Immobilization of Geobacillus pallidus RAPc8 nitrile hydratase (NHase) reduces substrate inhibition and enhances thermostability

Article abstract of DOI:10.1016/j.molcatb.2009.12.011

The nitrile hydratase (NHase) from the thermophilic strain Geobacillus pallidus RAPc8 was investigated for its potential application in the biocatalytic production of amides from nitriles. The recombinant NHase was immobilized to a range of insoluble matrices using various cross-linking agents. The immobilized preparation using EupergitC with 1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC) cross-linking exhibited the highest immobilization efficiency (93%). The pH range and optimal temperature for activity were unchanged by immobilization but the thermostability of the EupergitC-NHase was improved compared with the soluble enzyme; at 60°C the half-life of the immobilized NHase was 330min as compared with 54.5min for the soluble enzyme. Kinetic parameters V_max (4.5μmolmL^-1min^-1), K_m (17.3mM) and k_cat (3543.3min^-1) were obtained for the immobilized NHase at 50°C, as compared with 48.8μmolmL^-1min^-1, 10.2mM and 37777.1min^-1 respectively for the soluble enzyme. The operational stability was improved significantly by immobilization, with 85.7% of initial activity maintained after reuse for eight cycles. Most notably, the EupergitC-immobilized NHase showed substantially lower substrate inhibition (K_i=194.7mM) than the soluble enzyme (K_i=101.0mM). In the presence of various co-organic solvents, EupergitC-EDAC NHase showed statistically higher retention of activity than the non-immobilized control.

Full text of DOI:10.1016/j.molcatb.2009.12.011

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Immobilization of Geobacillus pallidus RAPc8 nitrile hydratase (NHase) reduces
substrate inhibition and enhances thermostability
a
b
c,∗
Idan Chiyanzu , Don A. Cowan , Stephanie G. Burton
a
Department of Chemical Engineering, University of Cape Town, Rondebosch 7701, Cape Town, South Africa
Institute for Microbial Biotechnology and Metagenomics, University of Western Cape, Bellville 7535, Cape Town, South Africa
Biocatalysis and Technical Biology Group, Cape Peninsula University of Technology, Bellville 7535, Cape Town, South Africa
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The nitrile hydratase (NHase) from the thermophilic strain Geobacillus pallidus RAPc8 was investigated
for its potential application in the biocatalytic production of amides from nitriles. The recombinant
NHase was immobilized to a range of insoluble matrices using various cross-linking agents. The
Received 26 September 2009
Received in revised form 9 December 2009
Accepted 10 December 2009
Available online 21 December 2009
®
immobilized preparation using Eupergit C with 1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC)
cross-linking exhibited the highest immobilization efficiency (93%). The pH range and optimal tempera-
®
ture for activity were unchanged by immobilization but the thermostability of the Eupergit C-NHase was
Keywords:
◦
improved compared with the soluble enzyme; at 60 C the half-life of the immobilized NHase was 330 min
Nitrile hydratase
Geobacillus pallidus RAPc8
Immobilization
Eupergit
−
1
−1
as compared with 54.5 min for the soluble enzyme. Kinetic parameters Vmax (4.5 ␮mol mL min ), Km
−
1
◦
(
17.3 mM) and kcat (3543.3 min ) were obtained for the immobilized NHase at 50 C, as compared with
48.8 ␮mol mL min , 10.2 mM and 37777.1 min respectively for the soluble enzyme. The operational
−1
−1
−1
Thermostable enzyme
Thermophilic
stability was improved significantly by immobilization, with 85.7% of initial activity maintained after
reuse for eight cycles. Most notably, the Eupergit C-immobilized NHase showed substantially lower
®
substrate inhibition (Ki = 194.7 mM) than the soluble enzyme (Ki = 101.0 mM). In the presence of various
®
co-organic solvents, Eupergit C-EDAC NHase showed statistically higher retention of activity than the
non-immobilized control.
©
2009 Elsevier B.V. All rights reserved.
1
. Introduction
More recently, isolated enzymes with high selectivity have been
used effectively to catalyse industrial reactions and high volumet-
ric productivity has been achieved by increased biocatalyst loading
[11]. However, in view of the noted instability of NHases, if isolated
and purified NHases are to be applied in industry, enhancing their
stability is seen as one prerequisite for their effective use [12]. The
use of thermostable enzymes isolated from thermophilic micro-
bial sources presents one opportunity to achieve higher stability.
