RESEARCH ARTICLE
Cyclomaltodextrinase
Genetic and Biochemical Characterization of a Novel
Thermostable Cyclomaltodextrinase From Anoxybacillus
flavithermus
Neda Aliakbari, Ziba Mirzaee, Vahab Jafarian,* Khosrow Khalifeh, and Mehdi Salehi
A strain of thermophilic Anoxybacillus flavithermus is characterized
bacteriologically and biochemically. Then, the gene responsible for encod-
ing cyclomaltodextrinase (CDase) in this bacterium is isolated, cloned, and
overexpressed. The sequence of the CDase is recorded in GenBank with
accession number KT633577.1. Biochemical and structural characterization
of the enzyme shows that CDase with molecular weight of 72 kDa is active
in the optimum temperature and pH of 65
C and 7.0, respectively and it
exists in oligomeric forms. It is observed that α-cyclodextrin (α-CD) has
higher kcat/km value as compared to starch, glycogen, β- and γ-CD and is
therefore a more specific substrate for the enzyme. Also, the addition of
metal ions such as K1þ and Na1þ can help to improve the catalytic
activity, while Fe2þ and Cuþ2 have inhibitory effects on the activity of the
enzyme. It is also found that the catalytic activity of the enzyme increases
in the presence of 5 mM β-mercaptoethanol, EDTA, DTT, and PMSF. Thin
layer chromatography (TLC) shows that glucose and maltose are the main
products of CDase-catalyzed reaction of α-, β-, and γ-CD hydrolysis.
Considering the modeling results, the CDase consists of three domains in
which the central domain is the catalytic core containing active site
residues. Based on its specificity for various substrates, the new
homologue of CDase can be considered as a suitable candidate for
industrial applications involving production of maltose and glucose from
α-, β-, and γ-CD or a mixture of these compounds in extreme conditions
of temperature.
N. Aliakbari, Z. Mirzaee, Dr. V. Jafarian, Dr. K. Khalifeh,
Dr. M. Salehi
Faculty of Sciences
Department of Biology
University of Zanjan
Zanjan, I. R. Iran
E-mail: v.jafarian@znu.ac.ir
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/star.201800133.
© 2018 The Authors. Starch - Stärke Published by WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim. This is an open access article under the
terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original
work is properly cited.
DOI: 10.1002/star.201800133
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1. Introduction
Thermophilic microorganisms include
proteins with unusual physico-chemical
properties which help to retain their
structure and activity at extreme condi-
tions.[1] Anoxybacillus flavothermus (previ-
ously known as Bacillus flavothermus) was
isolated from New Zealand’s hot springs in
1982 and it was revealed that the bacterium
grows at temperatures ranging from 30 to
70
C.[2,3] Glycoside hydrolase family 13
(GH13) is made up of several enzymes
such as α-amylases which are convention-
ally used in various industrial applications,
for example, in starch conversion pur-
poses. Enzymes with a spectrum of other
starch-modifying and hydrolyzing activities
also belong to families like glycogenase,
pullulanase, 1,6-glucosidase, branching
and debranching enzymes, maltogenic
amylase (MAase), neopullulanase (NPase),
and cyclomaltodextrinase (CDase).[4] The
biochemical properties of these enzymes
allow them to catalyze the hydrolysis of
starch, pullulan, glycogen, and mainly
cyclodextrin (CD). Different degrees of
resistance to hydrolysis by common amy-
lases and increasing application of the
latter substrate (CDs) have motivated
interest in the experiments involving CD-
degrading enzymes.[5] CDs include α-, ß-, and γ-CD and contain
6, 7, and 8 circular glucose units, respectively. Their structural
and functional stability, particularly under extreme conditions,
make them fit for applications such as dyeing, drug delivery, and
perfume production. Changes in ionic strength and temperature
in the cells can affect drug dissociation from CDs in drug
delivery applications of CDs.[6,7] It was also revealed that the size
of CDs has a profound effect on their activity.[8]
Cyclodextrinase from Bacillus macerans (CDase; EC 3.2.1.54) was
reported in 1968 and has proven to catalyze efficiently the
hydrolysis of CDs to create linear oligosaccharides of
α-1,4-linkages.[9] Many CDases including those from Thermoanaer-
obacter ethanolicus strain 39E,[10] Klebsiella oxytoca strain M5a1[11]
Bacillus species,[8] Flavobacterium species,[12] Paenibacillus spe-
cies,[13] and Thermococcus kodakarensis KOD1[14] have been
isolated and characterized. According to their substrate
specificity, they have been recommended for application in
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pharmaceutical and food industries including the production of
amylose-carried or amylose-free starch. Under high concen
tration of oligosaccharides, these enzymes also catalyze trans-
glycosylation of substrates to the C3-, C4-, or C6-hydroxyl groups
containing different acceptor sugar molecules.[8,15] Hence, their
isolation and characterization from thermophilic microorganisms
is of great importance.
CDase (EC No: 3.2.1.54) is also known as cyclohexaglucanase,
cyclohaptaglucanase, decyclizine, and cyclodextrinase.[16,17] They
have MW ranging from 62 to 90 kDa.[18–20] Depending on the
environmental conditions, these enzyme variants can exist either
in monomeric or oligomeric conformations.[18] In oligomeric
form, the preferred substrate is CDs and malto oligosaccharide;
while in monomeric form, the enzyme’s active site is more prone
to attract large sized substrates such as starch.[21]
Despite some similarity among different CDases isolated
from various types of bacteria, they have different properties
considering their thermal stability, response to temperature, pH
and substrate specificity.[8,10–14] In this study; for the first time, a
new strain of A. flavithermus isolated from the mineral water of
Gaynarge Nir hot spring, Ardabil, Iran and has bacteriologically
and molecularly been identified. Cloning, gene expression,
purification, and biochemical characterization of the CDase then
