Immobilization of E.

coli expressing
Bacillus pumilus CynD in three
organic polymer matrices

Maria L. Carmona-Orozco & Aram

J. Panay

Applied Microbiology and

Biotechnology

ISSN 0175-7598
Volume 103
Number 13

Appl Microbiol Biotechnol (2019)

103:5401-5410
DOI 10.1007/s00253-019-09859-z

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Applied Microbiology and Biotechnology (2019) 103:5401–5410
https://doi.org/10.1007/s00253-019-09859-z

ENVIRONMENTAL BIOTECHNOLOGY
Immobilization of E. coli expressing Bacillus pumilus CynD in three

organic polymer matrices

1 1
Maria L. Carmona-Orozco & Aram J. Panay

Received: 8 March 2019 / Revised: 12 April 2019 / Accepted: 15 April 2019 / Published online: 7 May 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Cyanide is toxic to most living organisms. The toxicity of cyanide derives from its ability to inhibit the enzyme
cytochrome C oxidase of the electronic transport chain. Despite its high toxicity, several industrial processes rely on the
use of cyanide, and considerable amounts of industrial waste must be adequately treated before discharge. Biological
treatments for the decontam- ination of cyanide waste include the use of microorganisms and enzymes. Regarding the use
of enzymes, cyanide dihydratase (CynD), which catalyzes the conversion of cyanide into ammonia and formate, is an
attractive candidate. Nevertheless, the main impediment to the effective use of this enzyme for the biodegradation of
cyanide is the marked intolerance to the alkaline pH at which cyanide waste is kept. In this work, we explore the
operational capabilities of whole E. coli cells overexpressing Bacillus pumilus CynD immobilized in three organic polymer
matrices: chitosan, polyacrylamide, and agar. Remarkably, the immobilized cells on agar and polyacrylamide retained more
than 80% activity even at pH 10 and displayed high reusability. Conversely, the cells immobilized on chitosan were not
active. Finally, the suitability of the active complexes for the degradation of free cyanide from a solution derived from the
gold processing industry was demonstrated.

Keywords Bioremediation . Cyanide . Cyanide dihydratase . Immobilization . Whole cells . Gold mining effluents

Introduction 1
Faculty of Natural Sciences, Universidad Icesi, Calle 18 No 122-
135, Cali, Colombia
Cyanide is a toxic compound that occurs in a variety of
natural and anthropogenic environments. The toxicity of
cyanide is primarily attributable to its ability to interrupt
the electronic transport chain by blocking the enzyme
cytochrome C oxidase (Cummings 2004). Despite its high
toxicity, more than 1 mil- lion tons of cyanide per year are
used in industrial processes, such as chemical synthesis,
electroplating, plastic manufactur- ing, paints, and gold and
silver extraction (Logue et al. 2010). All of these industrial
activities generate waste that must be adequately treated to
avoid possible environmental catastro- phes due to
accidental spills (Johnson 2015).

Electronic supplementary material The online version of this

article (https://doi.org/10.1007/s00253-019-09859-z) contains
supplementary material, which is available to authorized users.

* Aram J. Panay
joelpanay@gmail.com
 There are several available chemical treatments for
cyanide removal, most of which are based on the use of
sulfite oxida- tion, H2SO5 or hydrogen peroxide (Kuyucak
and Akcil 2013). In addition, biological treatments have
also been developed that are based on the use of
microorganisms and enzymes (Akcil 2003; Dash et al.
2009; Gupta et al. 2010). Regarding the use of enzymes
for the bioremediation of cyanide waste, cyanide
dihydratase (CynD) and cyanide hydratase (CHT) have
been suggested as viable alternatives (Thuku et al. 2009;
Martínková et al. 2015; Park et al. 2017).
Nevertheless, the intolerance of these enzymes to alkaline
pH (Jandhyala et al. 2005), a mandatory condition to
avoid cyanide volatilization (Mekuto et al. 2016), presents
the main impediment to the enzymatic biodegradation of
cyanide.
CynD enzymes are bacterial in origin and feature
helical quaternary structures that may reach molecular
masses of more than 400 kDa (Park et al. 2017). The
CynD enzyme from Bacillus pumilus recombinantly
expressed in Escherichia coli exhibits kinetic parameters
that are similar to the native en- zyme (Jandhyala et al.
2003), has an optimal temperature close to 38 °C, is
completely inactive at pH > 8.5, and is inhibited by Hg 2+
(Jandhyala et al. 2005). Recent advances in protein
engineering yielded CynD mutants displaying
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greater tolerance to an alkaline pH and thermostability Expression of CynD

(Crum et al. 2015; Crum et al. 2016). Nevertheless, the
most pH tolerant mutant retained 40% of its activity at pH Single colonies of E. coli BL21(DE3) transformed with the
9.5 and was inactive at pH 10. Therefore, an even more plasmid PD451-BpumCynD were grown in LB medium for
robust catalyst is required for the bioremediation of 16 h at 37 °C and 300 rpm. Fresh cultures were initiated
cyanide wastewater. with a
The immobilization of these enzymes or whole cells ex-
pressing them in polymer matrices may allow for
operational advantages, such as greater stability under a
very alkaline pH, and other extreme conditions associated
with industrial cya- nide solutions. Thus, we explored the
characteristics of immobilized complexes of whole E. coli
cells expressing the Bacillus pumilus CynD enzyme in
three organic polymer ma- trices: chitosan,
polyacrylamide, and agar. Additionally, we tested the
ability of active complexes to degrade free cyanide from a
solution derived from the gold processing industry.