Further, immobilized biocatalysts are often regarded as the appro-
priate form for application of enzymes in industry as they can be
reused, are readily separated from reaction mixtures, are conve-
nient to handle, and allow reduced effluent disposal problems [13].
In laboratory applications, NHases have commonly been used as
immobilized whole cell biocatalysts entrapped in calcium alginate
[14,15] or polyacrylamide gels [16].
The nitrile hydratases (NHases: EC3.5.5.1) catalyze the hydra-
tion of nitriles to their corresponding amides [1]. The versatile
nature of these enzymes, associated with their very broad substrate
specificity, has resulted in a number of commercial applications,
including manufacture of the commodity chemicals acrylamide
[
2], nicotinamide [3] and 5-cyanovaleramide [3], and in treatment
of organocyanide industrial effluents [4]. However, the instability
of many NHases currently limits successful application at com-
mercial scale, particularly in the production of acrylamide and
nicotinamide [5–7]. Consequently, whole-cells are often employed,
in processes which are operated at relatively low temperatures [3],
resulting in reduced reactivity due to diffusion limitation and low
specific activity [8]. Further, many NHases are inhibited at high
concentrations of substrate and product [9]. Attempts to overcome
substrate and product inhibition have been made by keeping these
concentrations low [10], but this compromises industrial capacity
for bulk amide production.
A thermostable NHase was isolated from the moderate ther-
mophile Geobacillus pallidus RAPc8 [17], the gene has been cloned
and over-expressed in E. coli BL21 (DE3) [18]. The NHase con-
sists of two subunits (␣ and ␤) having Mr of 28 and 29 kDa where
the functional structure is heterotetrameric (␣␤)2 [19]. The active
site of the G. pallidus RAPc8 NHase, which is located at interface
III
of the ␣- and ␤-subunits, contains a non-corrinoid cobalt (Co ),
∗ Corresponding author.
E-mail address: Burtons@cput.ac.za (S.G. Burton).
chelated cysteine sulfinic acid residues and main-chain nitrogen
1
381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.011

1
10
I. Chiyanzu et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
atoms (␣Ser114 and ␣Cys115) in what is known as a ‘claw set-
ting’ [20]. In preliminary studies, the enzyme was shown to exhibit
a broad specificity for aliphatic nitriles, and to be highly active
towards heteroaromatic substrates (such as 3-cyanopyridine), but
it showed no reaction with aromatic nitriles [17,18]. Both native
and recombinant enzymes are subjected to considerable substrate
and product inhibition [17,18].
against 25 mM potassium phosphate buffer, pH 7.2. The NHase
extract from HIC was loaded onto a pre-packed HiPrep 16/10
Q-Sepharose FF column equilibrated with 25 mM potassium phos-
phate buffer, pH 7.2. Bound protein was eluted with a linear
gradient of NaCl (0–0.5 M) in 20 mM potassium phosphate buffer,
pH 7.2. Fractions of 2 mL were collected and concentrated by dial-
ysis against 50 mM potassium phosphate buffer pH 7.2.
In this study, immobilization of the purified thermostable
G. pallidus RAPc8 NHase was investigated using a wide vari-
ety of support materials. Here, we show that G. pallidus NHase
2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
®
immobilized to Eupergit C and cross-linked with 1-ethyl-3-
(
dimethylamino-propyl) carbodiimide (EDAC) shows substantially
At each stage of purification, active proteins were analyzed by a
12% SDS-PAGE gel as described previously [17]. Protein bands were
stained with a solution of Coomassie Blue R-250 and the excess dye
was removed with destaining solution.
modified properties, including, importantly, approximately 50%
reduction in substrate inhibition.
2
. Materials and methods
2.5. NHase activity assay
2.1. Chemicals
The conversion of 3-cyanopyridine to nicotinamide was used
Carrier materials tested for immobilization included:
Eupergit C and CM (Röhm, Pharma Polymers, Germany),
Sepabeads Series EC (Resindion, Mitsubishi Chemicals Cor-
as the model reaction to monitor NHase activity. The standard
reaction mixture (3 mL) contained 50 mM of 3-cyanopyridine and
50 mM KH2PO4–K2HPO4 buffer (pH 7.2). The reaction was ini-
tiated by addition of 100 ␮L enzyme solution. All assays were
®
poration, Japan), SE and DEAE-Sephadex
A 50 (Pharmacia),
◦
Dowex (Serva Feinbiochemica), and Super-Q-Toyopearl (Tosoh
Corporation), glass beads (Supelco), and Bio-Beads (Bio-Rad).