were carried out. The differences in the amino acid sequence of
the new enzyme as compared to those of known CDases were
also described. Since this enzyme may have the potential to be
applied in pharmaceutical, biotechnology, and starch industries,
the aim of this study was to describe in detail this novel Ca2þ-
independent and thermostable cyclomaltodextrinase from A.
flavithermus.
2. Experimental Section
2.1. Chemicals and Enzymes
Substrates including starch, glycogen, α-, β-, and γ-CD as well
as dinitrosalicylic acid (DNS), phenyl methane sulfonyl fluoride
(PMSF), sodium dodecyl sulfate, and Tryptic soy broth (TSB)
were purchased from Merck (Darmstadt, Germany). Ni-NTA
agarose column was prepared from QIAGEN (Hilden,
Germany). Purification kit for PCR products, plasmid extrac-
tion kit, and Pfu PCR Permix was purchased from Bioneer
(South Korea), restriction enzymes EcoRI and BamHI were
purchased from Jena Bioscience (Germany), alkaline phospha-
tase and T4 DNA ligase were purchased from Thermo scientific
(UK), Taq DNA polymerase enzyme, pET28a plasmid for
cloning and gene expression were purchased from Invitrogen
company, and kanamycin was purchased from Madison (USA).
The silica gel plate was purchased from Qingdao Haiyang
Chemical Company (China).
2.2. Isolation and Screening of Microorganism
Water samples were taken from 50 cm depth of the main spring
of Gaynarge hot spring which is located in Ardabil province,
north-west of Iran. The temperature and pH of the spring were
63
C and 7.5, respectively. During repetitive sub-culturing, the
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temperature was raised from 30 to 75
C with a heating rate of
5
C. In order to screen the bacteria, Tryptic Soy Agar (TSB) was
used as growth medium for culturing. Then linear culture style
was used to obtain single colony from the medium. Afterwards,
the amylase-positive samples were identified by observing
apparent halo around the colonies as a result of starch
hydrolysis upon the addition of 5% lugol.[22] The identification
of isolates was first performed using bacteriological tests based
on Bergey’s Manual.[23] Besides, molecular biology tests were
performed to confirm the bacteriological tests. Morpho-
physiological and biochemical properties were analyzed and
16S rDNA gene sequence determination was carried out
on the promising strains. Acetoin and indole production assays
were performed via inoculation in Methyl Red-Voges-Proskauer
(MR-VP) and Sulphide Indole Motility (SIM) media, respec-
tively. Glucose, sucrose, and lactose were assayed by
inoculation into Triple Sugar Iron (TSI) medium. The latter
medium was also used to determine the H2S production.
Gram staining and enzyme tests including arginine dihydro-
lase, lysine decarboxylase, tryptophan deaminase, β-galactosi-
dase, catalase, oxidase, and ornithine decarboxylase; were
also carried out. Genomic DNA was extracted using the
Sambrook and Russell method to determine the 16S rDNA
sequence.[24] The amplification of 16S rDNA genes was
performed by 27F (50 AGTTTGATCCTGGCTCAG30 ) and
1492R (50 GGCTTACCTTGTTACGACTT-30 ) primers.[25] For
the PCR procedure, the temperature cycles were (a)
94
C (2 min, (b) 35 cycles in 94
C (1 min), 52
C (1 min),
72
C (90 s), and (c) 72
C (10 min). The product of amplification
was partially sequenced directly on the two strands through
Bioneer (Bioneer, South Korea) based on the super long run.[26]
The sequences of 16S rDNA were obtained from NCBI (http://
www.ncbi.nlm.nih.gov/) and multiple sequence alignment of
16S rDNA sequences was obtained from various Anoxybacillus
and Geobacillus species conducted with the Clustal W.[27]
Furthermore, homology search was carried out using
BLASTN and the phylogenetic tree was drawn using Phylip
software version 3.696 (http://evolution.genetics.washington.
edu/phylip.html).
2.3. Cloning and Sequence Analysis of CDase
Primers for CDase were designed using Gene Runner software
based on the CDase gene of A. flavithermus 52-1A (CP021838.1).
The primers for amplification of CDase are as follows:
50 -GCGGGATCCATGTTAAAAGAAGCGATTTACC-30 (For-
ward primer) and
50 -TTCGAATTCTTATACAGCGGACGTAGRYTG-30 (Reverse
primer)
and they consist of BamHI and EcoRI restriction sites (under-
lined sequence); respectively. The nucleotide encoding CDase
was amplified by PCR of a genomic DNA from A. flavithermus
ZNU-NGA. The PCR reaction was performed for sequence
amplification by Pfu PCR Permix. The temperature cycles of
PCR reaction of different stages are as follows: first stage:
95
C (2 min), second stage: 35 cycles in 95
C (1 min),
54
C (1 min), 72
C (90 s), and the third stage: 72
C (10 min).
After amplification and purification of CDase, the PCR products
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and vector pET28a were enzymatically digested in two separate
steps using restriction enzymes BamHI and EcoRI (single
digest). Then, the insertion of DNA fragment of CDase in
pET28a plasmid was performed using T4 DNA ligase. The cell of
Escherichia coli DH5α was made competent by calcium chloride
procedure[24] and recombinant plasmids were then transformed
into the competent cells. After extracting the plasmids, they were
confirmed by nucleotide sequencing. The sequencing was
performed by Bioneer Company (South Korea) through general
primers, T7 promoter, and T7 terminator. Sequence similarity
comparison was performed using BLASTN and BLASTP under
NCBI database.[28] Multiple sequence alignment was performed
using Clustal W and ESPript 3 programs.[27]
2.4. Heterologous Expression of CDase and Purification of
Recombinant CDase
For expression of the desired gene, the transformed E. coli BL21
(DE3) containing CDase was cultured in Terrific Broth (TB)
medium enriched with 35 μg mL1 kanamycine in 37
C and pH
7.0. The turbidity of growth at OD600 was monitored by UV-Vis
spectroscopy. Upon reaching 0.8–0.9 turbidity, the 0.5 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce
enzyme expression. Afterwards, the desired bacteria were shaken
at 180 rpm for 6 h in 30