Materials and methods

Culture media and reagents

All bacterial strains were grown in Luria-Bertani (LB)

broth (Merck Millipore, Burlington, MA, USA) or LB
supplement- ed with kanamycin (Gibco, Life Technologies,
Grand Island, NY, USA). Tris HCl, NaCN, KCN, and
TEMED were pur- chased from Thermo Fisher (Waltham,
MA, USA). Picric ac- id, glycine, isopropyl-β-D-
thiogalactopyranoside (IPTG), EDTA, sodium phosphate,
NaCl, glutaraldehyde (50%), acetic acid (99.7%), chitosan
(medium molecular weight), and Tris base were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
MOPS was purchased from Biomatik (Wilmington, DE,
USA). Miniprep kits were purchased from Omega Bio-Tek
(Norcross, GA, USA). Ultrapure MQ water was obtained
using an Arium® Pro Ultrapure system (Sartorius,
Gottingen, Germany).

Bacterial strains and plasmids

The bacterial strains Escherichia coli DH5α, used for

DNA replication, and Escherichia coli BL21(DE3), used
for protein production, were from Merck (Burlington, MA,
USA). The gene sequence coding for wildtype Bacillus
pumilus C1 cya- nide dihydratase (CynD) was codon-
optimized for E. coli, and cloned into vector PD451 by
DNA 2.0 (Atum, Newark, CA, USA) with a hexahistidine
affinity tag in its C-terminal region. This plasmid, named
PD451-BpumCynD, is available upon request.
100-fold dilution of the overnight culture into fresh LB 0.2 M KCl to obtain a uniform strand size of 3–7 mm in
medi- um supplemented with kanamycin at 25 μg/mL. diameter. Both the cubes and the strands were stored in
When the OD600nm reached 0.5–0.9, the cultures were 50 mM sodium phosphate buffer (pH 7.0) at 4 °C.
induced with
0.4 mM IPTG. The cells were harvested 4 h after
induction by centrifugation for 30 min at 4 °C and 5800
rpm using an Eppendorf 5804R centrifuge
(Eppendorf, Hamburg,
Germany). Cell pellets were immediately used or stored at
−
80 °C until further use. Untransformed E. coli BL21(DE3)
were grown following a similar procedure but without the
addition of antibiotics or IPTG. This culture served as a
neg- ative control for cyanide degradation. CynD
expression was monitored by SDS-PAGE and quantified
by densitometry using an Amersham Imager 600 imager.

Immobilization in chitosan

For immobilization of E. coli-CynD in chitosan spheres,

0.3 g of chitosan was diluted in a solution of 10 mL of 2%
acetic acid. To this solution, 0.3 g of cells resuspended in
1 mL of 20 mM Tris-base buffer pH 8.0 was added. This
mix was dropped into 50 mL of a solution that contained
methanol and 1 M sodium hydroxide (1:4) (Jyoti et al.
2017). The chi- tosan spheres were recovered by filtration
and incubated in a solution of 2% glutaraldehyde, as an
intercalating agent, for 10 min. Additionally, to test the
effect of the mesh on activity, spheres without
glutaraldehyde were synthesized. The spheres were
washed with 100 mM sodium phosphate buffer (pH 7.0)
and stored at 4 °C in the same buffer. To determine if
diffusion of the substrate was the cause of the low activity
observed, the pore size of the particles was increased by
reducing the chito- san to 1%. Additionally, permeabilized
(5% toluene, 6 mM EDTA in 50 mM Tris-base buffer pH
8.0) or sonicated E. coli- CynD cells were used for
immobilization on chitosan.

Immobilization in agar

The immobilization of E. coli-CynD on agar was

performed using the method of Woodward (1988). Cell
suspensions of
0.3 and 0.6 g of E. coli-CynD resuspended in 1 mL of
0.9% NaCl (w/v) were immobilized by entrapment in
0.4% agar. The agar solution was cooled to 50 °C before
the addition of the cells and polymerization at room
temperature, and the resulting gel was fragmented into 0.7
× 0.7 mm cubes. For the production of small agar beads,
the methodology of Ahamad and Kunhi (2011) was
followed. In this case, a 0.4% solution of agar in 4 mL of
0.9% NaCl (w/v), maintained at 50 °C, was mixed with
the cell suspension of 0.6 g of
E. coli-CynD. This mixture was dropped into 50 mL of
cold
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quantified at 520 nm, and a calibration curve is used to
Immobilization in acrylamide determine the cya- nide concentration in the sample. The
cyanide concentration was determined using a standard
Cell suspensions of 0.3 and 0.6 g of E. coli-CynD in 1 mL curve ranging between 0 and
of 20 mM sodium phosphate buffer (pH 7.0) were 4.0 mM cyanide. The samples from the solutions derived
immobilized by entrapment in polyacrylamide by mixing from
with 1.875 μL of acrylamide monomers and N,N-
methylenebisacrylamide at a molar ratio of 39:1, 50 μL of
10% ammonium persulfate, 3 mL of 20 mM sodium
phosphate buffer (pH 7.0), and 4 μL of TEMED. The
polymerization reaction was incubated for 20 min at room
temperature. The resulting gel was fragmented into cubes
of 0.7 × 0.7 mm or smaller beads and stored in 50 mM
sodium phosphate buffer (pH 7.0) at 4 °C.