Other reagents included 50% glutaraldehyde (Aldrich), 3-
aminopropyltriethoxysilane (Sigma), ethylenediamine (EDA)
Aldrich), 1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC)
Aldrich), trichloroacetic acid (Saarchem), ZnSO4 (Aldrich), AgSO4
Saarchem), CoCl2 (NT laboratories Suppliers) and CuCl2 (Holpro
carried out at 50 C, with stirring (150 rpm), in duplicate. Reactions
were terminated by addition of 0.2 mL of 3 M HCl. Nicotinamide
and 3-cyanopyridine concentrations in the reaction mixture were
determined by analytical HPLC using a Merck Hitachi LaChrom
system with 4.6 mm × 150 mm C18 column at a flow rate of
(
(
(
−
1
1.0 mL min . Separations were performed at room tempera-
ture with the following mobile phase system: acetonitrile-10 mM
KH2PO4–H3PO4 buffer (pH 2.8), 1:4 (v/v). The absorbance was mea-
sured at 235 nm using a UV detector.
Analytics (Pty) Ltd.
2
.2. Microorganism and cultivation
One unit of enzyme activity was defined as the amount of
enzyme that catalyzed the production of 1 ␮mol of nicotinamide
per minute under the standard assay conditions.
The G. pallidus RAPc8 NHase, cloned and expressed in E. coli
BL 21 (DE3) pLysS, was used throughout the study. Cultures were
grown in a medium consisting of 10 g/L bacto-tryptone, 5 g/L yeast-
2.6. Protein detection assay
extract and 10 g/L NaCl in dH O (pH 7.0). A 2 L flask containing
2
−1
1
000 mL medium with 50 ␮g mL ampicillin was inoculated and
Protein concentration was determined using the bovine serum
albumin as the standard and measuring absorbances at 595 nm
[21].
◦
incubated at 37 C with shaking at 220 rpm, then induced by addi-
tion of IPTG to 0.4 mM final concentration. Approximately 30 min
prior to induction, cobalt chloride solution was added to a final
concentration of 0.1 mM. At 30 min intervals, cell growth was esti-
mated spectrophotometrically at A600. After 4 h of cell growth
2.7. Methods of immobilization
(
4
OD
= 0.8), cells were harvested by centrifugation at 7000 × g,
Entrapment in Ca-alginate beads: 20 mL enzyme solution (con-
taining 10 mg protein) was added to 10 mL of 2 or 4% (w/v) sodium
alginate solutions as described previously [22].
Eupergit C and CM: 10 mg of enzyme dissolved in 20 mL of
50 mM potassium phosphate buffer (pH 7.2), were added to 5 g of
beads.
600
◦
C for 15 min and washed with 50 mM KH PO –K HPO buffer,
2 4 2 4
pH 7.2. Harvested cells were suspended in 50 mM KH PO –K HPO
2
4
◦
2
4
®
buffer total volume of 25 mL, pH 7.2, and sonicated at 0 C using a
VirTis Ultrasonic Cell Disrupter (for 10 min, at 30 s intervals breaks).
Disrupted cells were centrifuged at 7500 × g for 30 min, and the
supernatant was retained as cell-free enzyme extract.
Glutaraldehyde- and ethylene diamine-activated Amberlite XAD 4:
1
0 mg of NHase dissolved in 50 mM potassium phosphate buffer
2
.3. Protein purification
(pH 7.2) was added to 5 g of glutaraldehyde- or ethylene diamine-
activated Amberlite-XAD-4 beads (as described previously [23]).
