesized by biocatalytic transformation using penicillin acylase. Since the enzyme is soluble, immobilization on a solid porous support is necessary to make the catalyst recycleable and the process profitable. In this study, we developed a novel support of siliceous foam entrapped in a polymer matrix. Penicillin acylase was covalently immobilized on aminopropyl functionalized mesocellular foam silica (MCF) and was further cross-linked using glutaraldehyde without deactivation and upto 95% efficiency. The resulting biocatalyst had an activity of 1185 IU. mg^?1 and demonstrated improved resistance to the substrate and product inhibition. These parameters along with improvement in pH and thermal stability enhanced 6-APA yield by 20% in beads. Intrinsic kinetic parameters were calculated from the developed rate equation to deduce enzyme catalytic mechanism.

Full text of DOI:10.1016/j.mcat.2019.110484

MolecularCatalysis476(2019)110484
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Development of novel support for penicillin acylase and its application in 6-
aminopenicillanic acid production
Sonal R. Ayakar, Ganapati D. Yadav⁎
Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai, 400 019, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Penicillin acylase
6-Aminopenicillanic acid
Enzyme entrapment
Polyvinyl alcohol-alginate beads
Mesocellular foam silica (MCF)
There is an ever-increasing demand for the β-lactam bulk intermediate 6-aminopenicillanic acid (6-APA) that
has wide applications in the synthesis of newer generations of semisynthetic penicillins. It is commercially
synthesized by biocatalytic transformation using penicillin acylase. Since the enzyme is soluble, immobilization
on a solid porous support is necessary to make the catalyst recycleable and the process profitable. In this study,
we developed a novel support of siliceous foam entrapped in a polymer matrix. Penicillin acylase was covalently
immobilized on aminopropyl functionalized mesocellular foam silica (MCF) and was further cross-linked using
glutaraldehyde without deactivation and upto 95% efficiency. The resulting biocatalyst had an activity of 1185
IU. mg−1 and demonstrated improved resistance to the substrate and product inhibition. These parameters along
with improvement in pH and thermal stability enhanced 6-APA yield by 20% in beads. Intrinsic kinetic para-
meters were calculated from the developed rate equation to deduce enzyme catalytic mechanism.
1. Introduction
Techniques of immobilization on gels, films, foams, beads, and porous
materials as well as microbial aggregates have been reported. The most
A β-lactam bulk intermediate, 6-aminopenicillanic acid (6-APA) is
used in the synthesis of newer generations of semisynthetic penicillin
[1,2]. 6-APA is synthesized from penicillin G or penicillin V by side
chain hydrolysis. There are two routes for 6-APA synthesis. The entirely
chemical route was used until the 1980s known as ‘Delft cleavage’ [3].
This route posed several sustainability hazards and lost its popularity
due to the involvement of hazardous chemicals, reactions conducted at
very low temperatures; and generation of hazardous waste [4]. The
second route is a single step enzymatic transformation using microbial
penicillin acylase in an aqueous environment [5]. Efficient enzyme
production and recovery have aided the successful implementation of
biocatalysis for 6-APA synthesis. Despite these advantages, the soluble
nature of the enzyme, penicillin G and co-products – 6-APA and phe-
nylacetic acid (PAA) make enzyme reuse and product purification dif-
ficult. Though penicillin acylase is a very well studied class of enzymes,
it suffers from various stability issues. Wild type enzyme lacks thermal
stability and wide pH activity [6]. The reaction of 6-APA formation is
reversible and hence it is difficult to reach 100% conversion (Scheme
1). The products PAA and 6-APA inhibit the enzyme further reducing
the enzyme activity [7]. These problems are eliminated or dealt by
immobilizing the enzyme and recovery as heterogeneous phase [8–13].
A continuous reactor system has also been investigated [14,15].
recent advancements in PGA immobilization strategy have been in the
selection of mode and support for immobilization. One approach that is
gaining popularity is the employment of thermo-sensitive block copo-
lymers. They provide an advantage of carrying out the catalysis at the
solution stage and raising temperature beyond lower critical solution
temperature (LCST) to cement the polymer with an enzyme for se-
paration. The polymers studied for PGA bear LCST of 39 °C [16] which
is lower than optimal temperature (above 40 °C) observed for PGA ac-
tivity [17]. Magnetic supports have been studied for PGA immobiliza-
tion that offer ease of separation post reaction [17–21]. The iron oxide-
based supports suffer from low surface area and pore volume limiting
enzyme loading capacity [17]. To impart higher porosity to the support
structure various other template-based approaches have been at-
tempted [19] wherein, the immobilization ceiling point was reached by
the highest reported surface area (323 m2. g−1). Moreover, the mag-
netic support particles show high-degraded reactivity, which leads to
their instability and aggregation [22]. Silica has been extensively stu-
died as the support material for PGA immobilization [23–26] due to its
hydrophilic nature and ease of surface modification. Particulate silica
shows lower surface area leading to very low activity retention and
reusability. One such approach involving adsorption, crosslinking and
covalent linking led to activity recovery of maximum 62% [23].
⁎ Corresponding author.
E-mail addresses: gdyadav@yahoo.com, gd.yadav@ictmumbai.edu.in (G.D. Yadav).
https://doi.org/10.1016/j.mcat.2019.110484
Received 24 February 2019; Received in revised form 15 June 2019; Accepted 20 June 2019
2468-8231/©2019ElsevierB.V.Allrightsreserved.