Activity determinations

For free cyanide degradation capacity, 0.3 g of free or

immobilized E. coli-CynD cells was incubated with 5 mM
NaCN in 20 mM sodium phosphate buffer (pH 8.0). All as-
says were performed at room temperature with constant
mixing. Aliquots from the solutions were withdrawn at
30 min, and the content of cyanide was determined using
picric acid methodology. Cyanide concentrations were used
to calculate the percentage of cyanide removed by the
catalyst. All experiments were performed in triplicate.
Additionally, cyanide removal activity experiments were
performed for free
E. coli-CynD cells in the presence of 2% acetic acid and
2% glutaraldehyde and for chitosan-immobilized E. coli-
CynD in the presence of 1 M NaCl. Control experiments
included de- termination of cyanide removal after addition
of E.coli trans- formed with an empty vector and addition
of polymers with- out cells.
For initial velocity studies with free or immobilized E.
coli- CynD, cell suspensions of 100 μL that contained
0.092 mg of total protein were used. For the kinetic assays,
the polyacryl- amide support was fragmented into small
particles to reduce its radius, and the agar support was
polymerized in KCl to obtain small strands. Reactions
were performed in 1 mL of the solutions with different
cyanide concentrations. Each re- action was run for 5 min
and was sampled every minute. The variations in cyanide
concentrations with time were used to calculate initial
reaction rates. Graphs of initial velocity vs cyanide
concentration were fitted to the Michaelis-Menten equation
to obtain KM and Vmax values.

Cyanide determination

The cyanide concentration was determined using the

method of Fisher and Brown (1952). In this method, the
reaction product between cyanide and picric acid is
the activity assays were mixed with equal volumes of
picric acid 1% and 0.5 M sodium carbonate. The reaction
was incu- bated at 90 °C for 5 min and stopped by the
addition of dis- tilled water. The absorbance was
measured at 520 nm using a GENESYS 10S
spectrophotometer (Thermo Scientific, Waltham, MA,
USA). The assay solution was diluted when the sample
concentration was > 4.0 mM so that the absorbance at 520
nm fell within the linear range of the standard curve.
The percent cyanide removal was calculated as follows:

½Initial cyanide]−½Final cyanide]

%cyanide removal ½Initial cyanide] ¼

×
100

Reusability

For the reusability experiments of E. coli-CynD

immobilized on agar and polyacrylamide, 0.3 g of cells
was immobilized on each support. Each reaction cycle
was performed using a solution containing 5 mM NaCN
and 20 mM Tris-base buffer (pH 8.0). The content of
cyanide in the solution was deter- mined after 120 min of
reaction using the picric acid method- ology and the
results used to calculate the percent cyanide degraded.
After each cycle, the solid support was removed from the
solution and washed, and a new reaction cycle was
initiated by the addition of fresh 5 mM NaCN in 20 mM
Tris- base buffer (pH 8.0). When not in use, the
immobilized sys- tem was stored in 50 mM sodium
phosphate buffer (pH 7.0) at 4 °C.

Leaching from polymers

The leaching of E. coli-CynD from the agar and

polyacryl- amide supports was tested using the method of
Bagal and Karve (2006). For this purpose, 0.3 g of cells
immobilized on each support was suspended in 5 mL of
20 mM Tris-base buffer (pH 8.0) for 5 h. Then, in
independent assays, 100 μL of the solution in which each
polymer had been suspended were assayed in 1 mL of 5
mM NaCN in 20 mM Tris-base buffer (pH 8.0). After 30
min of reaction, the percent cyanide degradation was
determined.

Activity at alkaline pH

For these assays, 100 μL of E. coli-CynD cell suspension

containing 0.092 mg of total protein was used either free
or immobilized on agar droplets and small
polyacrylamide beads. Small particles were preferred to
minimize the effect of diffusion on activity. The CynD
activity was tested using solutions of 5 mM cyanide in
either 20 mM Tris-base buffer pH 8.0 or 20 mM glycine
buffer at pH 9.0 and 10.0. The
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reactions were performed in a final volume of 1 mL, at
room temperature and with constant mixing. Reaction
rates, at each pH, were determined by measuring the
cyanide concentration every 2 min for 10 min. Each
reaction was performed in trip- licate, and the cyanide
concentration was determined using the picric acid
method. For either free or immobilized E. coli- CynD, the
activity was reported relative to that at pH 8.0.
presence of a prominent band around 38 kDa, the expected
molecular weight of CynD (Fig. 1). The maximal
expression of CynD occurred 4 h after induction with IPTG,
and based on densitometric analysis, the enzyme represents
approximately 20% of the total protein content of E. coli.

Biodegradation of industrial cyanide wastewater

The wastewater resulting from a cyanidation process

(barren solution) was obtained from Sociedad Minera del
Sur S.A, a gold mine located in Buenos Aires, Cauca,
Colombia. For bioremediation, 0.6-g cell weight of E.
coli-CynD was immobilized on agar and polyacrylamide.
The immobilization systems were added to the barren
solution either diluted 30- fold or undiluted, and the
cyanide content was determined 4 h after addition of the
catalyst. The percent cyanide degradation was calculated
using the concentration in a control kept at similar
conditions but without the addition of catalyst.

Physicochemical characterization

The characterization of the systems of E. coli-CynD

immobilized on agar and polyacrylamide was carried out
by scanning electron micrography (SEM) performed at the
labo- ratory for nanoscience and nanotechnology at
Universidad Pontificia Bolivariana (Bucaramanga,
Colombia) using a Tescan MIRAN 3 FEG-SEM
microscope.

Software and sequences

GraphPad Prism 7 was used for the kinetics analysis and

con- struction of graphics. The GenBank accession number
for the Bacillus pumilus C1 cyanide dihydratase gene is
AF492815. Additionally, the nucleotide sequence for the
CynD gene, co- don optimized for expression in E. coli
and with a C-terminal six-histidine tag, was deposited in
GenBank under accession number MH917689.