C. Subsequently, for extraction of CDase,
the cultured medium was centrifuged at 4
C and 4000 rpm for
20 min. The sediment of bacteria was suspended again by adding
5 mL lysis buffer prepared at pH 8.0 using 10 mM imidazole,
50 mM Tris, and 300 mM sodium chloride. Then, the bacterial
suspension was sonicated for 30 stages (each stage 20 s). To obtain
the crude extract, the disrupted cells were centrifuged in 4
C and
12 000 rpm for 20 min. Since the desired proteins contain histidine
tail, they were transferred to Ni-NTA agarose column for
purification and eluted by elution buffer. The elution buffer was
prepared by mixing 300 mM imidazole, 50 mM Tris, and 300 mM
sodium chloride at pH 8.0. The pooling, desalting, and
concentration of active elutions were performed by Amicon
ultra-15 centrifugal filter units (Merck, Germany). 12% SDS-PAGE
was used according to Laemmli[29] and protein concentration was
determined by Bradford assays.[30] In order to determine the MW
of His-tag tail upon running SDS-PAGE, the sequence of protein
was used as input for ProtParam program[31] and the MW of
protein was estimated. To elucidate the oligomeric state of the
enzyme, native PAGE was also performed.
2.5. CDase Assay
The CDase activity was measured by DNS according to Bernfeld
method[32] using different substrates including α-, β-, and γ-CD,
starch, and glycogen. The reaction mixture includes 50 mL of
1% substrate dissolved at HEPES buffer (50 mM, pH 7.0) and
50 mL of the purified enzyme dissolved in the same buffer
solution. The enzyme concentration of the solution was 1 Unit
which is defined as the amount of enzyme that released 1 mmol
of product per minute.[33] The mixture was incubated at
65
C for 20 min. After incubation, the reaction was stopped by
adding 100 mL of DNS and the mixture was then boiled for
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5 min in water bath. After cooling, 900 mL of distilled water was
added. The release of reducing sugar was then assessed by
measuring the absorbance at 540 nm against the control
sample. One unit of CDase activity in the assay condition was
referred to the amount of enzyme producing 1 mmol reducing
sugar per minute.
2.6. Activity and Stability Studies on Recombinant CDase
CDase activity was determined using β-CD based on Bernfeld
method.[32] pH profile of the enzyme was determined with 50 mM
mixed buffer containing HEPES (pK: 7.48  1), sodium acetate
(pK: 4.76  1), sodium phosphate (pK1: 2.15 ; pK2: 7.20  1) and
glycine (pK: 9.78  1) tuned to different pH values (2.0–11.0) at
65
C.[34] The temperature profile for the activity of CDase was also
determined. The HEPES buffer was generally used at pH 7.0 and at
different incubation temperatures (20–100
C).
For thermostability measurement, the enzyme solution was
first prepared in 50 mM HEPES at pH 7.0 and it was incubated at
65, 70, and 75
C for various periods of time (1, 5, 10, 20, 30, 60,
90, and 120 min). Subsequently, after snap-cooling with ice water
bath for half an hour, the relative activity of enzyme was assayed.
The kinetic parameters such as Michaelis-Menten constant
(Km), maximal velocity (Vmax), and kcat values of the enzyme were
determined by Lineweaver-Burk plot under various concen-
trations of α-, β-, and γ-CD at constant pH 7.0 and 65
C.[35]
The effects of different metal ions (Kþ, Naþ, Zn2þ, Fe3þ, Co2þ,
Mg2þ, Ca2þ, and Cu2þ) and chemical compounds (EDTA, PMSF,
DTT and β-mercaptoethanol) on the activity of CDase were also
determined. The concentrations used for all the studied metal
ions and chemicals were 5 and 10 mM. The conditions for
assaying the residual activity of CDase were the same as those
stated earlier (section 2.5). The activity of CDase in control
samples with no metal ions and chemicals was regarded as
100%. All the experiments were performed thrice and data were
reported as means  standard deviation (SD).
2.7. Hydrolysis Products
Thin layer chromatography (TLC) was used to identify hydrolysis
products which resulted from CDase activity. Analysis of the
hydrolysis products of CDase on 1% (w/v) of α-, β- and γ-CD was
performed by their incubation with 1 mg of purified enzyme in
HEPES buffer (50 mM, pH 7.0) at 65
C for 6 h. The reaction
processes were stopped by boiling the solutions for 10 min. Then,
TLC of the reaction mixtures was analyzed on a silica gel plate
(Qingdao Haiyang Chemical company, China), with a mobile phase
system containing water:n-butanol:ethanol (2:5:3, v/v/v). After
drying the gel, 0.5% (w/v) of thymol in 5% (v/v) of a sulfuric acid
solution in ethanol was sprayed on the plate and it was heated at
105
C for 5 min in order to visualize the saccharide spots.
2.8. Homology Modeling
Three-dimensional (3D) structural models of CDase were made
with MODELLER program, version 9.17.[36] The crystal structure
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of maltogenic amylase (PDB ID: 1SMA) was used as template to
build the 3D structural models. The quality of the models was
assessed using the Structure Analysis and Verification (SVAES)
Server[37–40] and ModEval program at SaliLab Model Evaluation
Server (https://modbase.compbio.ucsf.edu/evaluation//).[41–43]
The validity of the selected model was confirmed based on
the scores of ERRAT and Verify 3D programs under the SAVES
server and Discrete Optimized Protein Energy (DOPE) score of
the ModEval program. The structure was also visualized and
analyzed with Swiss-PdbViewer software (version 3.7) to obtain
its root-mean-square deviation (RMSD).
grew at an optimum temperature of 55
C and pH of 8.5.
Moreover, the amplification and sequencing of 16S rDNA were
also performed. After being transferred to agarose gel, the
product of PCR was about 1500 bp. However, direct sequencing
showed that the 16S rDNA sequence was edited to a total length
of 1494 bp. The output of sequence blast for the 16S rDNA
showed that it had 99% homology with A. flavithermus strain 52-
1A and it was named as A. flavithermus ZNU-NGA. Figure 1
shows the result of phylogenetic analysis of A. flavithermus ZNU-
NGA and related sequences.