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
Nomenclature
CE
w
k’
concentration of free catalytic sites on E (mol. cm−3
biocatalyst loading (g. cm3)
reaction rate constant for forward reaction of A and B
giving P and Q (cm3. mol−1. min−1
reaction rate constant for reverse reaction of P and Q
giving A and B (cm3. mol−1. min−1
equilibrium constant for complexation of
(cm3. mol−1
equilibrium constant for complexation of
(cm3. mol−1
)
A
penicillin G
B
water
)
P
6-APA
k’1
KA
KB
KP
KQ
D
PAA
)
E
are active sites on the biocatalyst
A
B
with
with
from
from
E
E
E
E
CA
CB
CP
CQ
CP
CA.E
CB.E
CP.E
CQ.E
Ct
concentration of A in bulk (mol. cm−3
)
)
)
)
)
concentration of B in bulk (mol. cm−3
concentration of P in bulk (mol. cm−3
concentration of Q in bulk (mol. cm−3
concentration of P in bulk (mol. cm−3
concentration of A complexed with E (mol. cm−3
concentration of B complexed with E (mol. cm−3
concentration of P complexed with E (mol. cm−3
concentration of Q complexed with E (mol. cm−3
concentration of total catalytic sites on E (mol. cm−3
)
equilibrium constant for dissociation of
P
)
(mol. cm−3)
)
)
)
)
equilibrium constant for dissociation of Q
(mol. cm−3
)
k”
k
actual reaction rate constant for reaction of A and B (cm3
apparent rate constant for reaction of A and B (min−1
)
)
Scheme 1. Enzymatic hydrolysis of penicillin
G to 6-aminopenicillanic acid (APA) and phe-
nylacetic acid (PAA).
In this study we selected MCF as a porous support for immobiliza-
tion due to its high surface area and pore volume available for penicillin
G acylase (PGA). PGA is a large periplasmic protein with a high mo-
lecular weight (86 kDa) and 7 nm × 50 nm × 5.5 nm in size. The active
site is located deep inside the PGA structure at the bottom of cone-
shaped depression formed by intertwining of the two chains of the
enzyme molecule [27]. PGA demonstrates attraction for electronegative
groups through common surface amino acid residues like tyrosine, ar-
ginine, histidine, lysine, serine and threonine. The effects of nature of
porous supports for PGA immobilization [9,25,28] and pore char-
acteristics [28] have been studied in detail. Higher pore size and pore
volume were demonstrated to improve PGA immobilization [28]. The
choice of porous support was based on its having suitable surface and
pore characteristics to provide accommodation and formation of a
monolayer of PGA. Ordered mesoporous silica has well controlled and
uniformly sized pores ranging from 2 to 50 nm; synthesized by the in-
telligent arrangement of the block copolymer (or organic surfactant)
and an inorganic silica source and are employed in enzyme im-
mobilization [29]. Mesocellular foam silica (MCF) among these has
tunable > 15 nm pores making it a suitable candidate as support for
penicillin acylase [30]. MCF has been reported as good support for
various biocatalytic applications [24,31,32]. Another advantage of
employing MCF as support is its flexibility towards surface modification
due to the reactive chemistry of silanol groups [29]. It can be used
directly for immobilization aided by adsorption or coupled with various
techniques like covalent binding or crosslinking. The covalent binding
provides selective and stronger interactions without substantially al-
tering the diffusional properties of the foam. PGA has exhibited inter-
actions with aminopropyl functionalized foam [25]. This work involves
the development of an efficient immobilization strategy to tackle these
issues and to optimize the bioprocess through enzyme kinetic studies. It
provides a comparative study of different modes of immobilization on
MCF. We employed PGA immobilization by covalent attachment on
amino-modified MCF followed by glutaraldehyde crosslinking to
achieve stronger binding. It led to high enzyme loading and activity
retention but lost the enzyme by leaching through repeated use. To
combat this, the pore size of MCF could not be reduced as it has shown
to lower the enzyme loading in the past [33]. Increasing the cross-
linking also negatively impacted the enzyme activity due to loss of
flexibility [34–36]. Entrapment in the polymeric matrix has not only
reported to prevent enzyme leaching but also to improve enzyme sta-
bility through micro-environment regulation [36–39]. Hence, the final
step of entrapment was integrated, and the resulting biocatalyst showed
improved reusability. We also developed a new kinetic model that can
predict the reaction course and parameters.
2. Materials and methods
2.1. Enzyme and reagents
Penicillin G acylase (PGA, EC 3.5.1.11) was obtained as a gift
sample from KDL Biotech Ltd., India in phosphate buffer (pH 7.5). The
source organism of the enzyme is E. coli. Penicillin G potassium salt was
procured from Unimark Remedies, India. Chemicals involved in MCF
support synthesis were tetraethyl orthosilicate (TEOS, Merck, India),
Pluronic P123 triblock copolymer (poly (ethylene oxide)-block–poly
(propylene oxide)-block-poly (ethylene oxide), EO20–PO70–EO20,
MW = 5800) (Sigma Aldrich, India), 1,3,5-trimethylbenzene (TMB)/
mesitylene (Sigma Aldrich, India), ammonium fluoride (Thomas Baker,
India), 3-aminopropyltriethoxysilane (APTS) (Sigma Aldrich, India),
glutaraldehyde 25% w/v aqueous solution (SDFCL, India) and hy-
drogen chloride 37% w/v aqueous solution (Sigma Aldrich, India).
Chemicals used for entrapment were sodium alginate and polyvinyl
alcohol (S.D. Fine Chemicals, Mumbai India), gelatine and starch
(Himedia, Mumbai), calcium chloride, boric acid potassium dihydrogen
phosphate, dipotassium hydrogen phosphate and tris HCl (all Sigma
Aldrich, India). Buffers were suitably prepared in deionized water.