Results

CynD expression in E. coli

CynD was efficiently expressed in E. coli, as shown by the

Activity of immobilized E. coli-CynD chitosan did not appear to be related to the pore size,
because neither the variation in the percentage of chitosan
The activity of an immobilized biocatalyst can be (from 1 to
influenced by the characteristics of the immobilization
matrix. Therefore, the ability of immobilized E. coli-
CynD cells to degrade cya- nide in three different organic
matrices was determined (Table 1). After 30 min of
reaction time, E. coli-CynD immobilized on agar and
polyacrylamide degraded approxi- mately 70% of the
cyanide contained in the solution. In con- trast, E. coli-
CynD immobilized in chitosan only degraded 9% of the
cyanide contained in the solution. Subsequent experi-
ments were conducted to determine the possible causes of
the low activity observed for E. coli-CynD immobilized in
chito- san and to further characterize the active systems of
E. coli- CynD in agar and polyacrylamide.

Kinetic characterization

The dependence of the initial reaction velocity on the

cyanide concentration showed saturation kinetics for
free and immobilized E. coli-CynD (Fig. 2). The kinetic
parameters for whole E. coli-CynD cells are reported in
Table 2. The Vmax value is reported in relation to the
number of whole cells used in the reaction. The kinetic
analysis of immobilized
E. coli-CynD allows the calculation of stationary and
opera- tional effectiveness factors (Table 2). The
stationary (η) and operational (η°) effectiveness factors
can be calculated accord- ing to Eqs. 1 and 2, respectively
(Buchholz et al. 2012). In Eq. 1, Vimm is the velocity of the
immobilized catalyst, while Vf represents the velocity of
the free catalyst. In Eq. 2, the oper- ational effectiveness
factor is calculated based on the time required for the
same amount of free (t) and immobilized (ť) catalyst to
drive the reaction to 90% completion.

η ¼ V imm=V f

ð1Þ
0
η∘ ¼ t= t

ð2Þ

E. coli-CynD immobilization on chitosan

During the experiments in which E. coli-CynD is

immobilized on chitosan, the medium is acidified with
acetic acid to in- crease the solubility of the polymer
(Jyoti et al. 2017). In addition, the stability of the polymer
can be improved with a cross-linking agent such as
glutaraldehyde. Although these reagents have the
potential to affect the activity of E. coli- CynD, at the
concentrations used in the polymerization pro- cess,
neither acetic acid nor glutaraldehyde affected the activ-
ity of E. coli-CynD (Fig. 3).
The loss of E. coli-CynD activity when immobilized on
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Fig. 1 Temporal analysis of

CynD expression in E. coli cells.
Top: SDS-PAGE of the total pro-
tein content of E. coli-CynD.
Bottom: Densitometric analysis
of the band corresponding to
CynD (38 kDa) monitored at dif-
ferent times after induction with
IPTG

3%) nor the absence of glutaraldehyde in the
polymerization reaction increased the catalytic activity
(Fig. 3). Furthermore,
concentration was determined by the picric acid method
b
The cyanide removed by each polymer was calculated in three
indepen- dent experiments. We report the average value for the three
polymers

Table 1 Cyanide removal by E. coli-CynD cells immobilized on

three different organic polymersa

Organic polymer Cyanide removed

(%)

Agar 75 ± 10
Polyacrylamide 61.9 ± 3.5
Chitosan 9.4 ± 2.9
Free E. coli-CynD (intact cells not immobilized) 87.3 ± 6.6
Control (E. coli empty vector) 0.42 ± 0.7
Control (polymers only)b 8.4 ± 4.3
a
The assay solution contained 5 mM NaCN, 20 mM Tris buffer (pH
8.0), and 0.3 g of E. coli-CynD cells immobilized on each matrix.
Reactions were performed for 30 min at 25 °C. The percentage of
cyanide removed was determined by comparing the cyanide content
with a control without addition of any biocatalyst. Data represent the
mean ± SD of three inde- pendent experiments (n = 3). The cyanide
it was impossible to increase the activity of E. coli-CynD
immobilized on chitosan using permeabilized or sonicated
cells or by the addition of NaCl, the latter of which was
added in an attempt to reduce repulsive electrostatic
interactions.
Finally, it was ruled out that the lack of activity in the
chitosan matrix was due to cell leaching during the
immobili- zation process, because there was no evidence
of enzymatic activity in the polymerization solutions (data
not shown).