Epoxy-Sepabeads (EC-EP), amino-epoxy Sepabeads (EC-HFA),
ethylendiamino Sepabeads (EC-EA), hexamethylendiamino Sepa-
beads (EC-HA) and metal ion activated Sepabeads (EC-IDA): 10 mg
of enzyme solution in 50 mM potassium phosphate buffer (pH 7.2),
◦
The cell-free enzyme extract was heat-treated at 55 C for
5 min and centrifuged (7500 × g, at 4 C for 15 min) to remove
◦
4
precipitated protein. The supernatant was fractionated with
(
NH ) SO . Proteins precipitating at 20% saturation were collected
4 2 4
◦
2+
2+
2+
2+
by centrifugation (7500 × g, at 4 C for 15 min) and discarded,
and the supernatant was loaded onto a pre-packed Hiload 16/10
Phenyl-Sepharose column on a Pharmacia FPLC system. The column
was equilibrated with 1.0 M ammonium sulphate in 50 mM potas-
sium phosphate buffer, pH 7.2. Elution of bound protein was with
a 1.0–0 M linear gradient of ammonium sulphate in 50 mM potas-
was added to 5 g of each, pre-activated by Ag , Zn , Cu or Co
ions (Resindion, Mitsibishi Chemical Corporation).
Cation and anion exchangers: 10 mg of enzyme solution in 50 mM
potassium phosphate buffer (pH 7.2) was added to 5 g each of Super
Q-Toyopearl, DEAE-Sephadex, SE-Sephadex C 50 and Dowex beads.
Glass beads: 10 mg of enzyme solution in 20 mL of
potassium phosphate buffer was added to 5 g of 3-
aminopropyltrimethoxysilane-activated glass beads [24].
−
1
sium phosphate buffer, pH 7.2 at a flow rate of 3.0 mL min . The
NHase-containing fractions were combined and dialysed overnight

I. Chiyanzu et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
111
®
In all experiments, the ratio of enzyme solution to beads
was kept at 4:1 and the mixtures were incubated with stir-
ring at 150 rpm (in an orbital shaker) at room temperature for
specified time periods: 72–96 h for Eupergit C, Eupergit CM,
EDA-Amberlite XAD-4, glutaraldehyde-Amberlite XAD-4, Epoxy-
Sepabeads (EC-EP), amino-epoxy Sepabeads (EC-HFA), ethylen-
diamino Sepabeads (EC-EA), hexamethylendiamino Sepabeads
2.10. Effect of temperature on soluble and Eupergit C
(EDAC)-immobilized NHase
®
®
◦
Reactions were carried out between 20 and 70 C. 100 ␮L of
purified soluble or immobilized NHase (100 ␮L or 0.15 mg pro-
tein) was added to 2.9 mL of 50 mM phosphate buffer, pH 7.2.
The reaction mixture was pre-incubated for 1 min at the desired
temperature. 3-Cyanopyridine was then added to a final concen-
tration of 50 mM and the reaction mixture incubated for a further
5 min. Reactions were terminated and reaction mixtures analysed
as described above. Relative activity is expressed as percentage of
(
EC-HA) and metal ion-activated Sepabeads (EC-IDA); 42–48 h for
cationic and anionic exchanger resins, glass beads and bio-beads
and 30 min for Ca-alginate. During incubation, residual activity
was assayed periodically. After incubation the supernatant was
removed by decanting, the residual activity re-assayed and the
immobilized biocatalyst washed three times with 50 mM potas-
sium phosphate buffer, pH 7.2. In a separate procedure, EDAC or
◦
the enzyme activity at 50 C, taken as 100%.
®
glutaraldehyde 0.5 w/v (%) were added to the Eupergit C-enzyme
2.11. Effect of co-organic solvents on the NHase activity
biocatalyst to allow cross-linking of the immobilized NHase [23].
The mixture was further incubated for an additional 3 h. Filtrates
were assayed for protein concentration and enzyme activity to
determine the effects of cross-linking on binding and activity yields.
Purified soluble or immobilized NHase (100 ␮L or 0.15 mg pro-
tein) was added to 2.9 mL of 50 mM phosphate buffer, pH 7.2
containing the specified organic solvent at 10 (v/v)%. Reactions
were incubated, terminated and reaction mixtures analysed as
described above. The residual activity in the samples without
organic solvent (buffer only) was taken as 100%.
®
2
.8. Characterization of Eupergit C (EDAC)-immobilized G.
pallidus RAPc8 NHase
2
.8.1. Loading efficiency
Protein loading was determined by measuring the amount of
®
2
.12. Thermal stability of the soluble and Eupergit C
(
EDAC)-immobilized NHase
protein before and after immobilization, and was expressed as load-
ing efficiency (%). The loading efficiencies were calculated as:
Enzyme thermostability was determined by pre-incubating sol-
◦
ubleandimmobilized NHaseat40, 50, 60, and 70 C for upto 90 min.