2.2. MCF synthesis and amino-functionalization
MCF was prepared by the hydrothermal method [32]; 2 g of
pluronic P123 was dissolved in 75 cm−3 of 1.6 M aqueous hydro-
chloride solution at room temperature. 4 g of TMB was then added and
the resulting solution was stirred vigorously at 39 °C for 3 h. 4.6 cm3 of
TEOS was added to the mixture and stirring was continued for 24 h. 2.5
cm3 of 0.27 M aqueous ammonium fluoride solution was then added,
and the resulting mixture was kept at 100 °C for 30 h for aging. The
white solid obtained was filtered and washed with deionized water
2

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
until the filtrate was neutral. It was again washed thrice with ethanol,
dried at 100 °C for 12 h and calcined at 550 °C for 6 h. 0.25 g of APTS
was added to 200 cm3 of 1 M aqueous hydrochloride solution at room
temperature and was stirred for hydrolysis. 1 g of MCF was added to it
and the mixture was shaken in an incubator shaker at 25 °C for 8 h at
160 rpm. The suspension obtained was then kept at 100 °C for 24 h. The
resulting powder was filtered and washed with water and ethanol. It
was dried at 80 °C for 12 h. This product is labeled as NH2-MCF.
suspension, glutaraldehyde solution was added, and shaking was con-
tinued for one more hour. The solid obtained was centrifuged and
washed several times with phosphate buffer (0.05 M, pH 7.9) till su-
pernatant was free of protein (confirmed by Bradford protein assay).
The product obtained was named as Crs-Enz-NH2-MCF. Effect of con-
centration of glutaraldehyde was tested from 0.05 M to 0.2 M. For en-
trapment into beads, 5 cm3 Crs-Enz-NH2-MCF was mixed with 10 cm3
polymer solution and the resulting suspension was filled in a syringe
fitted with 22 G × 1¼" (0.7 mm × 30 mm) needle. Beads were cast by
controlled release of the suspension in chilled congealing solution from
a height of 15 cm. The beads obtained were cured in the same solution
for 12 h at 4 °C. Beads were then filtered and washed several times with
water.
2.3. Enzyme crosslinking and entrapment
0.2 g of NH2-MCF was mixed with 10 cm3 of 0.05 M phosphate
buffer of pH 7.9 and wetted under shaking for 1 h. PGA was added to
the mixture and shaken in an incubator shaker at 25 °C or 3 h at
200 rpm. This stage was designated as Enz-NH2-MCF. To the same
Alginate and its composites with starch, gelatine and polyvinyl al-
cohol (PVA) were tested of physical stability using NH2-MCF foam as a
b
d
Fig. 1. (a) Effect of modes of penicillin G acylase immobilization on activity. 1: adsorption on mesocellular foam (MCF), 2: adsorption on MCF followed by
glutaraldehyde crosslinking (Crs-Enz-MCF), 3: covalent attachment on aminopropyl functionalized MCF (Enz-NH2-MCF), 4: covalent attachment on aminopropyl
functionalized MCF followed by glutaraldehyde crosslinking (Crs-Enz-NH2-MCF), 5: covalent attachment on aminopropyl functionalized MCF followed by glutar-
aldehyde crosslinking and subsequent entrapment in alginate-polyvinylchloride beads (SA/PVA-Crs-Enz-NH2-MCF). (b) Effect of support matrix on penicillin G
acylase activity on immobilization and time course study. Free enzyme,
Crs-Enz-NH2-MCF,
SA/PVA-Crs-Enz-NH2-MCF,
Permeabilized SA/PVA-Crs-Enz-
NH2-MCF. (c) Effect of enzyme loading on PGA immobilization as Crs-Enz-NH2-MCF. Percent penicillin G acylase amount retained on immobilization (data label
shows value in mg of protein immobilized per g of the support), percent penicillin G acylase activity retained on immobilization (data label shows value of activity
obtained in IU per g of the support). (d) Effect of glutaraldehyde comcentration on PGA immobilization as Crs-Enz-NH2-MCF. Percent penicillin G acylase amount
retained on immobilization (data label shows value in mg of protein immobilized per g of the support), percent penicillin G acylase activity retained on im-
mobilization (data label shows value of activity obtained in IU per g of the support).
3

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
control. Detailed protocols for making individual beads were: a) algi-
nate beads- sodium alginate (SA) solution in water congealed with
0.2 M calcium chloride solution; b) SA/starch beads- 2% (w/v) SA and
10% (w/v) starch (gelatinized at 70 °C) and congealed with 0.2 M cal-
cium chloride solution; c) SA/gelatine beads- 2% (w/v) SA and 10%
(w/v) gelatine (gelatinized at 70 °C) and congealed with 0.2 M calcium
chloride solution; d) SA/PVA 1–2 % (w/v) SA and 2–10 % (w/v) PVA
(gelatinized at 70 °C) and congealed with 0.2 M calcium chloride and
0.5 M boric acid solution. Permeabilization of beads was carried out by
washing them with phosphate buffer (0.05 M pH 8) and water. They
were then hardened in the congealing solution for 1 h prior to air drying
and stored at 4 °C until use.
the isocratic elution mode in the ratio of 7:3, respectively. Buffer A
contained 5 mM potassium dihydrogen phosphate and 2.4 mM sodium
dodecyl sulphate, a solution of which was adjusted to pH 3 with
phosphoric acid. The column temperature was maintained at 30 °C and
diode array detector set at 215 nm wavelength. The retention time for
penicillin G was 8.2 min, 6-APA was 4.1 min and PAA was 9.9 min. To
study the hydrolysis of penicillin G to yield 6-APA various parameters
(agitation, pH, temperature and substrate concentration) were studied.
Enzyme kinetic analysis was also performed.
