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Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea
Research Division for Biotechnology, Korea Atomic Energy Research Institute (KAERI), Jeongeup 580-185, Republic of Korea
c
Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA
b
a r t i c l e
i n f o
Article history:
Received 15 November 2013
Received in revised form 17 March 2014
Accepted 21 March 2014
Available online 30 March 2014
Keywords:
Glycoside hydrolase family 57 (GH57)
Hyperthermophile
Maltose-forming -amylase
Thermococcus sp. CL1
a b s t r a c t
Maltose-forming -amylase is a glycoside hydrolase family 57 (GH57) member that is unique because it
displays dual hydrolysis activity toward -1,4- and -1,6-glycosidic linkages and only recognizes maltose.
This enzyme was previously identied only in Pyrococcus sp. ST04 (PSMA); however, we recently found
two homologs subgroups in Thermococcus species. One subgroup (subgroup A) showed relatively high
amino acid sequence similarity to PSMA (>71%), while the other subgroup (subgroup B) showed lower
homology with PSMA (<59%). To characterize the subgroup B maltose-forming -amylase from Thermococcus species (TCMA), we cloned the CL1 0868 gene from Thermococcus sp. CL1 and then successfully
expressed the gene in Escherichia coli. Although TCMA has a different oligomeric state relative to PSMA,
TCMA showed similar substrate specicity. However, TCMA was shown to hydrolyze maltooligosaccharides more easily than PSMA. Also, TCMA displayed different optimum conditions depending on the
glycosidic linkage of the substrate. TCMA had the highest activity at 85 C and at pH 5.0 for -1,4-glycosidic
linkage hydrolysis whereas it showed its maximal activity to cleave -1,6-glycosidic linkages at 98 C and
pH 6.0.
2014 Elsevier Inc. All rights reserved.
1. Introduction
Starch is one of the most abundant storage polymers in nature
and is a resource for many industrial processes including the production of biological materials for foods, textiles, and detergents [1].
Various living organisms produce amylolytic enzymes that act on
starch and related oligo- and maltooligosaccharides [2]. These amylolytic enzymes are mainly classied into the glycoside hydrolase
family 13 (GH13), also known as the -amylase superfamily. The
GH13 family contains various hydrolases, transglycosidases and
isomerases, including -amylase, -glucosidase, cyclomaltodextrinase (CDase), and cyclomaltodextrin glucanotransferase (CGTase)
[3] It is known that GH13 enzymes have a predicted (/)8 -barrel
(i.e. TIM-barrel) fold and possess a catalytic triad formed by three
Corresponding author at: Department of Food Science & Biotechnology and Institute of Life Science & Resources, Kyung Hee University, Yongin 446-701, Republic of
Korea. Tel.: +82 31 201 2631; fax: +82 31 204 8116.
E-mail address: cspark@khu.ac.kr (C.-S. Park).
http://dx.doi.org/10.1016/j.enzmictec.2014.03.009
0141-0229/ 2014 Elsevier Inc. All rights reserved.
residues corresponding to Asp206, Glu230, and Asp297 of Takaamylase A (the -amylase from Aspergillus oryzae) [4].
Recently, some -amylases and related enzymes have been classied in a second -amylase family, designated as GH57, because
their protein structures are composed of a (/)7 barrel [5]. More
than 900 genes originating from both eubacteria and archaea are
included in the GH57 family and their enzymatic activities are
assigned as -amylases, amylopullulanases, branching enzymes,
-galactosidases, and 4--glucanotransferases [6,7]. However, relatively few enzymes (19 genes or enzymes) have been isolated
and characterized from the GH57 family. In addition to the structural differences between GH13 and GH57 family members, there
are also distinctive substrate specicities between the families.
For example, the GH57 amylopullulanase from Staphylothermus
marinus shows unique substrate specicity displaying cyclodextrin
hydrolysis, which was not found in any amylopullulanase from the
GH13 family since they are actually inhibited by cyclodextrin [8].