3.2. Nucleotide Cloning, Expression, and Purification of

3. Results CDase
3.1. Isolation and Identification of Microorganism and 16S
The CDase was amplified (Figure 2A) and cloned successfully
rDNA Sequence Analysis
into the pET-28a(þ). The sequencing results of the CDase
extracted from A. flavithermus ZNU-NGA was recorded in
The morpho-physiological and biochemical results for identifi- GenBank (accession no. KT633577.1). The nucleotide sequence
cation of the isolated bacterium from Gaynarge Nir hot spring in of the reported CDase had the highest identity (86%) with that of
Ardabil are shown in Table 1. From the comparison of these data A. flavithermus 52-1A, 85% with that of A. flavithermus WK1, 79%
with those obtained from Bergey’s Manual bacteriological with that of Geobacillus subterraneus KCTC 3922, and 77% with
tests,[23] the isolate has been identified as A. flavithermus. It CDase of A. flavithermus cda13. The current CDase contains 1764
nucleotides encoding 587 amino acid residues with TAA
Table 1. Morphological, biochemical, and physiological features of termination codon. According to ProtParam program, the
Anoxybacillus flavithermus ZNU-NGA. isoelectric point and molecular weight (MW) of CDase-ZNU
is predicted as 5.27 and 68.8 kDa, respectively. In the open-
Experiments Results
reading frame encoding the enzyme, no signal peptide was
Colony features Yellow, found showing its intracellular location in the original host.
Round, When compared with another enzymes in the GH13 family, the
Smooth
amino acid sequence of CDase-ZNU (AMB26774.1) showed
76.46% identity with A. flavithermus (AAX29991.1),[44] 70.03%
Spore status Terminal
Gram þ
Optimum pH for growth 8.5
Optimum temperature for growth 55
C
Growth in 2.5% NaCl þ
Hydrolysis of Starch þ
Gelatin –
Utilization of þ
D-glucose þ
D-lactose þ
D-sucrose þ
Enzymes
Arginine dihydrolase þ
Lysine decarboxylase þ
Tryptophan deaminase þ
β-galactosidase þ
Catalase þ
Oxidase þ
Ornithine decarboxylase –
H2S –
SIM –
MR-VP þ
Figure 1. The phylogenetic tree of Anoxybacillus flavithermus ZNU-NGA
Nitrate to Nitrite þ
and related species constructed by Phylip package.