3. Results and discussion
3.1. Mode of PGA immobilization
2.4. Protein quantification and enzyme assay
MCF was synthesized with a gravimetric yield of 96% calculated
based on the template. Aminopropyl functionalization had a gravi-
metric yield of 90%. Protein content and activity of the PGA solution
was 23.0 mg.cm−3 and 200 IU. cm−3, respectively. The modes of PGA
immobilization studied were: adsorption on MCF (Enz-MCF), adsorp-
tion on MCF followed by glutaraldehyde crosslinking (Crs-Enz-MCF),
covalent binding on aminopropyl functionalized MCF (Enz-NH2-MCF)
and covalent binding on aminopropyl functionalized MCF followed by
glutaraldehyde crosslinking (Crs-Enz-NH2-MCF). For this purpose, 600
IU PGA was added to 1 g of the support and 0.1 M glutaraldehyde was
used for crosslinking. Results showed that the nature of forces utilized
for immobilization had a significant effect on activity (Fig. 1a). Weaker
interactions of adsorption gave low protein loading and subsequent
activity (26.4 mg protein loading and 220 IU g-1 of Enz-MCF activity).
Stronger interactions of covalent binding achieved higher enzyme
loading and activity (45.6 mg protein loading and 380 IU.g-1 of Enz-
NH2-MCF activity). Glutaraldehyde crosslinking had a positive effect on
activity retention (53.0 mg protein loading and 420 IU.g-1 of Crs-Enz-
MCF activity). Both covalent binding and crosslinking resulted in
higher activity and hence was selected for further studies (67.5 mg
protein loading and 550 IU.g-1 of Crs-Enz-NH2-MCF activity). Reactions
of penicillin G hydrolysis with Crs-Enz-NH2-MCF lost 47% of the ac-
tivity on 4th reuse. The reaction mixture was then tested for the pre-
sence of PGA by Bradford assay. The mixture showed the presence of
protein and hence confirmed the loss in activity due to enzyme leaching
on reuse. Crs-Enz-NH2-MCF was entrapped into polymeric beads (SA/
PVA-Crs-Enz-NH2-MCF) to prevent PGA leaching. The entrapment
caused no change in the activity (570 IU. g−1 of SA/PVA-Crs-Enz-NH2-
MCF activity) and retained 91% activity after 4th reuse. These results
are summarized in Table 2.
The protein content was determined by Bradford protein assay
(bovine serum albumin was used to plot a standard calibration curve).
Enzyme activity assay was performed as reported [25].
2.5. Characterization of immobilized PGA
FT-IR studies were conducted for MCF, NH2-MCF, Crs-Enz-NH2-MCF
to analyze functionalization by using a Bruker IFS-66 single-channel
Fourier transform spectrophotometer. The thin pellet was prepared by
mixing the sample with spectroscopic grade potassium bromide. The
pellet was subjected to a number of scans to record the spectra from
4000 cm−1 to 400 cm−1. XRD analysis was carried out on Bruker dif-
fractometer D8 with Cu-Kα (1.54 Å) radiation for MCF, NH2-MCF. The
X-ray diffraction patterns were recorded in the 2θ from 5 to 50°. The
surface properties were measured by the Brunauer–Emmett–Teller
(BET) method using ASAP 2020 (Micromeritics, USA). Degassing was
necessary prior to analysis with conditions: MCF (at 623 K for 4 h);
NH2-MCF (at 373 K for 4 h); Crs-Enz-NH2-MCF, PVA/SA-Crs-Enz-NH2-
MCF and permeabilized PVA/SA-Crs-Enz-NH2-MCF (at 323 K for 6 h).
Morphological characteristics of MCF, NH2-MCF, Crs-Enz-NH2-MCF and
PVA/SA-Crs-Enz-NH2-MCF were captured with a scanning electron
microscope (Camera SU 30 microscope, JEOL, Japan). TEM was per-
formed for the foam and the beads using JEOL JEM 2100, Japan mi-
croscope. Thermogravimetric analysis (TGA) and differential scanning
calorimetry (DSC) was carried out on STA 6000 Pyris (Perkin Elmer)
series analyzer. About 20 mg of sample was placed in the analyzer and
the analysis was programmed from 30–200 °C with 10 °C/min rise in
temperature. Crs-Enz-NH2-MCF and PVA/SA-Crs-Enz-NH2-MCF were
analyzed by this technique.
The above study concluded that the entrapment improved the reu-
sability as well as the activity of the biocatalyst. There are reports that
the microenvironmental conditions around the enzyme influence its
activity [36,37]. The entrapment has also reported to increase the dif-
fusional limitations and hence a comparative study was planned to
judge the influence of entrapment on the course of the reaction
(Fig. 1b). These reactions were carried out at the following conditions:
6 IU PGA in 30 cm−3 reaction buffer pH 7 containing 10 mM penicillin
G, agitated at 200 rpm and at 50 °C. The free enzyme had the lowest
conversion and immobilization improved the activity of the enzyme.
Crs-Enz-NH2-MCF, SA/PVA-Crs-Enz-NH2-MCF and permeabilized SA/
PVA-Crs-Enz-NH2-MCF reached similar substrate conversion (67% -
68%). Free enzyme reached 56% conversion in 180 min. This verified
that the entrapment had no substantial effect on the activity as well as
mass transfer. Crs-Enz-NH2-MCF had the fastest initial rate
(0.121 mM.min-1) followed by permeabilized PVA/SA-Crs-Enz-NH2-
MCF (0.116 mM.min-1) and PVA/SA-Crs-Enz-NH2-MCF (0.108 mM.min-
1) suggesting time lag to set up uniform concentration profile inside the
beads than that of foam. But after the achievement of uniform con-
centration profile in beads, the rate matched with that of the foam.
Permeabilization of beads did not have an effect on conversion due to
the high catalytic activity of the enzyme and the mass transfer
2.6. Reaction set up and conditions
Cylindrical, flat bottom, baffled glass reactor with an aspect ratio
(H/D) of 2 and of capacity 50 cm3 was used to carry out reactions. The
reactor had an internal diameter of 2 cm. A standard 6 blade-pitched
turbine impeller of 0.7 cm diameter was used for stirring. The impeller
was placed at a height of 0.7 cm from the bottom. The temperature of
reaction mass was maintained using a thermostatic water bath with an
accuracy of 1 °C. A typical reaction consisted of 30 cm3 of 30 mM
penicillin
G potassium salt, 0.3 g PVA/SA-Crs-Enz-NH2-MCF bead
loading in phosphate buffer (10 mM, pH 7.0). The course of reaction for
this study was 3 h in most of the cases. Effect of temperature was tested
from 30 to 60 °C. For all the comparative experiments, activity
equivalent of free PGA and immobilized PGA (on foam or in beads) was
used.