Characterization of E. coli-CynD immobilized on

agar and polyacrylamide

E. coli-CynD cells immobilized on agar and

polyacrylamide showed a high capacity to degrade
cyanide (Table 1). One of the advantages of immobilizing
biocatalysts is that it promotes their continuous use,
reducing operating costs. Using a 5 mM cyanide solution
at pH 8.0, the E. coli-CynD system immobilized on
agar demonstrated high reusability (Fig. 4). Remarkably,
at cycle 22, it was still able to degrade 91% of the added
cyanide. However, by cycle 30, the cyanide removal
capacity had decreased to approximately 60%. In
addition, an
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Fig. 2 Dependence of the initial velocity on the cyanide
concentration for free and immobilized E. coli-CynD. All assays were
performed using a solution of E. coli-CynD containing 0.092 mg of
total protein. Free cells (circle), immobilized on agar (black
circle), or immobilized on polyacrylamide (black triangle). Initial
velocities were calculated by monitoring the cyanide concentration
every minute for 5 min. The symbols represent the average of three
experimental replicates, and the line represents the result of the data
fitted to the Michaelis-Menten equa- tion. The cyanide concentration
was determined by the picric acid method
average lifetime of 30 days at 4 °C was determined for this
system. In contrast, the reusability of the E. coli-CynD
immobilized on polyacrylamide was much lower. The
activity of this system fell off rapidly after 5 cycles. The
loss of activity appeared to be associated with greater cell
leaching in the latter matrix (Online Resource 1 Fig. S1).
In industrial processes, cyanide solutions are maintained
at pH 10 or greater to avoid the formation of volatile HCN
(Johnson 2015). For E. coli-CynD free cells, we observed a
clear loss of activity at a pH > 9 (Fig. 5). The characteristic
loss of activiy at alkaline pH for the purified enzyme was
previously reported (Jandhyala et al. 2005). In stark
contrast,
E. coli-CynD immobilized on agar or polyacrylamide
main- tained high levels of activity even at pH 10 (Fig. 5).
Table 2 Kinetic parameters
for Catalyst
E. coli-CynD in solution or
immobilizeda on agar or
polyacrylamide E. coli-CynD
Purified CynDb
E. coli-CynD-agar
E. coli-CynD-polyacrylamide
a
The kinetic parameters were calculated by graphing the initial velocity vs the cyanide concentration for E.
Appl Microbiol Biotechnol (2019) 103:5401–
Bioremediation of wastewater from the mining
industry
Free cyanide in a barren solution from a Colombian gold
processing plant was quantified at 528 mM (13.760 ppm)
and the solution was very alkaline (pH 11). We assayed the
ability of E. coli-CynD cells immobilized on agar or poly-
acrylamide to degrade the free cyanide in the barren
solution
diluted 30-fold (17.6 mM CN−) or the undiluted barren solu-
tion (Fig. 6). Both immobilization systems were able to de-
grade 98% of the free cyanide in the diluted solution, while
only 43% of the free cyanide (227 mM) was degraded
when the undiluted solution was used. In the latter
instance, the catalyst completely lost its activity during the
first cycle. Therefore, an additional aliquot of fresh catalyst
and another reaction cycle were required to achieve
total cyanide degradation.
Discussion
The enzymes, cyanide dihydratase (CynD), are attractive
biological alternatives for the bioremediation of cyanide
waste. However, industrial cyanidation solutions present
a challenge for enzymatic degradation, because these solu-
tions are kept at highly basic pH values, contain high con-
centrations of cyanide, and (in the case of gold processing)
contain heavy metals (Johnson 2015) that can inhibit
CynD. Therefore, the use of whole cells overexpressing
wildtype CynD for the bioremediation of these solutions
is an attractive alternative, because they have been shown
to be more suitable for industrial processes under extreme
conditions (Wachtmeister and Rother 2016; Carvalho
2017). Moreover, the abundant overexpression of CynD
in E. coli is a desirable characteristic for biotransformation
processes, which allows the separation of the production
stage of the biocatalyst (cell growth) and the transforma-
tion stage of substrates into products (Lin and Tao 2017).
KM (mM) Vmax η (Vimm/Vf) η°
(μmol min−1 mg−1) (t/ť)
2.1 8.5 ND ND
2.5 88 ND ND
1.3 3.6 0.44 0.5
0.5 1.0 0.09 0.5
coli- CynD in solution or
immobilized in agar and
polyacrylamide. η, ratio of
the initial velocities (Vmax) of
the immobilized and free
catalyst; η°, ratio of the time
needed to drive the reaction
to 90% completion by the
immobilized and free
catalyst (Buchholz et al.
2012)
b
Taken from Meyers et al.
(1993)
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circle) and polyacrylamide (black triangle). Each reaction cycle
contained 5 mM NaCN and 20 mM Tris buffer (pH 8.0). Cyanide
degradation was determined after 120 min of reaction time. The
symbols represent the average of three experimental replicates. The
cyanide content was determined by the picric acid method

Fig. 3 Activity of free and chitosan-immobilized E. coli-CynD under

different conditions. All reactions were performed starting with 5 mM
NaCN in 20 mM Tris buffer (pH 8.0) and 0.3 g of free or
immobilized
E. coli-CynD cells. Cyanide removal was determined after 30 min of
reaction time. A. E. coli-CynD in solution. B. E. coli-CynD in
solution plus 2% acetic acid. C. E. coli-CynD in solution plus 2%
glutaraldehyde.
D. E. coli-CynD on chitosan cross-linked with glutaraldehyde. E. E.
coli- CynD on chitosan not cross-linked. F. E. coli-CynD on 1%
chitosan G. Sonicated E. coli-CynD immobilized on chitosan. H. E.
coli-CynD on chitosan cross-linked with glutaraldehyde plus 1 M
NaCl

Initial velocity studies showed that E. coli-CynD

followed saturation kinetics, with a KM value similar to the
value pre- viously reported for the purified enzyme
(Meyers et al. 1993; Jandhyala et al. 2003). This result
suggests that cyanide can rapidly penetrate the cell
membrane of E. coli by equalizing intra- and extracellular
concentrations. Therefore, diffusion through the cell
membrane is not a rate-limiting step, a char- acteristic that
can make cyanide very cytotoxic. Regarding the value of
Vmax, the uncertainty of the CynD concentration in- side E.
coli-CynD makes comparisons with the purified en- zyme
impossible. Nevertheless, the Vmax for free whole cells