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Figure 2. A) PCR product of the nucleotide sequence of the CDase coding gene on the agarose gel electrophoresis; M refers to the marker. B) Expression
analysis of CDase by SDS-PAGE; supernatant of non-induced protein 1), induced protein 2), precipitation of induced protein 3), elution of purified
protein by means of nickel affinity column chromatography 4), and protein molecular mass markers (M). C) The results of PAGE analysis showing the
oligomeric states of the enzyme.
with maltogenic amylase (MAase) of Thermus spp. IM6501
(WP021322528.1),[20] 68.66% with neopullulanase (NPase) of
Geobacillus strearothermophilus (AAK15003.1),[45] and 69.35%
with MAase of Geobacillus spp. (ADM86931.1).[46] The analysis of
amino acid sequence showed that CDase-ZNU has four
conserved regions[21,47] which are the common conserved
regions in the CD-hydrolyzing enzymes and they include
CDase, NPase, and MAase. The catalytic residues including
Asp328, Glu357, and Asp424 were also identified in CDase-ZNU.
Additionally, the conserved region (WLRGDQFDAVMNYP) as
the CD-binding site was observed in the CDase between the
conserved regions III and IV (Figure 3). These results confirmed
that CDase obtained from A. flavithermus ZNU-NGA belongs to
the GH13 family of CD-hydrolyzing enzymes. As shown in
Figure 3, contrary to the N-terminal and central domains, the
sequence of C-terminal domain in CDase-ZNU does not
show high homology with its counterparts in CDases, MAases,
and NPases. The N-terminal domain has been proposed to be
involved in determining substrate specificity. It was found
that the N-terminal domain (residues 1–118), catalytic module
(residues 119–507), and C-terminal domain (residues 508–587)
of CDase-ZNU had 71.19, 84.06, and 45.33% identity with their
counterparts of the CDase of A. flavithermus (AAX29991.1),
respectively. Therefore, the lowest degree of sequence conserva-
tion was observed in the C-terminal domain. Moreover, CDase-
ZNU has three extra residues (SAV) in the C-terminal domain as
compared with the aforementioned enzymes (Figure 3). It has
been assumed that the C-domain acts as a catalytic module by
exposing hydrophobic residues to the solvent.[48] However, as
shown in Figure 3, few residues in the domain were observed to
be conserved and there were completely conserved residues only
at four positions (I, II, III, and IV).
On the other hand, the reported CDase was expressed in E. coli
BL21(DE3) with a high level of expression, such that it produces
a remarkable amount of soluble protein. The specific activity of
the interacellular purified CDase-ZNU was 48.11 U mg1
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(Table 2). Meanwhile, the extracellular activitiy of the CDase
was not observed in the supernatants of E. coli BL21 (DE3). The
SDS-PAGE showed a single protein band with a MW of
approximately 72 kDa (Figure 2B). PAGE analysis showed that
the enzyme exists in oligomeric form (Figure 2C).
3.3. Effect of pH and Temperature on the Enzyme
Assay of the CDase under various conditions of pH showed that
it is active in a wide range of pH with the maximum activity
at pH 7.0. However, up to 60% of the enzyme activity was
measured at pH 6.5–8.0 and the activity decreased dramatically
at pHs lower than 6.5 and upper than 8.0, such that the
optimum pH for the enzyme activity is 7.0 (Figure 4A).
Although the enzyme was active at temperatures ranging from
20 to 100
C, the higher activities (>60%) were observed at
temperatures between 45 and 70
C. The optimum temperature
for the enzyme activity is 65
C (Figure 4B).
3.4. Thermostability of the Enzyme
As shown in Figure 4C, the enzyme showed high thermal
stability at 65
C. The remaining activity of the enzyme was 70%
of the original activity upon incubation at 65
C for 60 min, but it
reduces to half of the original activity after incubation at 65
C for
100 min. After incubation at 70
C for 30 min, the enzyme kept
50% of its activity. However, the enzyme completely lost its
activity after 75
C incubation for 5 min.
3.5. Substrate Specificity and Hydrolysis Product of CDase
To find a suitable substrate for biochemical function of the
enzyme, its activity was also assayed using various substrates
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Figure 3. Alignment of the amino acid sequence of CDase-ZNU (AMB26774.1) (CD2) with CDase from Anoxybacillus flavithermus
(AAX29991.1) (CD1), MAase from Thermus spp. IM6501 (WP021322528.1) (MA1), NPase from Geobacillus strearothermophilus
(AAK15003.1) (NP), MAase of Geobacillus spp. (ADM86931.1) (MA2). The position of secondary structure elements: α -helix (α ),
β -strand ( β ), 3 10 -helix ( η ), turn (T), active site residues (
), and catalytic residues Asp 328 , Glu 357 , and Asp 424 ( Δ ). There are four conserved
regions (I–IV) commonly among enzymes in the glycoside hydrolase subfamily 13 of the α -amylase family. CD-binding site conserved regions
is boxed.

Table 2. Purification data for purified CDase-ZNU.