2.7. HPLC analysis of the reaction mixture
The analysis was carried out by reverse phase HPLC (Agilent 1260
infinity) using Agilent Zorbax Eclipse C18 column (250 mm × 4.6 mm
×5 μm). The mobile phase composition was buffer A and acetonitrile in
4

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
inhibitions were already taken care at the speed of agitation of 200 rpm.
of the SieOH chains. The peaks at 1090 cm−1 and 800 cm−1 corre-
spond to the antisymmetrical and symmetrical stretch vibrations of the
Si–O–Si bond, respectively. The peak at 460 cm−1 results from the
Si–O–Si bend vibrations. The peaks at 1635 cm−1 and 3500 cm−1 are
assigned to the bend and stretch vibrations, respectively, of the NeH
bond. The spectra of NH2-MCF showed a drop in transmittance at
1650 cm−1 and 3500 cm−1 indicating amino functionalization. For Crs-
Enz-NH2-MCF, there appeared shrinkage in OeH and NeH stretch
(3500-3300 cm−1) region indicating occupancy of the enzyme at these
sites with covalent binding.
3.2. Effect of enzyme loading
The enzyme distribution affects the specific activity of the im-
mobilized enzyme [25]. Densely distributed enzyme molecules cannot
transition easily to their active form. Hence, it was required to estimate
the optimal loading and determined by varying enzyme concentration
(2.5 to 7.5 cm−3 or 500 to 1500 IU) keeping immobilization conditions
the same. The protein loading and activity went on increasing till en-
zyme loading of 6.25 cm−3. g-1 of NH2-MCF beyond which it did not
change. At this PGA concentration, all sites on support were saturated
(Fig. 1c). Immobilization efficiency was 98% and around 3% enzyme
deactivation occurred from mass balance calculations leading to 95%
activity recovery. The deactivation was higher at lower enzyme loading
due to excessive crosslinking by glutaraldehyde per PGA molecule.
Average crosslinks per enzyme molecule determined the degree of de-
activation. So, PGA loading was optimized to 6.25 cm3. g-1 that
achieved 140 mg.g-1 protein loading and 1185 IU. g-1 activity.
3.6. XRD analysis of the biocatalyst
Fig. 2b gives XRD patterns. A single peak was obtained at 2θ value
of 22.4°. The broad nature of the peak indicates the amorphous nature
of MCF, NH2-MCF and Crs-Enz-NH2-MCF. The full width half maximum
(FWHM) was 6.0° suggesting the high degree of amorphous attitude.
Reduction in the peak intensity from MCF to NH2-MCF to Crs-Enz-NH2-
MCF was observed. The average spacing between the two layers (d) was
observed to be 4.0 Å which suggest the thickness of a single cell layer of
3.3. Effect of glutaraldehyde concentration
Lysine residues on the surface of an enzyme are involved in forming
crosslinks with glutaraldehyde [34] preventing enzyme leaching. Glu-
taraldehyde concentration was varied from 0.01 to 0.2 M (Fig. 1d).
Lower glutaraldehyde concentration leads to insufficient crosslinking
leading to lower activity retention (verified by the presence of PGA in
washings). Higher glutaraldehyde concentration leads to higher cross-
linking resulting in loss of flexibility in the enzyme molecule required
for catalytic activity [34–36]. The enzyme reached maximum im-
mobilization efficiency at 0.1 M. At higher glutaraldehyde concentra-
tion (0.2 M), excessive crosslinking decreased PGA activity. 0.1 M glu-
taraldehyde was therefore employed in the later stages of work. Crs-
Enz-NH2-MCF generated as a result of these steps did leach PGA on use
and hence required entrapment in a polymeric matrix.
3.4. Bead polymer stability
Polymer concentration influences two parameters – bead stability
and mass transfer resistance for reactant, products and enzyme.
Optimum polymer concentration will impart stability to beads, ease
movement of reactant and products, but hinder escape of enzyme mo-
lecules. We tested alginate and composite polymer blends for entrap-
ment. SA concentration was varied from 0.5 to 4 % w/w. 2% SA beads
lost their shape on washings, but 4% SA beads retained their shape.
When they were tested in reaction, the alginate polymer solubilized at
reaction conditions. Hence SA was blended with other polymers to
improve stability. Polymer SA/starch beads leached foam on storage
due to starch solubilization. SA/gelatine beads had high surface in-
stability and aggregated on storage. SA/PVA beads had better stability
and were selected for further perturbations to optimize polymer blend
ratio. 2% w/v SA and 5% w/v PVA led to optimal stability. Crs-Enz-
NH2-MCF trapped in these SA/PVA beads (SA/PVA-Crs-Enz-NH2-MCF)
had an activity of 20 IU. g−1 and exhibited higher thermal stability than
Crs- Enz-NH2-MCF (Fig. 6b). Improvement in the thermal stability of
the enzyme on immobilization has been studied for PGA and other
enzymes [5,31,35,40] that highlight the involvement of favourable
interactions of the enzyme with the support [41]. The beads obtained
were spherical (1–1.5 mm diameter) in shape and stable in 10 mM
phosphate buffer at pH range from 6 to 9.
3.5. FT-IR characterization of the biocatalyst
FT-IR spectra showed (Fig. 2a) a broad peak at 3400-3300 cm−1 is
ascribed to the OeH stretch vibrations of the SieOH chains, whereas
the peak at around 970 cm−1 is attributed to the OeH bend vibrations
Fig. 2. (a) FT-IR spectrum of MCF, NH2-MCF and Crs-Enz-NH2-MCF. (b)
XRD pattern of MCF, NH2-MCF and Crs-Enz-NH2-MCF.