Hyperthermophilic archaea are microorganisms that thrive in
extremely hot environments optimally at and above 80 C [9]. They
are attractive in biological industries because they produce many
highly heat-stable enzymes that are active under harsh conditions
10
constructed using the MEGA 5.0 program based on the neighbor-joining method
and was evaluated by a bootstrap test of 1000 replicates [19,20].
2.4. Cloning and expression of the gene encoding TCMA from Thermococcus sp.
CL1
Thermococcus sp. CL1 genomic DNA was prepared using genomic DNA isolation methods [21]. The gene encoding TCMA (CL1 0868) was identied in the
whole genome sequence based on BLAST searches. The specic oligonucleotide
primers, CL57 F NdeI (5 -CAT ATG CTG ATG AAG TAC GCC CA-3 ) and CL57 R XhoI
(5 -CTC GAG TAA CCT GTG CTC ACA CCA CT-3 ) were designed containing NdeI and
XhoI restriction sites (underlined), and the gene was amplied by PCR using PrimeSTAR DNA polymerase and Thermococcus sp. CL1 genomic DNA as a template. The
standard conditions for PCR were as follows: one cycle of denaturation at 94 C for
5 min, 25 cycles of denaturation at 94 C for 40 s, annealing at 55 C for 40 s, extension at 72 C for 2 min, and an extra extension at 72 C for 5 min. The amplied PCR
product was puried using the Axygen Gel extraction kit and cloned into the pGEMT easy vector (Promega, Madison, WI, USA). For the overproduction of TCMA, the
insert was excised from the pGEM T-easy vector using NdeI and XhoI and ligated
into the pET-21a(+) vector (Novagen, Darmastadt, Germany) treated with the same
restriction enzymes to create pET-TCMA.
The recombinant E. coli BL21-CodonPlus(DE3)-RP strain carrying pET-TCMA was
grown in 250 mL LB with ampicillin (100 g/mL) and chloramphenicol (34 g/mL)
at 37 C. Gene expression was induced by the addition of 0.5 mM isopropyl-1-thio-d-galactoside (IPTG) when the optical density (600 nm) of the culture reached
0.55. After a 20 h induction at 37 C, the cells were harvested by centrifugation
(10,000 g, 20 min, 4 C) and resuspended in lysis buffer (20 mM sodium phosphate,
500 mM NaCl, and 20 mM imidazole, pH 7.0). The cell suspensions were disrupted
by sonication (Sonier 450, Branson, Danbury, CT, USA; output 4, 60 times for 10 s,
constant duty) in an ice bath. Clear lysates containing crude enzyme was obtained
by centrifugation (10,000 g, 20 min, 4 C). The crude extract was passed through a
fast protein liquid chromatography (FPLC) system (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden) with a HisTrapTM HP chromatography column. The recombinant
TCMA was eluted with elution buffer (50 mM NaH2 PO4 , 250 mM NaCl, and 500 mM
imidazole at pH 7.0). The puried TCMA was identied by gel electrophoresis in a
10% (w/v) sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel. The protein concentration of the enzyme was determined using the bicinchoninic acid (BCA) protein
assay kit (Thermo Fisher Scientic, Waltham, MA, USA) with bovine serum albumin
(BSA) as the standard.
2.5. Enzyme assays
-1,4-Hydrolysis activity of TCMA was measured in 20 mM sodium citrate (pH
5.0) and determined by measuring the amount of released glucose using a GLzyme
kit (Shinyang, Seoul, Korea). One unit of -1,4-hydrolysis activity was dened as
the amount of enzyme that released 1.0 mol of glucose per minute. The reaction
mixture (100 L) was composed of 10 mM G3 as a substrate and 2.0 g of enzyme
solution (8.29 U/mg). The -1,4-hydrolysis reaction was performed for 5 min at
85 C. The reaction mixture was mixed with 190 l GLzyme glucose oxidase solution
and incubated at 37 C for 15 min prior to measuring absorbance at 505 nm using an
Innite 200 PRO spectrophotometer (TECAN, Mnnedorf, Switzerland). The activity of -1,6-bond hydrolysis was determined by dinitrosalicylic acid (DNS) [22] One
unit of activity was dened as 1.0 mol of reducing sugars produced per minute. The
reaction mixture was composed of 10 mM substrate (G2--CD) and 2.0 g enzyme
solution (11.23 U/mg) in 20 mM sodium citrate (pH 6.0) that was incubated at 98 C
for 5 min. The addition of DNS solution (7.06 g 3,5-dinitrosalicylic acid, 13.2 g NaOH,
204 g Rochelle salt, 5.06 mL phenol, 5.53 g Na-metabisulte, and 944 mL water) followed by boiling the mixture for 5 min stopped the reaction. The absorbance of
the reaction mixture was then measured spectrophotometrically at 550 nm. G2 was
used as the standard molecule for the DNS assay [22].