Fig. 4 Reaction cycles for E. coli-CynD immobilized on agar (black

Fig. 5 Activity of free and immobilized E. coli-CynD at alkaline pH
values. All assays were performed using a solution of E. coli-CynD
containing 0.092 mg of total protein. Assays were conducted in a
volume of 1 mL consisting of 5 mM NaCN in either 20 mM Tris-
base buffer pH 8.0 or 20 mM glycine buffer at pH 9.0 and 10.0.
Symbols denote free (circle), immobilized on agar (black circle), or
immobilized on polyacrylamide (black triangle) systems. The
activity was determined by measuring the cyanide concentration in
the reaction every 2 min for 10 min. The mean and standard
deviation of three experiments are report- ed. In all cases, the
activity is reported relative to the same catalyst at pH 8.0

sets a reference to compare the kinetic characteristics of

the immobilized biocatalysts in the different polymer
matrices assayed.
Some advantages of trapping native or recombinant
whole cells in polymer matrices, such as hydrogels
(chitosan),

Fig. 6 Removal of free cyanide from mining cyanidation wastewater

(barren solution) by immobilized E. coli-CynD. A. Diluted barren
solution treated with E. coli-CynD immobilized on agar. B. Diluted
barren solution treated with E. coli-CynD immobilized on
polyacrylamide. C. Undiluted barren solution treated with E. coli-
CynD immobilized on polyacrylamide. D. Undiluted barren solution
after two reaction cycles with E. coli-CynD immobilized on
polyacrylamide. All reactions were performed for 4 h. The
cyanide degradation was determined relative to the cyanide content
in the solution without added catalyst. The error bars indicate the
standard deviation of two biological replicates
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5410

thermogels (agar), and synthetic polymers protection to the catalyst against alkaline denaturation (Fig.
(polyacrylamide), are their low production cost, high yield, 6). Altogether, even at pH 10, both immobilization systems
and long lifespan (Trelles and Rivero 2013). However, for retained approx- imately 80% activity relative to free cells
any immobilization system, because the particular at pH 8.0. These results contrast with those reported for
characteristics depend on the biocatalyst, the matrix, and purified CynD, which loses activity at pH > 8.4 (Jandhyala
the substrate, the optimal combi- nation must be et al. 2005). These results
determined experimentally (Sheldon 2007; Zucca et al.
2016).
The kinetic analysis of immobilized E. coli-CynD in
agar and polyacrylamide allowed the stationary and
operational effectiveness factors for these systems to be
calculated (Table 2). These values are important to
optimize the opera- tion of bioreactors with these
immobilized systems (Kasche et al. 1979; Buchholz and
Klein 1987). The stationary effec- tiveness factor reflects
the substrate gradient inside and out- side the particles. The
significantly larger value observed for the system of E.
coli-CynD on agar most likely corresponds to the larger
pores of this material. The difference in porosity is evident
on SEM images of both polymers (Online Resource 1 Fig.
S2). In contrast, the operational effectiveness factor re-
flects how fast the end point of an enzymatic process is
reached. In both cases, immobilized E. coli-CynD required
twice as long as free cells to degrade the same amount of
substrate under similar conditions. In conclusion, the
kinetic behavior of E. coli-CynD immobilized on agar and
polyacryl- amide is optimal for a bioremediation process.
The physicochemical characteristics of the matrices
deter- mine the performance of the immobilized systems
and the choice of the reactor to be used in a particular
process (e.g., stirred tank, fluidized, or fixed beds). Agar
remains one of the most commonly used polymers because
of its desirable char- acteristics, such as high
hydrophilicity, porosity, and mechan- ical strength. In
addition, agar is physically and chemically inert (Zucca et
al. 2016). The thermoresistance of intracellular CynD was
a key factor for the successful immobilization of this
enzyme on agar, as the purified enzyme loses activity at
temperatures higher than 42 °C (Jandhyala et al. 2005).
E. coli-CynD immobilized on agar showed high reusability,
retaining close to 60% of its ability to degrade cyanide for
30 cycles and a half-life longer than 30 days.
In contrast, the catalyst immobilized on polyacrylamide
(another organic matrix widely used to immobilize
catalysts) (Datta et al. 2013) lost its capacity to degrade
cyanide after five cycles. This behavior correlates with
an increased leaching of the CynD activity into the storage
buffer in this system (Online Resource 1 Fig. S1).
One of the most remarkable characteristics of the
immobilized systems of E. coli-CynD on agar and
polyacryl- amide is the ability of these polymers to provide
suggest that by using immobilized cyanide-degrading en-
zymes, the enzymatic degradation of industrial cyanide
solu- tions is feasible.
Chitosan is a natural polymer that is widely used for
the immobilization of E. coli for different industrial and
laborato- ry biocatalysis applications (Vorlop and Klein
1987; Zajkoska et al. 2013). The low activity observed for
E. coli-CynD immobilized on chitosan contrasted with the
high activity ob- served for the biocatalyst immobilized
on the other two organ- ic polymers. Unfortunately, the
experimental results did not allow us to determine the
exact cause of the low activity for the former complex.
However, it was possible to rule out inactivation due to
the polymerization conditions or diffusion of the substrate
due to the pore size of the matrix or the cell membrane.
Another possibility, which is difficult to demon- strate, is
the electrostatic repulsion between the hydroxyl groups of
chitosan, which is deprotonated at the basic pH used
in the reactions, and the negative charge of the CN−
substrate
prevented diffusion of the substrate to the particle.
Nevertheless, similar effects have been observed for other
polymer systems (González-Sáiz and Pizarro 2001;
Valderruten et al. 2014). Overall, the low activity observed
for E. coli-CynD immobilized on chitosan demonstrates
that although immobilization on solid matrices is well
established, there are no general rules to predict the best
matrix for a specific application. Thus, the selection of a
matrix must be based on experimental evidence.
The ability of an immobilized system to degrade free
cya- nide in a real sample was determined using a residual
mining cyanidation solution. When the sample was
diluted, both sys- tems degraded 100% of the free cyanide
in a relatively short time of 4 h. Although dilution of the
sample is not optimal, rapid degradation of cyanide is
important, avoiding its accu- mulation in cyanidation
dams and possible environmental ca- tastrophes resulting
from accidental or intentional spills of this waste (Tarras-
Wahlberg et al. 2001; Rico et al. 2008; Johnson 2015). On
the other hand, the enzymatic degradation of cya- nide in
untreated industrial waste required two reaction cycles
and the addition of fresh catalyst for the second cycle.
This result reflects the complexity of the sample and the
challenge associated with achieving bioremediation in real
industrial samples.
Recent reports have summarized efforts focused on the
enzymatic degradation of industrial cyanide waste
(Martínková et al. 2015; Park et al. 2017). Whole cells and
purified enzymes have been used in laboratories for the
bio- remediation of these solutions. Here, we contribute to
those efforts by reporting the characteristics of
immobilization sys- tems of E. coli-CynD on organic
polymers and confirming their ability to degrade free
cyanide in complex solutions from the mining industry.
The immobilized E. coli-CynD systems reported in this
study present optimal characteristics for their use in
bioreactors. Additionally, these immobilized
 Author's personal
copy
Appl Microbiol Biotechnol (2019) 103:5401–5410 5409
https://doi. org/10.1021/ac60069a014
González-Sáiz JM, Pizarro C (2001) Polyacrylamide gels as support
biocatalysts could operate in tandem with other systems di- for enzyme immobilization by entrapment. Effect of
rected at transforming the degradation products formate polyelectrolyte carrier, pH and temperature on enzyme action
and ammonia (Mekuto et al., 2016). Such a combination and kinetics
could add value to industrial cyanide waste.