5

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
mesocellular foam (MCF).
3.10. Effect of pH
An enzyme is a polymer of amino acids and its activity is highly
dependent on the pH of the solution. The activity profiles for free and
immobilized PGA (SA/PVA-Crs-Enz-NH2-MCF) at different pH values
were found to vary greatly (Fig. 5b). The reaction catalyzed with free
enzyme attained maximum conversion at 120 min and later tended to
be reversible at higher pH values of 8 and 8.5. Whereas, reaction with
immobilized PGA reached maxima at 180 min and was irreversible at
all pH range tested. Thus pH 7 was used for the further study.
3.7. Surface and pore structure characterization of the biocatalyst
Surface property characterization using ASAP (Table 1) revealed
that the MCF synthesized had
a high surface area (more than
600 m2 g−1) with high pore volume (3.2 cm3. g−1) and large pore size
(28 nm). Surface area decreased on aminopropyl functionalization and
subsequent enzyme immobilization suggesting occupancy of the groups
in pores. Aminopropylation of MCF did not alter the pore volume due to
thin layer binding; but average pore size increased due to blockade of
small pores. Beads had smaller pore size and hence prevented enzyme
leaching. Permeabilization of the beads opened the pores due to swel-
ling but the pore volume remained unchanged. Isotherms of all the
samples showed hysteresis loop between adsorption isotherm and
desorption isotherm depicting mesoporous nature.
3.11. Effect of temperature
The next intrinsic parameter tested was the reaction temperature.
Immobilized PGA had higher activity than a free enzyme, but the trend
remained the same for both. The conversion increased with tempera-
ture and reached maxima at 50 °C. Conversion dropped for immobilized
PGA but remained stable for the free enzyme at 60 °C (Fig. 5c). Im-
mobilized PGA from reaction carried out at 60 °C was recovered and
reused at 40 °C, at which regain in activity was observed. Hence, loss of
activity at higher temperature is attributed to irreversible PGA in-
activation. The reaction temperature of 50 °C was chosen for further
study. Overall higher conversion by the enzyme on immobilization
could be attributed to either improvement in the activity or reduction in
enzyme deactivation or reduction in negative feedback by the products.
To decipher it more, we calculated the energy of activation from Ar-
rhenius plots (Fig. 5d). The energy of activation for the free enzyme was
3.8. Electron microscopic analysis of the biocatalyst
SEM images (Fig. 3a–f) of MCF and Crs-Enz-NH2-MCF showed no
difference in the surface morphology. SA/PVA-Crs-Enz-NH2-MCF image
showed a coating of SA and PVA composite matrix on the foam. The
surface thus appeared bulgy and porous. To have more elaboration
about the topography, high-resolution images were taken. MCF was
made up of spherical particles of particle size of 3–4 μm. The spherical
particles were in an agglomerated state with a smooth surface. The TEM
images (Fig. 4a and b) of Crs-Enz-NH2-MCF showed the presence of
three-dimensional pore system which was composed of uniformly sized
large hexagonal cells interconnected by uniform windows. The particles
had defined and thick wall structure. This justifies the high adsorption
properties of MCF. TEM images of SA/PVA-Crs-Enz-NH2-MCF (Fig. 4c
and d) showed a polymer coat around the foam particles. The film
formed by PVA/SA composite matrix is thin and the foam is successfully
entrapped into it.
6.35 kcal mol−1
and
for
PVA/SA-Crs-Enz-NH2-MCF
was
8.42 kcal mol−1. These values are reasonable for enzymatic reactions.
The higher energy of activation for beads means there is more tem-
perature dependence and the rate of reaction changes significantly for
beads than that for the free enzyme with a change in temperature. It
also reflects the diffusional limitation of the substrate in immobilized
PGA.
3.12. Enzyme reusability and thermal stability
3.9. Effect of speed of agitation
The aim of our study was to improve the reusability of the bioca-
talyst making the process economical. To prove the advantage of PGA
immobilized beads over the previous step of immobilization (Crs-Enz-
NH2-MCF), catalyst reusability study was carried out for both (Fig. 6a).
Reusability was determined for five cycles with similar reaction con-
ditions. Crs-Enz-NH2-MCF addition was done on activity equivalent
basis. SA/PVA-Crs-Enz-NH2-MCF had better reusability than Crs-Enz-
NH2-MCF (Fig. 6a). The formation of polymer matrix layer entrapping
PGA immobilized foam particles prevented leaching of the enzyme. The
protein estimation of reaction mixture filtrate was also done to verify
leaching. PVA/SA-Crs-Enz-NH2-MCF retained 94.3% activity retention
even after fifth use (and 80% after 10th use) where Crs-Enz-NH2-MCF
lost half of its activity after fifth use.
The substrate (penicillin G) molecules are required to reach the
enzyme active site situated inside the support overcoming interparticle
and intraparticle mass transfer resistance for the progress of the reac-
tion. To ensure that the reaction was not diffusion controlled, the
support porosity was kept higher. It was thus assumed that the con-
centration of the substrate on the surface of the supports is the same as
that present inside the pores. To overcome the mass transfer limitation,
an influence of speed of agitation on the progress of the reaction was
studied (Fig. 5a) at a reaction temperature of 40ᵒ C. Penicillin G con-
version increased with increasing agitation until 200 rpm; at speed
higher than 200 rpm, the conversion remained constant. At 300 rpm,
the beads underwent physical damage due to shear and a steep rise in
the conversion was observed. Reusability of broken biocatalyst reduced
drastically. Hence optimal agitation speed was 200 rpm.