Aliquots of 100 ␮L of soluble enzyme or biocatalyst (equivalent
to 0.15 mg) were withdrawn at 10 min intervals and the residual
activities measured under the standard assay conditions. Relative
activity is expressed as percentage of the enzyme activity prior to
incubation, taken as 100%.
ꢀ
ꢁ
P − P
i
f
Loading efficiency (%) =
× 100
(1)
P
i
i
where P is the initial amount of protein in the enzyme solution, Pf
is the final amount of protein in enzyme solution after immobiliza-
tion.
®
2
.13. Recycled use of Eupergit C (EDAC)-immobilized NHase
2.8.2. Immobilization efficiency
The extent of immobilization (immobilization efficiency) was
®
The operational stability of the Eupergit C (EDAC)-immobilized
determined by measuring the NHase activity before and after the
immobilization process. The immobilization efficiencies were cal-
culated as:
◦
NHase was measured by repeated 5 min reactions at 50 C using a 3-
cyanopyridinesubstratewithsinglebatches ofbiocatalyst prepared
as described above. At the end of each cycle, a 1 mL sample was
withdrawn, centrifuged, the amount of product formed analyzed
by HPLC and residual activity calculated. The immobilized enzyme
sample was washed with 100 mL KH PO –K HPO buffer (pH 7.2)
ꢀ
ꢁ
aimm
afree
Immobilization effeciency (%) =
× 100
(2)
2
4
2
4
before re-use in a subsequent cycle. Volumetric productivity of each
cycle was measured as expressed as g/L/h.
where aimm is apparent specific activity of immobilized enzyme
(
(
U/mg bound protein) and afree is specific activity of free enzyme
U/mg protein).
2.14. Effect of substrate concentration on NHase activity
®
2
.9. Effect of pH on soluble and Eupergit C (EDAC)-immobilized
G. pallidus RAPc8 NHase
The effect of substrate concentration on activity for both soluble
and immobilized NHase was measured using the 3-cyanopyridine
at various concentrations under otherwise standard reaction con-
ditions.
The effect of pH on the stability of soluble and immobilized
NHase was determined using four buffer systems: acetate/citric
acid (pH 4–6), potassium phosphate (pH 6–8), Tris–HCl (pH 8–10),
and sodium hydrogen phosphate/sodium hydroxide (pH 10–12).
Purified soluble or immobilized NHase (100 ␮L or 0.15 mg pro-
tein) was added to 3.0 mL of 50 mM buffer of specified pH. The
2.15. Substrate specificity and kinetic analysis of NHase
◦
The kinetic constants Km, and Vmax for conversion of het-
mixture was pre-incubated at 50 C for 1 min. 3-Cyanopyridine
eroaromatic nitriles by the soluble and immobilized NHase were
determined by measuring initial reaction rates over a sub-
strate concentration range of 5–100 mM using 2-cyanopyridine,
was then added to a final concentration of 50 mM and the reac-
tion mixture incubated for a further 5 min. The reaction was
stopped by addition of 200 ␮L of 3 M HCl. Samples were centrifuged,
and the supernatant was analyzed by HPLC. Relative activity is
expressed as a percentage of the enzyme activity at pH 7, taken as
3
-cyanopyridine and 4-cyanopyridine. Kinetic constants were cal-
culated using the Hanes–Woolf plot. Experiments were carried out
in duplicate and experimental error was no more than ± 4% (S.D.).
1
00%.

1
12
I. Chiyanzu et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
Fig. 1. pH-activity profiles of soluble and Eupergit®C (EDAC)-immobilized NHase.
Buffers used include: CH3COOH/CH3COONa for pH 4–6 (ꢀ); K2HPO4/KH2PO4 pH 6–8
(ꢁ); Tris–HCl pH 8–10 (ꢂ) and NaHPO4–NaOH pH 10–12 (᭹).
3
. Results and discussion
3
.1. Purification and molecular weight determination of G.
Fig. 2. Temperature-activity profiles for soluble and Eupergit®
immobilized NHase at pH 7.2.
C (EDAC)-
pallidus RAPc8 NHase
Based on methods published previously [17,18] the recombi-
nant G. pallidus RAPc8 NHase was purified to homogeneity from
E. coli cells with a 67% yield and 1.2-fold purification. SDS-PAGE
showed that the enzyme to be more than 95% homogeneous (data
not shown). The enzyme was immobilized and the biocatalyst
characterised with the objective of developing its application for
production of amides of industrial interest.