The optimum temperature for hydrolysis activity was examined in 20 mM
sodium citrate buffer (pH 6.0) at temperatures ranging from 80 C to 95 C for 5 min
with 1.88 g of TCMA (0.02 U for G2--CD and 0.08 U for G3). The effect of pH was
determined at various pH values between 4.0 and 9.0 with 60 mM universal BrittonRobinson buffer [equal mixture of 120 mM acetic acid, phosphoric acid and boric
acid, pH value is adjusted using a NaOH solution].
2.6. Analysis of TCMA hydrolytic patterns
To analyze the hydrolysis pattern of various substrates, 0.5% amylose, amylopectin, soluble starch, pullulan, branched cyclodextrin (G2--CD, G3--CD, and
G4--CD), or 10 mM maltooligosaccharides (from G2 to G7) were used as substrates.
The substrates were reacted with 1.88 g TCMA (0.02 U for G2--CD and 0.08 U
for G3) in 20 mM sodium citrate buffer (pH 5.0) at 75 C for 12 h. The patterns of
hydrolysis products were analyzed using TLC and HPAEC.
2.7. Thin-layer chromatography
Thin layer chromatography (TLC) analysis was performed using Whatman silica
gel K5F thin-layer chromatography (TLC) plates (Kent, UK). After baking each TLC
11
listed (Table 1). Among them, ten homologs are from Thermococcus genera, whereas ve homologs are from Pyrococcus genera.
In phylogenetic analysis, these homologs clearly divided into two
groups (Fig. 1A). One contains homologs from Thermococcus zilligii AN1 (TzilA 08562), T. barophilus MP (TERMP 02139), T. litoralis
DSM 5473 (OCC 00020), and T. sibiricus MM 739 (TSIB 1138) along
with those from Pyrococcus genera (Table 1). Another subgroup
includes proteins from Thermococcus sp. 4557, T. onnurineus NA1
(TON 1811), T. kodakarensis KOD1 (TK1743), Thermococcus sp. CL1
(CL1 0868), Thermococcus sp. AM4 (TAM4 1086), and T. gammatolerans EJ3 (TGAM 0418). Homologs in subgroup A were more closely
related to those of Pyrococcus genera than of subgroup B. The amino
acid homology of the sequences in subgroup A with PSMA was
approximately within the range of 7187%, while homology of the
sequences in subgroup B was below 59%. Also, the proteins in subgroup B were about 20 amino acids shorter than those in subgroup
A. The sequence differences between the two subgroups were also
observed in a multiple sequence alignment (supplemental Fig. S1).
However, they commonly have ve conserved regions, which are
distinguished from other GH57 enzymes (Fig. 1B).