Purification step Total protein [mg L1] Total activity [U mg1] Specific activity [U mg1] Purification folds Recovery [%]

Crude enzyme 6078.3  38 5118.5  102 0.84  0.1 1 100

Chromatography 71.01  0.2 3417.68  17 48.11  0.1 57.27 66.7

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Figure 4. Effects of pH (A) and temperature (B) on the CDase activity of Anoxybacillus flavithermus ZNU-NGA. Irreversible heat inactivation of
Anoxybacillus flavithermus ZNU-NGA CDase (C) and relative activity of Anoxybacillus flavithermus ZNU-NGA CDase in the presence of different substrates
(D). Data are means of three independent replicates SD.

including different types of CDs, glycogen and starch
(Figure 4D). The enzyme exhibited 1.8 and 1.6% activity in
the presence of glycogen and starch, respectively In when
compared with α-CD.
The kinetic parameters of the CDase in the presence of α-, β-,
and γ-CD are shown in Table 3. Kinetic studies showed a
decrease of Km for CDs in the order of α-CD>β-CD>γ-CD. In
addition, intrinsic catalytic rate constant (kcat) was also the
highest for α-CD; however, kcat for α-CD hydrolysis was
approximately 52 and 88% higher than that for β- and γ-CD,
respectively. The kcat/Km value for α-CD was also higher than the
others. These data together showed that CDase-ZNU has more
specificity for α-CD.
The formation of product by the enzymatic digestion of α, β and
γ-CD, glycogen and starch at different time intervals was evaluated
using TLC and it was shown that using of α-, β-, and γ-CD as
substrate could lead to the production of maltose and glucose
(Figure 5). These substrates differ from each other depending on
the presence of glucose moiety. The rates of hydrolysis for starch
and glycogen by the enzyme were too low to detect.
3.6. Effect of Metal Ions and Some Chemicals
The activity of CDase was also assayed upon incubation of
enzyme at various concentrations of metal ions and chemicals.
As shown in Table 4, at the concentration of 5 mM of Kþ, Naþ,
and Zn2þ, the enzyme activity increases by about 49, 34, and
16%; whereas, at the concentration of 10 mM, these metal ions
enhance the enzyme activity by about 36.5, 23, and 40%,
respectively. Inversely, at 5 and 10 mM concentration of Co2þ,
Ca2þ, and Mg2þ, its activity decreases by about 91, 66, and 31%
and 84.5, 73, and 82%, respectively. Moreover, addition of Cu2þ
and Fe2þ leads to inhibition of the activity, while, adding DTT,
EDTA, PMSF, and β-mercaptoethanol in 5 mM leads to an
increase in the enzyme activity by about 95, 34, 28, and 24%,
respectively (Table 4). Also, the enzyme activity increases by
about 91, 38, and 48% in the presence of 10 mM DTT, EDTA, and
β-mercaptoethanol, respectively. Among the chemicals, only
10 mM of PMSF reduces the enzyme activity by 50% as
compared to the control.

Table 3. Kinetic parameters of CDase-ZNU with different substrate. 3.7. Homology Modeling

Substrate Km [mg mL1] kcat [s1] kcat/Km [mg mL1 S]1 Upon running the MODELLER program, several 3D structures
α-CD 3.99  0.3 580  0.9 145.36  11.16 of protein were constructed. Table 5 contains quality scores of the
selected model provided by different quality control programs.
β-CD 3.45  0.5 382  0.5 110.72  16.20
According to quality scores, the validity of the selected model was
γ-CD 2.98  0.4 308  0.8 103.36  14.14
confirmed and it was used for further analysis. The selected

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Table 4. Effect of representative metal ions (in their chloride form)

and Chemicals on the relative activity of CDase.

Metal ions/Chemicals Concentration (mM) Relative activity (%)