3.2. Immobilization of G. pallidus RAPc8 NHase
The G. pallidus RAPc8 NHase exhibited its highest activity at
◦
pH 7 (Fig. 1) and temperature 60 C, and the activity was demon-
◦
strated over the temperature range 40–70 C (Fig. 2). However,
the activity was not retained for more than 20 min at tempera-
◦
tures above 50 C (Fig. 3a). Since immobilization is one approach
to enhance the stability of enzymes for use as biocatalysts, this
was investigated in the case of the G. pallidus RAPc8 NHase.
In order to select an optimal immobilization method, various
®
support materials were investigated, including Eupergit C and
CM, Sepabeads Series EC, SE and DEAE-Sephadex A 50 (Pharma-
cia), Dowex, and Super-Q-Toyopearl, glass beads, and Bio-Beads.
Cross-linking and activating reagents including 50% glutaralde-
hyde, 3-aminopropyltriethoxysilane, ethylenediamine (EDA), and
1
-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC) were also
investigated. Loading and immobilization efficiency data for each
support are given in Table 1.
Hexamethylendiamino-activated
Sepabeads
(EC-HA),
(
100%), amino epoxy-activated Sepabeads (EC-HFA) (96.2%),
ethylendiamino-activated Sepabeads (EC-EA) (91.5%) and
®
Eupergit C (90.5%) showed the highest loading efficiencies,
respectively. We attribute this to the presence of the extended
spacer arm affording increased access for the protein. However,
the total protein loading did not correlate with immobilization
efficiency of the NHase (Table 1). With respect to immobilization
®
efficiency, Eupergit C with either EDAC or glutaraldehyde cross-
Fig. 3. Thermostability of soluble [A] and Eupergit®C (EDAC)-immobilized [B]
linking was the optimum support material tested. The higher
immobilization efficiencies obtained with cross-linked matrices
NHase.

I. Chiyanzu et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
113
Table 1
Loading and immobilization efficiencies for G. pallidus RAPc8 NHase prepared by various techniques.
Support
Technique
Total protein loading (mg)
Loading efficiency (%)
Immobilization efficiency (%)
Eupergit C (EDAC)
Eupergit C (GA)
EDA-Amberlite XAD 4
GA-Amberlite XAD 4
Eupergit C
DEAE-Sephadex A 50
SE-Sephadex A 50
Eupergit CM
Dowex®50WX8
EC-IDA-Ag
EC-IDA-Co
EC-EA
Controlled pore glass
EC-HFA
EC-EP
Covalent
Covalent
Covalent
Covalent
Covalent
Anionic
Cationic
Covalent
7.7
5.8
1.8
1.6
9.1
3.5
7.2
8.2
7.4
8.1
3.3
9.2
8.8
9.6
8.1
6.2
8.5
10
77.1
58
92.3
83.4
65.4
53.5
53.1
47.2
42.8
32.1
36.9
34.6
32.4
30.1
28.7
28.5
28.4
25.6
24.8
24.6
24
18.5
15.5
90.5
35.2
72.2
82.3
73.6
81.2
32.5
91.5
88.4
96.2
81.2
61.7
85
Cationic
Metal chelation
Metal chelation
Covalent
Simple adsorption
Covalent
Covalent
Bio-beads
EC-IDA-Cu
EC-HA
EC-IDA-Zn
Simple adsorption
Metal chelation
Covalent
Metal chelation
Anionic
100
7.3
9.5
9.6
8.1
73.2
95.2
95.8
80.8
Super-Q-Toyopearl
20.9
a
4
2
% Sodium alginate
% Sodium alginate
Entrapment
Entrapment
nd
nd
a
nd: not determined.