To study the thermal stability (Fig. 6b) of biocatalysts, they were
subjected to 50ᵒ C in phosphate buffer (10 mM, pH 7) under static
conditions for a few days until the activity drop was significant. Free
Table 1
Surface area and pore characteristics of different stages of penicillin G acylase immobilization.
Steps →
1
2
3
4
5
Parameter
MCF
605.4
3.2
NH2-MCF
484.6
3.3
Crs-Enz-NH2-MCF
203.6
0.7
SA/PVA- Crs-Enz-NH2-MCF
193.0
0.6
Permeabilized SA/PVA- Crs-Enz-NH2-MCF
158.5
0.6
BET surface area (m2 g−1
Pore volume
)
(BJH adsorption)
(cm3 g−1
)
Pore size
28.0
33.1
13.5
13.8
15.0
(BJH adsorption)
(nm)
6

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
Table 2
Immobilization parameters for biocatalysts (refer section 3.1).
Mode/s of immobilization
Immobilization yield
(% w/w)
% Activity retained
% Enzyme deactivation
Enz-MCF
Crs-Enz-MCF
Enz-NH2-MCF
Crs-Enz-NH2-MCF
SA/PVA-Crs-Enz-NH2-MCF
Adsorption
Adsorption followed by crosslinking
Covalent attachment
Covalent attachment followed by crosslinking
Covalent attachment followed by crosslinking followed by entrapment
38.3
76.8
66.1
97.8
97.8
36.7
70.0
63.3
91.7
94.8
1.6
6.8
2.8
6.1
3.0
Fig. 3. SEM image (a) MCF. (b) MCF. (c) Crs-Enz-NH2-MCF. (d) Crs-Enz-NH2-MCF. (e) SA/PVA-Crs-Enz-NH2-MCF. (f) SA/PVA-Crs-Enz-NH2-MCF.
enzyme lost 90% activity in 24 h. SA/PVA-Crs-Enz-NH2-MCF and Crs-
Enz-NH2-MCF retained 68% and 55% of their activity respectively until
the 7th day suggesting improvement in the thermal stability due to
immobilization.
enzyme showed more activity and conversion. At higher penicillin G
concentration (30 and 50 mM), the PGA/SA-Crs-Enz-NH2-MCF had
more conversion and activity. This suggests that the immobilized en-
zyme has increased resistance to the substrate inhibition even at higher
substrate loading. The lower activity at a low concentration of substrate
can be justified by diffusional limitations that were even evident from
Arrhenius plot. The reaction of hydrolysis is highly selective, and no
other side-products were seen. Conversion decreased with increasing
substrate concentration in both cases (Table 3).
3.13. Effect of substrate concentration
The enzymatic action of PGA involves two reactants- penicillin G
salt and water. Water being solvent, is in excess and penicillin G salt is
the limiting reactant. The conversion and reaction rate decreased with
an increase in the concentration of penicillin G due to the inhibitory
effect of substrate on biocatalyst [5] (Fig. 7a and 7b). This inhibitory
effect is more dominant in the case of free enzyme than SA/PGA-Crs-
Enz-NH2-MCF. At low concentration (10 mM) of penicillin G, the free
3.14. Reaction kinetics
Initial rates of reactions were determined for varying substrate
concentration. PGA is non- competitively inhibited by the substrate-
7

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
Fig. 4. TEM images (a) Crs-Enz-NH2-MCF. (b) Crs-Enz-NH2-MCF. (c) SA/PVA-Crs-Enz-NH2-MCF. (d) SA/PVA-Crs-Enz-NH2-MCF.
Fig. 5. (a) Effect of speed of agitation on SA/
PVA-Crs-Enz-NH2-MCF reaction progress.
Speeds of agitation: 150 rpm, 200 rpm,
250 rpm and
300 rpm. (b) Effect of pH of
the reaction mixture on penicillin G hydrolysis
progress catalyzed by free enzyme and by
SA/PVA-Crs-Enz-NH2-MCF. (c) Effect of tem-
perature on penicillin G hydrolysis reaction
catalyzed by
free enzyme and by
SA/
PVA-Crs-Enz-NH2-MCF. (d) Arrhenius plots.
Free enzyme and by
MCF.
SA/PVA-Crs-Enz-NH2-
b
d
8

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
b
b
Fig. 6. (a) Reusability of biocatalyst.
SA/PVA-Crs-Enz-NH2-MCF and by
Crs-Enz-NH2-MCF. (b) Thermal stability of biocatalysts. Free enzyme, Crs-
Enz-NH2-MCF and SA/PVA-Crs-Enz-NH2-MCF.
Fig. 7. (a) Initial rates of penicillin G hydrolysis reactions catalyzed by free
enzyme at penicillin G concentrations: 10 mM, 20 mM and 30 mM. (b)
Initial rates of penicillin G hydrolysis reactions catalyzed by SA/PVA-Crs-Enz-
penicillin G [5] and Lineweaver Burk (Fig. 8a) plots for free and im-
mobilized PGA did not cross at the same point. Michaelis Menten
constants (Km) for free and immobilized enzyme were 20.71 mM−3 and
19.31 mM, respectively. The low value of Km suggests that immobilized
enzyme has more affinity than that of free enzyme towards the sub-
strate. Maximum velocity of reaction (Vmax) is 0.38 mM min-1 and
0.29 mM min-1. For all these experiments the enzyme concentration was
0.2 IU. cm−3 of the reaction mixture.
NH2-MCF at penicillin G concentrations: 10 mM,
20 mM and
30 mM.
Table 3
Yield of 6-amino penicillanic acid.