control – 100.0
Ca2þ 5 34.1  0.04
10 27.3  0.6
K1þ 5 149.1  0.4
10 136.5  0.7
Fe2þ 5 1  0.01
10 2  0.07
Zn2þ 5 116.9  0.8
10 140.5  0.9
Co2þ 5 9.7  0.2
10 15.4  0.1
Cu2þ 5 0.0
10 0.0
Na1þ 5 134.5  0.5
10 122.7  0.75
Mg2þ 5 68.9  0.3
10 17.9  0.08
EDTA 5 134.8  0.5
10 138.1  0.2
PMSF 5 128.2  0.3
10 50.5  0.1
DTT 5 194.9  0.6
10 190.9  0.2
Figure 5. The hydrolysis products by recombinant CDase derived from
Anoxybacillus flavithermus ZNU-NGA with a substrate concentration of β-mercaptoethanol 5 124  0.3
1 g/L at 65
C for 6 h through TLC. M, standards of oligosaccharide ([G1] 10 148.9  0.7
glucose, [G2] maltose, [G3] maltotriose, [G4] maltotetraose, [G5]
maltopentaose, and [G6] maltohexaose); L1, α-CD; L2, β-CD; L3, γ-CD;
L4, starch; and L5, glycogen.
As a result of overexpression of recombinant enzymes and
accumulation of high concentration of folding intermediates,
molecular aggregates were formed in bacterial cell which could
model was visualized and further analyzed by spdbv software
not be recovered.[49] Therefore, soluble proteins were purified for
(version 3.7). Figure 6 shows the whole structure of the enzyme
further studies. As shown in SDS-PAGE (Figure 2B, lane 4), the
and a close up view of the active site.
soluble protein had appropriate level of purity essential for
biochemical characterization of the protein.
4. Discussion The specific activity of the purified CDase was 48.11 U mg1
which is higher than that of some other reported intracellular
Members of the subfamily GH13-20 can at least hydrolyze two CDase such as CDase derived from E. faecium K-1.[48]
out of the three kinds of starchy substrates. CDases are very The optimum temperature and pH ranges of the reported
unique since they have multi-substrate specificity for pullulan, thermophilic bacteria are in the range of 50–65
C and pH
starch, and in particular CDs.[44] In the present study, the 5.9–7.5, respectively [13,18,44,50,51]. In the present study, the
gene for CDase from A. flavuthermus as a native thermophilic optimum temperature for growth of A. flavithermus ZNU-NGA
strain was cloned and expressed in E. coli BL21 (DE3). Its was 55
C, while it was 65
C for the isolated CDase. As
kinetics and enzymatic features and its thermal stability were reported for several species of bacteria, the optimum tempera-
then studied. From the present results, CDase sequence from ture for the growth of bacteria may be lower than that for its
A. flavithermus ZNU-NGA had 76.46% identity with that of A. constituent’s enzymes.[3] According to this study, the optimum
flavithermus (AAX29991.1). The MW of CDase-ZNU is 72 and temperature and pH for CDase are 65
C and 7.0, respectively.
68 kDa with and without His-tag, respectively, which is fairly They are similar to those for CDase-Tk obtained from T.
similar to CDase from Enterococcus faecium K-1 (69.4 kD).[48] kodakarensis KOD1,[14] while they are different from CDase-Tc
According to other studies, the MW for CDase has been (from Thermococcus species CL1)[11] and CDase-Tp (Thermofilum
reported to be in the range of 62–90 kDa.[12–14,19] pendens Hrk 5)[52] in which their optimum temperature and pH

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Table 5. The quality scores of selected structural model of CDase

provided by different quality control programs.

SAVESa) ModEvalb) Swiss-pdbViewer

Verify 3D ERRAT z-DOPE RMSD

Model 97.96 60.806 1.176 0.54

a)
http://services.mbi.ucla.edu/SAVES/
b)
https://modbase.compbio.ucsf.edu/evaluation//

are 85 and 95
C and 5.0 and 5.5, respectively. CDase-ZNU had

also high activity (>60%) at pHs ranging from 6.5 to 8.0
(compared to 100% at pH 7.0) showing that the enzyme can
hydrolyze its substrates approximately in neutral pH. It was also
observed that the activity of CDase-ZNU is more than 60% at
temperatures ranging from 45 to 70
C showing its thermophilic
nature and it has a remarkable thermostability. From thermo-
stability point of view, the incubation conditions caused CDase-
ZNU to retain a high value (70%) of its activity at 65
C for
60 min, while it was 50
C for 30 min in CDase from Paenibacillus
spp.[13] and 60
C for 60 min in MAase from Geobacillus spp.[46]
It has been reported that the preferred substrates for the
oligomeric form of CDase family are cyclodextrin and malto-
oligosaccharide; while starch is a more appropriate substrate for
the monomeric form in which the active site is selective for big
substrates.[8] The affinity of CDase-ZNU for substrates increases
in the order of α-CD>β-CD>γ-CD>starch>glycogen. The ratio
of kcat/Km as an indication of substrate specificity for different
substrates increases as α-CD>β-CD>γ-CD. The resulting
data of this study show that the recombinant CDase had more
specificity toward α-CD.[53] Similar to CDase-ZNU, α-CD is the
specific substrate for the CDase isolated from A. flavithermus
(AAX29991.1).[44] The CDases from E. faecium K-1,[47] Bacillus
sphaericus ATCC7055[54] and T. ethanolicus 39E[10] also had more
specificity for α-CD as compared to β-CD and γ-CD, while the
CDases from Bacillus sphaericus E-244,[51] Bacillus spp. I–5,[8] and
Paenibacillus spp.[13] uses β-CD as preferred substrate. These
data can also support the classification of CDase obtained from Figure 6. Structural description of CDase from Anoxybacillus flavithermous
A. flavuthermus ZNU.NGA in the CDase group. ZNU-NGA showing the symmetrical orientation of monomers relative to
According to these findings, the CDase is more specific with each other. A) Ribbon diagram representation of the enzyme in dimer
all CD types when compared with starch and glycogen (by about form in which C- and N-terminal domain as well as the position of active
site are provided. B) Representation of protein as Van Der Waals
99 times). It has been reported that the CDs specificity of
surface of the residues. The predicted subsequences in active site by
recombinant CDase isolated from E. faecium K-1 was about 96
ESPript 3 program including residues 323–332, 352–360, and 419–424 are
times higher as compared to starch[48] in which non-significant represented as white, red, and yellow color, respectively. C) Close-up view
hydrolysis was observed with polysaccharides including starch of the residues in the active site of the enzyme. Three-dimensional
and glycogen. The idea was that the α-1,6 glycosidic linkage could structural models of CDase-NGA was constructed by MODELLER
not be attacked by the enzyme.[46] Therefore, the substrate program using crystal structure of Maltogenic Amylase (PDB ID:
specificity of CDase-ZNU for branched substrates is less than 1SMA) as template and the best model was selected by structural
that for CDs. The significant differences of CDase-ZNU quality scores as described in the main text of manuscript.
specificity for CDs and starch or glycogen may be attributed
to the difficulty in accessing large-sized substrate to the active
site as a result of steric hindrance which originated from the be explained by assuming the higher stability of the enzyme-
presence of oligomeric forms of the enzyme in the solution[45,53] substrate complex when α-CD is used as substrate.
in which the maltose and glucose are the main products of In this study, command lines of MODELLER program were
CDase-degraded α-, β-, and γ-CDs. These patterns of hydrolysis used for construction of a 3D dimeric form of CDase. Structural
obtained by CDase-ZNU are similar to those of some other examination of the modelled enzyme confirmed that the central
CDases [13,54–57]. When the values of Km of CDase-ZNU were domain in the CDase has the functional property as described by
compared with various preferences to α-, β-, and γ-CD, this can Fritzsche et al.[12] and Unban et al.[47] Structural features of the