Table 2
Comparative thermostability of thermophilic NHases (expressed as % residual activity following incubation at given temperatures).
o
Microorganism
Temperature ( C)
Incubation time (min)
Residual activity (% initial)
Reference
Bacillus BR 449
G. pallidus RAPc8
G. pallidus DAC521
Bacillus smithii
Ps. thermophila
Eupergit®C (EDAC)-immobilized G. pallidus RAPc8
60
50
50
55
50
60
120
150
–
90
120
90
100
50
50
50
100
80
[44]
[17]
[45]
[46]
[47]
(This paper)
might be attributed to stabilization of the NHase quaternary struc-
ture. It has been established that chemical cross-linking, either
by use of bifunctional reagents or multipoint covalent immobi-
lization, will commonly stabilize both monomeric and multimeric
protein structures [25–28]. Based on immobilization efficiency as
both free and immobilized NHase showed first order kinetics
and were used to estimate deactivation constant and half-life
values (data not shown). The estimated parameters were k
d
−
1
(immobilized) = 0.0021 min , t1/2 (immobilized) = 330.1 min, kd
−
1
(soluble) = 0.0127 min
and t1/2 (soluble) = 54.5 min. While it is
®
a criterion for production of an efficient biocatalyst, Eupergit C
cross-linked with EDAC was selected for further characterization.
noted that comparisons of thermal stability in NHases are com-
plicated by the fact that researchers have used diverse approaches
[
29], comparative data shown in Table 2 indicate that the immo-
®
3
.3. Performance of the Eupergit C (EDAC)-immobilized NHase
bilized G. pallidus RAPc8 NHase compares favourably with other
thermophilic NHases.
A comparison of the pH-activity profiles of both soluble and
®
immobilized G. pallidus RAPc8 NHase showed no significant differ-
ences, with an optimum pH of approximately 7.0 (Fig. 1), consistent
with the report of Pereira et al. [17].
3.5. Reusability of Eupergit C (EDAC)-immobilized NHase
The potential for recycling of the immobilized biocatalyst was
measured by conducting repeated reactions with the same batch of
biocatalyst under otherwise constant conditions. The Eupergit C
The effect of temperature on the activity of soluble and immobi-
◦
®
lized NHase was determined over the temperature range 20–70 C
(
Fig. 2). The immobilized preparation showed better retention
(EDAC)-immobilized NHase showed only a small decrease in
volumetric productivity after repeated use, from 45 to 40 g/L/h
(50–44 mM/h) after 8 cycles; the retention of activity indicates only
approximately 10% loss of activity in this protein over the 8 cycles.
These reactions were conducted using the equivalent of 5 g pro-
tein per litre of solution in the reactor, which compares favourably
with other reports [10]. In general, this result is consistent with the
of activity after incubation at higher temperatures. This behav-
ior is consistent with conformation stabilization associated with
immobilization/cross-linking. The temperature optima for the sol-
◦
uble and immobilized enzyme preparations were similar (60 C).
®
3
(
.4. Thermostability of soluble and Eupergit C
◦
EDAC)-immobilized NHases
stability of the immobilized enzyme at 50 C (Fig. 3).
®
The thermostability of the soluble and Eupergit C (EDAC)-
3.6. Effect of substrate concentration on G. pallidus RAPc8 NHase
immobilized NHases was compared over a temperature range of
◦
4
0–70 C. In both cases, NHase was functionally stable in the tem-
The effect of substrate concentration on NHase activity was
studied by using varying concentrations of 3-cyanopyridine (5, 10,
20, 50, 100, 200, 300, 400, and 500 mM) under standard assay con-
ditions. Profiles of soluble and immobilized NHase are shown in
Fig. 5. Maximal rates were achieved at 100 mM substrate for the
◦
perature range of 40–50 C over 80 min of incubation (Fig. 3a
and b). However, at 60 C, the immobilized NHase exhibited sig-
nificantly higher stability than that of the soluble enzyme. Plots
of the natural log of relative activity as a function of time for
◦

1
14
I. Chiyanzu et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
Fig. 6. The effect of organic solvents on the activity of soluble and Eupergit®
EDAC)-immobilized G. pallidus RAPc8 NHase.
C
(
ally exhibited reduced activity compared to the co-solvent free
control (Fig. 6). Solvent-dependent enzyme inactivation is gener-
ally attributed to disruption of the protein hydration layer, either
directly impacting catalytic function or reducing observed catalytic
rates as a result of protein denaturation [31–33]. G. pallidus RAPc8
NHase has seven bridging water molecules involved in mainte-
nance of the geometry of the ␣ and ␤ dimeric structures [34]. It
is suggested that disruption of water molecules involved either
the dimer interface or the dimer–dimer association may accelerate
inactivation via dissociation of the quaternary structure. Notably,
bi-phasic organic/aqueous systems (such as with benzene and
toluene) did not result in significant losses of enzyme activity
(Fig. 6). In almost all cases, the immobilized enzyme preparation
retained higher levels of activity under identical incubation con-
ditions (Fig. 6). These data are consistent with the stabilization of
quaternary structure resulting from multi-point covalent linkages
and intra-protein crosslinking [28].