Acknowledgments The authors would like to thank Dr. Eduardo

Ruiz- Durántez for his valuable help with the chitosan
immobilization experiments.

Funding Icesi University grant number COL0093872-639 funded

this study.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict

of interest.

Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors.

References
Ahamad A, Kunhi M (2011) Enhanced degradation of phenol by
Pseudomonas Sp. CP4 entrapped in agar and calcium alginate
beads in batch and continuous processes. Biodegradation
22:253–265. https://doi.org/10.1007/s10532-010-9392-6
Akcil A (2003) Destruction of cyanide in gold mill effluents:
biological versus chemical treatments. Biotechnol Adv
21(6):501–511. https:// doi.org/10.1016/S0734-9750(03)00099-5
Bagal D, Karve MS (2006) Entrapment of plant invertase within
novel composite of agarose-guar gum biopolymer membrane.
Anal Chim Acta 555:316–321.
https://doi.org/10.1016/j.aca.2005.09.010
Buchholz K, Klein J (1987) Characterization of immobilized
biocatalysts.
Method Enzymol 135:3–30
Buchholz K, Kasche V, Bornscheuer UT (2012) Biocatalysts and
enzyme technology, 2nd edn. Blackwell, London
Carvalho C (2017) Whole cell biocatalysts : essential workers from
nature to the industry. Microb Biotechnol 10:250–263.
https://doi.org/10. 1111/1751-7915.12363
Crum MA, Park JM, Sewell BT, Benedik MJ (2015) C-Terminal
hybrid mutant of Bacillus pumilus cyanide dihydratase
dramatically en- hances thermal stability and ph tolerance by
reinforcing oligomeri- zation. J Appl Microbiol 118:881–889.
https://doi.org/10.1111/jam. 12754
Crum MA, Sewell BT, Benedik MJ (2016) Bacillus pumilus cyanide
dihydratase mutants with higher catalytic activity. Front
Microbiol 7:1–10. https://doi.org/10.3389/fmicb.2016.01264
Cummings TF (2004) The treatment of cyanide poisoning. Occup
Med-C 54(2):82–85. https://doi.org/10.1093/occmed/kqh020
Dash RR, Gaur A, Balomajumder C (2009) Cyanide in industrial
waste- waters and its removal: a review on biotreatment. J
Hazard Mater 163(1):1–11.
https://doi.org/10.1016/j.jhazmat.2008.06.051
Datta S, Rene Christena L, Rajaram YRS (2013) Enzyme
immobiliza- tion: an overview on techniques and support
materials. 3 Biotech 3: 1–9. https://doi.org/10.1007/s13205-012-
0071-7
Fisher FB, Brown JS (1952) Colorimetric determination of cyanide in
stack gas and waste water. Anal Chem 24:1440–1444.
 Humana Press, New York, pp 365–374. https://doi.org/10.1007/
parameters. Eur Polym J 37(3):435–444. 978-1-62703-550-7_1.
https://doi.org/10.1016/ S0014-3057(00)00151-8
Gupta N, Balomajumder C, Agarwal VK (2010) Enzymatic
mechanism and biochemistry for cyanide degradation: a review.
J Hazard Mater 176:1–13.
https://doi.org/10.1016/j.jhazmat.2009.11.038
Jandhyala D, Berman M, Meyers PR, Sewell BT, Willson RC,
Benedik MJ (2003) Cynd, the cyanide dihydratase from
Bacillus pumilus: gene cloning and structural studies. App
Environ Microbiol 69(8): 4794–4805.
https://doi.org/10.1128/AEM.69.8.4794-4805.2003
Jandhyala D, Willson RC, Sewell BT, Benedik MJ (2005)
Comparison of cyanide-degrading nitrilases. App Microbiol
Biotechnol 68(3):327– 335. https://doi.org/10.1007/s00253-005-
1903-8
Johnson CA (2015) The fate of cyanide in leach wastes at gold
mines: an environmental perspective. Appl Geochem 57:194–
t2p0s5:/./dhot i. org/10.1016/j.apgeochem.2014.05.023
Jyoti KB, Chauhan K, Attri C, Seth A (2017) Improving stability
and reusability of Rhodococcus pyridinivorans nit-36 nitrilase
by whole cell immobilization using chitosan. Int J Biol
Macromol 103:8–15.
https://doi.org/10.1016/j.ijbiomac.2017.05.012
Kasche V, Kapune A, Schwegler H (1979) Operational effectiveness
factors of immobilized enzyme systems. Enzym Microb