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enzyme show that the position of active sites in the dimeric form
of the enzyme allows small-sized substrates better entrance to
the reaction site.[53]
It has been reported that metal ions play a significant role in
the activity of α-amylase family.[58] Investigating the effect of
representative metal ions on the activity of the enzyme
showed that the presence of some metal ions led to the
decrease of the enzyme activity. This finding is in agreement
with that of CDase enzyme isolated from hyper thermophilic
bacteria Thermococcus spp. CL1.[59] For a better understanding
of the effect of metal ions on the enzyme activity, EDTA was
also used as a calcium ion chelating agent. It was observed
that EDTA had remarkable effect on the CDase-ZNU activity
suggesting that this CDase is Ca2þ independent.[52] Ca2þ-
independent behavior can be considered as a useful property
for starch processing. This is because calcium ion has been
evidenced to inhibit the activity of glucose isomerase which
is a main feature in fructose syrup production from starch.[60] It
was observed that the remover of some metal ions leads to
increase in the enzyme activity. Based on these data, it
appears that the CDase is functionally regulated by the ion
type in a manner that the elimination of some metals from
the reaction environment can leads to enhancement of
enzyme activity and vice versa. In a similar report, Cu2þ has
been reported to inhibit the activity of the CDase isolated from
E. faecium K-1.[48]
The present study shows that chemical reducing agents
including DTT and β-mercaptoethanol can enhance enzyme
activity. The activation of CDase-ZNU by β-mercaptoethanol
could be ascribed to the reduction of protein aggregation by
destroying the intermolecular disulfide linkages and/or the
protection of thiol groups that stabilize the 3D structure of
enzyme.[61] These compounds may reduce thiol groups near the
active site residues of the CDase-ZNU.[62] It seems that
structural rearrangement facilitates the insertion of substrate in
the active site and the accessibility of substrate to the
catalytic residues as well as exertion of products in the unit
of time.[51] Overall, it was observed that DTT could be
considered as an excellent additive that increases the activity
of CDase to about 2-fold comparted to the control. Examining
the sequence of CDase-ZNU showed that there are nine
cysteine residues in the enzyme structure. Moreover, it has
been reported that DTT as an antioxidant agent is able to
stabilize enzyme by protecting sulfhyidryl groups, thus leading
to enhancement of the enzyme activity.[63] Enzyme activity is
dramatically reduced by adding high concentration of PMSF,
while surprisingly, in low concentrations of PMSF, the
functional features of the enzyme are improved. Based on
ESPript 3 results and 3D structure of protein, it can be
concluded that PMSF has probably a direct effect on Ser422
which is predicted as one of the main residues in the catalytic
site of protein.[64] According to bioinformatics studies and the
results of ESPript 3 program, three conserved residues
were identified in the CDase sequence when compared with
CDase of A. flavithermus (AAX29991.1),[43] MAase in Thermus
spp. IM6501 (WP021322528.1),[20] NPase in G. strearothermo-
philus (AAK15003.1)[45] and MAase in Geobacillus spp.
(ADM86931.1).[46] These residues include Asp328, Glu357, and
Asp424 which are conserved in all α-amylase families.
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In conclusion, the recombinant enzyme showed high
degrees of thermostability and appropriate strict substrate
specificity toward CDs rather than starch and glycogen
and the main hydrolysis product is glucose and maltose. Of
note, this is the first report describing the nucleotide
sequence, biochemical characteristics, and structural
features of CDase obtained from A. flavithermus ZNU-NGA.
Due to the novel features of the enzyme, it may have
the potential to be applied in industrial biotechnology in
the future.
Abbreviations
CDase, Cyclomaltodextrinase; CDs, Cyclodextrins; MAase, Maltogenic
amylase; NPase, Neopullulanase.
Acknowledgement
This work was supported by a grant from the research council of the
University of Zanjan.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
catalytic efficiency, characterization, cyclomaltodextrinase, molecular
cloning, molecular modeling
Received: May 3, 2018
Revised: May 14, 2018
Published online:
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