Fig. 4. 3-Cyanopyridine substrate inhibition of soluble and Eupergit®C (EDAC)-
immobilized NHase.
soluble enzyme, with higher substrate concentrations leading to a
rate reduction. This is consistent with findings reported for other
NHases which also exhibit substrate inhibition at high substrate
concentrations [36].
Experiments using immobilized G. pallidus RAPc8 NHase over
the same substrate range (5–500 mM) showed
a markedly
increased optimal substrate concentration (300 mM) with
enhanced enzyme performance (compared with the soluble
enzyme) at even higher substrate concentrations (Fig. 4). The
mechanism by which immobilization reduces substrate inhibition
is unclear. We speculate that conformational stabilization resulting
from covalent immobilization and cross-linking may be responsi-
ble for preventing deleterious conformational changes that occur
in the NHase structure during the substrate–enzyme binding
process [33], particularly at higher substrate concentrations.
Experimental data were fitted to the Haldane model (Fig. 5a and
b) and inhibition constants were determined using GraphPad prism
3.8. Kinetic parameters for G. pallidus RAPc8 NHase
Kinetic constants for G. pallidus RAPc8 NHase were determined
using three heteroaromatic nitrile substrates; 2-cyano-, 3-cyano-
and 4-cyanopyridine over a concentration range of 5–100 mM
5
software (Hearn Scientific). Calculated inhibition constants for the
®
soluble and Eupergit C (EDAC)-immobilized NHase preparations
were 110 and 195 mM, respectively.
(
4
Table 3). Vmax values for the soluble NHase were estimated as
5.6, 31.3, and 13.1 ␮mol mL min respectively for substrates 3-
− −1
1
3
.7. Effect of biphasic organic and aqueous solvents on NHase
cyanopyridine, 2-cyanopyridine, and 4-cyanopyridine. The relative
values for the three substrates are consistent with the extent of acti-
vation of the nitrile carbon resulting from electron delocalization
across the heteroaromatic ring.
activity
The use of organic solvents may be desirable in biocatalysis
®
involving the conversion of non-polar substrates such as aromatic
nitriles. Since the G. pallidus RAPc8 NHase showed selectivity for
aromatic substrates [18], its activity in a range of organic media
was investigated. When different water–miscible organic solvents
were used as co-solvents (at 10%, v/v), the soluble NHase gener-
A comparison of constants of the soluble and Eupergit C
(EDAC)-immobilized NHase preparation on 3-cyanopyridine
(Table 3) shows approximately 10-fold reduction in Vmax and
2-fold increase in Km. Such a substantial reduction in catalytic effi-
ciency is attributable to the immobilization of enzyme molecules
Fig. 5. Non-linear regression Haldene model fitted to substrate inhibition data of (a) soluble [R2 = 0.857, Sy. x = 1 2.8] and (b) Eupergit®C (EDAC)-immobilized [R2 = 0.9504,
Sy. x = 2.85] G. pallidus RAPc8 NHAse.

I. Chiyanzu et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115
115
Table 3
Kinetic parameters for soluble and Eupergit®C (EDAC)-immobilized G. pallidus RAPc8 NHase.
Vmax (ꢀmol mL 1 min
−
−1
)
Km (mM)
kcat (min
−1
)
kcat/Km (mM min
−1
−1
)
R2
Substrate
2
3
4
3
-Cyanopyridine
-Cyanopyridine
-Cyanopyridine
-Cyanopyridine (immobilized NHase)
33.8
48.1
15.3
4.5
5.3
10.2
8.7
26598
37777
12078
3543
5018
3703
1388
204
0.9894
0.9951
0.9802
0.9830
17.3
in inactive or substrate-blocked configurations, and to diffusional
limitations typically associated with the use of high surface area
porous immobilization matrices [41–43].
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4
. Conclusion
The G. pallidus RAPc8 NHase, previously found to be active on
[21] M. Bradford, Anal Biochem. 72 (1976) 248–254.
[
[
[
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production of nicotinamide and related aromatic amides. However,
its relatively poor thermostability and notable substrate inhibi-
tion are disadvantageous in this regard. The results presented
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