Penicillin G concentration
Yield
(mg of 6-APA per 30 mL of
the reaction mixture)
Usually hydrolysis reactions are treated as uni-bi reactions [42]
indicating that one reactant gives rise to two products, when carried out
in aqueous solutions. The proposed reaction rate equation is as follows:
mM
mg of Penicillin G per 30 cm3 of the
reaction mixture
112.0
335.0
559.0
Free Enzyme
SA/PGA-Crs-Enz-
NH2-MCF
68.1
113.9
152.0
10
30
50
86.2
95.5
109.6
E
A + B → P + Q
(1)
For the above reaction,
K
1
K
A
A. E + B. E ↔ P. E + Q. E
(4)
(2)
(3)
A + E ←→ A. E
P. E ↔ P + E
K
B
K
P
(5)
B + E ←→ B. E
9

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
c
Fig. 8. (a) Lineweaver Burk plot for
concentration. (b) Reaction rate plot for free enzyme at temperatures: 30 °C,
free enzyme and SA/PVA-Crs-Enz-NH2-MCF. V: initial rate or velocity of the reaction, S: substrate-penicillin G acylase
40 °C and 50 °C. x is fractional conversion of penicillin G. (c) Reaction rate plot
for SA/PVA-Crs-Enz-NH2-MCF at temperatures: : 30 °C,
40 °C and
50 °C. x is fractional conversion of penicillin G.
Q. E ←→ Q + E
k = k "e; w
(11)
K
Q
(6)
Expression 9 becomes,
Rate′ expression for the overall reaction can be written as,
Ct2 k1KAKB CA CB
− rA = k CA.E CB.E − k1′ CP.E CQ.E
− r
=
(7)
(8)
initial
(1 + KA CA + KB CB)2
(12)
Also, the total catalytic sites,
Since, KA and KB < < < 1, hence (1 + KA CA + KB CB) is almost
Ct = CA.E + CB.E + CP.E + CQ.E + CE
equal to 1
Also, B is in excess, hence,
Substituting this in the rate expression (8) we get,
− r
= k CA
(13)
initial
2
′
Ct (k1KAKB CACB − k1 KP KQ CP CQ)
− rA
=
Here, k = Ct2 k1 kA kB
(1 + KACA + KB CB + KP CP + KQ CQ)
(9)
Hence, for the initial kinetically controlled reaction of penicillin G
hydrolysis using PGA can be deduced to first (pseudo-first) order re-
action mechanism. Initial reaction rate plots (Fig. 8b and 8c) verified
that both free and immobilized PGA (SA/PGA-Crs-Enz-NH2-MCF) ex-
hibited first order rate kinetics. The apparent rate constants were cal-
culated and reported in Table 4. A higher value of the apparent rate
constant for SA/PGA-Crs-Enz-NH2-MCF indicated a faster reaction.
Only initial rate is considered when adsorption is weak, the con-
centration of reactants is very large than that of products and therefore,
substrate inhibition can also be ignored.
− r
= k CA CB
(10)
initial
Where,
10

S.R. Ayakar and G.D. Yadav
MolecularCatalysis476(2019)110484
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Kinetic constants for penicillin G hydrolysis reaction.
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k
k"
(cm3
k
k"
(min−1
)
g
−1 min−1
)
(min−1
)
(cm3
g
−1 min−1
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4. Conclusions
Support of siliceous foam entrapped in the polymer matrix was
developed. A systematic study was undertaken to understand the role of
each component. Penicillin acylase was covalently immobilized on
aminopropyl functionalized mesocellular foam and was further cross-
linked using glutaraldehyde, which was highly stable and active, with
only 3% deactivation and 94.8% efficiency. At this step, 140.8 g of
protein loading and 1185 IU of penicillin acylase activity was obtained
per gram of foam. Mesocellular foam silica (MCF) has high porosity and
wide interconnected pore structures, and hence has a disadvantage of
enzyme leaching. Hence it was then entrapped into polyvinyl alcohol
(5%) – alginate (2%) bead with a polymer to enzyme immobilized foam
ratio of 2:1 volumetrically. The enzymatic activity was 20 IU/g- beads.
Enzyme immobilization increased penicillin G conversion from 56% to
68%. The immobilized enzyme also showed improved resistance to
substrate inhibition and exhibited an absence of reversible reaction at
high pH (above 8) unlike free enzyme. All these improvements in in-
trinsic parameters were the result of favorable conformational changes
induced and resistance to deactivation mechanisms due to im-
mobilization. The immobilized enzyme in bead showed improved pH
stability and highest thermal activity at 50 °C. Both enzyme im-
mobilized foam and the foam entrapped bead showed the same con-
version on first use without much difference in rate indicating the ab-
sence of diffusional limitations. But, the activity of foam decreased to
52.4% due to enzyme leaching whereas, 90.6% enzyme activity was
retained for beads. Beads had improved thermal stability and could be
stored with 70% retention of activity for a week at elevated tempera-
ture. The novel supported biocatalyst was characterized at each step.
Finally, a rate expression was also deduced for penicillin G hydrolysis to
6-APA and kinetic parameters were calculated. This is an example of
biocatalysis where smart construction of the immobilized enzyme not
only yielded improved physical parameters but also reduced mechan-
isms of enzyme deactivation of catalytic promiscuity.
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nanobiocatalyst by immobilizing penicillin acylase onto magnetic nanocrystalline
cellulose and its use for efficient synthesis of cefaclor, Chem. Eng. J. 346 (2018)
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lytic properties of acylase I from Aspergillus melleus immobilized on amino- and
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template removal methods and their effects on enzyme immobilization, J. Porous
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Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
S.R.A. acknowledges junior research fellowship (DBT, GoI). G.D.Y.
gratefully acknowledges support as R.T. Mody Distinguished Professor
Tata Chemicals Darbari Seth Distinguished Professor of Leadership and
Innovation and J.C. Bose National Fellow (DST, GoI).
[25] J. Zhao, Y. Wang, G. Luo, S. Zhu, Covalent immobilization of penicillin G acylase on
aminopropyl-functionalized mesostructured cellular foams, Bioresour. Technol. 101
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jmbi.2001.5043.
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