Technol 1:41–46. https://doi.org/10.1016/0141-
0229(79)90010-3
Kuyucak N, Akcil A (2013) Cyanide and removal options from
effluents in gold mining and metallurgical processes. Miner Eng
50:13–29. https://doi.org/10.1016/j.mineng.2013.05.027
Lin B, Tao Y (2017) Whole-cell biocatalysts by design. Microb Cell
Factories 16(1):1–12. https://doi.org/10.1186/s12934-017-0724-7
Logue B, Hinkens DM, Baskin SI, Rockwood GA (2010) The
analysis of cyanide and its breakdown products in biological
samples. Crit Rev Anal Chem 40(2):122 – 147.
https://doi.org/10 . 1080 /
10408340903535315
Martínková L, Veselá AB, Rinágelová A, Chmátal M (2015)
Cyanide hydratases and cyanide dihydratases: emerging tools in
the biodeg- radation and biodetection of cyanide. Appl
Microbiol Biotechnol 99:8875–8882.
https://doi.org/10.1007/s00253-015-6899-0
Mekuto L, Ntwampe SKO, Akcil A (2016) An integrated biological
approach for treatment of cyanidation wastewater. Sci Total
Environ 571:711–720. https://doi.org/10.1016/j.scitotenv.2016.07.
040
Meyers PR, Rawlings DE, Woods DR, Lindsey GG (1993) Isolation
and characterization of a cyanide dihydratase from Bacillus
pumilus C1. J Bacteriol 175(19):6105–6112.
https://doi.org/10.1128/jb.175.19. 6105-6112.1993
Park JM, Sewell BT, Benedik MJ (2017) Cyanide bioremediation:
the potential of engineered nitrilases. Appl Microbiol
Biotechnol 101(8):3029–3042. https://doi.org/10.1007/s00253-
017-8204-x
Rico M, Benito G, Salgueiro AR, Díez-Herrero A, Pereira HG
(2008) Reported tailings dam failures: a review of the european
incidents in the worldwide context. J Hazard Mater 152:846–
852. https://doi. org/10.1016/j.jhazmat.2007.07.050
Sheldon RA (2007) Enzyme immobilization: the quest for optimum
per- formance. Adv Synth Catal 349:1289–1307.
https://doi.org/10. 1002/adsc.200700082
Tarras-Wahlberg NH, Flachier A, Lane SN, Sangfors O (2001)
Environmental impacts and metal exposure of aquatic
ecosystems in rivers contaminated by small scale gold mining:
the Puyango river basin, southern Ecuador. Sci Total Environ
278:239–261. https:// doi.org/10.1016/S0048-9697(01)00655-6
Thuku RN, Brady D, Benedik MJ, Sewell BT (2009) Microbial
nitrilases: versatile, spiral forming, industrial enzymes. J Appl
Microbiol 106: 703–727. https://doi.org/10.1111/j.1365-
2672.2008.03941.x
Trelles JA, Rivero CW (2013) Whole cell entrapment techniques.
In: Guisan J (ed) Immobilization of enzymes and cells, 3rd edn.
 Author's personal
copy
5410 Appl Microbiol Biotechnol (2019) 103:5401–

5410

Valderruten NE, Valverde JD, Zuluaga F, Ruiz-durántez E (2014)

Synthesis and characterization of chitosan hydrogels cross-
linked with dicarboxylic acids. React Funct Polym 84:21–28.
https://doi. org/10.1016/j.reactfunctpolym.2014.08.006
Vorlop KD, Klein J (1987) Entrapment of microbial cells in chitosan.
In: Immobilized enzymes and cells part b. Method Enzymol.
Academic Press. New York, pp 259–268
Wachtmeister J, Rother D (2016) Recent advances in whole cell
bioca- talysis techniques bridging from investigative to
industrial scale. Curr Opin Biotechnol 42:169–177.
https://doi.org/10.1016/j. copbio.2016.05.005
Woodward J (1988) Methods of immobilization of microbial cells. J
Microbiol Methods 8:91–102. https://doi.org/10.1016/0167-
7012(88)90041-3
Zajkoska P, Rebro M, Rosenberg M (2013) Biocatalysis with
immobilized Escherichia coli. Appl Microbiol Biotechnol 97:
1441–1455. https://doi.org/10.1007/s00253-012-4651-6
Zucca P, Fernandez-lafuente R, Sanjust E (2016) Agarose and its
deriv- atives as supports for enzyme immobilization. Molecules
21:1–25. https://doi.org/10.3390/molecules21111577
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