3.2. Sequence analysis of the gene encoding TCMA (CL1 0868) in
Thermococcus sp. CL1
3. Results
3.1. In silico study of G2-forming -amylase homologs in
Thermococcales
G2-forming -amylase is a new member of the GH57 amylase
family that hydrolyzes -1,6- as well as -1,4-glycosidic linkages
and produces only G2. To date, the characterization of this type
enzyme has only been reported in Pyrococcus sp. ST04 [16]. Based
on BLAST analysis, the G2-forming -amylase homologs are only
observed in Thermococcales including Thermococcus and Pyrococcus genera (E-value < 1e10). These two genera are closely related
to each other. Only few differences such as the average GC content and optimum growth temperature are identied. In NCBI
database, total fteen G2-forming -amylase homologs have been
The gene encoding TCMA was cloned into the pET-21a(+) vector
and used to transform E. coli BL21 CodonPlus(DE3) RP for efcient
heterologous production of the archaeal protein. The addition of
0.5 mM IPTG to transformants harboring pET-TCMA induced gene
expression. TCMA is a thermostable enzyme that originates from
a hyperthermophilic archaea. Therefore, heat-treatment (70 C for
20 min) efciently removed most of the heat-labile E. coli proteins
from the crude extract. The recombinant TCMA with a C-terminal
histidine tag was rapidly puried by Ni-NTA afnity chromatography. In SDS-PAGE analysis, puried TCMA showed as a single
band of approximately 70 kDa, which is consistent with the estimated size deduced from the primary amino acid sequence of TCMA
(Fig. 2). The molecular mass of TCMA under non-denaturing conditions was determined as 67,115 Da, which indicated that TCMA
12
Table 1
List of proposed maltose-forming -amylases in Thermococcales.
Group
Organism
Accession number
Homologya (%)
Subgroup A
597
597
597
599
598
597
596
599
597
599
YP 006354539
NP 578599
YP 004424466
YP 004624034
NP 142934
NP 126637
YP 004072336
WP 004070095
WP 010480007
YP 002994541
100
86
86
82
86
87
77
71
73
71
Subgroup B
575
575
578
575
578
575
YP
YP
YP
YP
YP
YP
002958784
006424867
002308198
004762801
184156
002581296
57
57
55
57
59
57
Fig. 1. (A) Phylogenetic tree analysis of maltose-forming -amylases in Thermococcales. The tree was constructed using the neighbor-joining method of the MEGA 5.0 program
based on multiple sequence alignments of amino acid sequences obtained from the NCBI database. The number near the branch indicates the bootstrap value based on 1000
replicates. (B) Sequence logos of family GH57 maltose-forming -amylases from Thermococcus and Pyrococcus genera (16 strains). The sequences analyzed as PSMA homologs
were obtained from the NCBI database through the BLASTP tool and the sequence logo was created using the Weblogo 3.0 server [34]. The asterisk indicated location of
catalytic residues.
Fig. 2. SDS-PAGE analysis of puried TCMA. Lane M, protein size marker; Lane 1, proteins from crude extract (7 g); Lane 2, proteins (3 g) after heat treatment (70 C,
20 min); Lane 3, puried TCMA (1 g).
To investigate the characteristics of TCMA, various maltooligosaccharides and branched cyclodextrins were reacted with
TCMA at 75 C for 12 h (Fig. 3). Similar to PSMA, TCMA had
bifunctional specicity for -1,4- and -1,6-glycosidic linkages in
its substrates. The highest -1,4-glycosidic bond hydrolysis activity
was observed when G3 was reacted with TCMA (8.29 0.22 U/mg).
TCMA cleaved G3 into G2 and glucose. The reaction with
other maltooligosaccharides such as G4 (1.25 0.06 U/mg) and
G5 (1.32 0.05 U/mg) also produced G2 as the main product,
but the activity was relatively weak. -1,6-Glycosidic linkage
hydrolysis was determined using branched cyclodextrins that had
-1,6-glycosidic linkages between the maltooligosaccharides ranging from G2 to G4 and -CD. Among these substrates, TCMA
efciently hydrolyzed G2--CD and G4--CD into cyclodextrin
and G2. The strongest specic activity was seen with G2--CD
(11.23 0.16 U/mg). Other substrates, including G1--CD and G3-CD, were cleaved by TCMA at low levels (Fig. 3B). In addition,
TCMA only released G2 from polymers such as soluble starch, amylopectin, and glycogen, while G2 from reaction with amylose and
pullulan was rarely detected (Fig. 3C).
13
Fig. 3. TLC analysis of -1,4-hydrolyzing (A), -1,6-hydrolyzing (B), and polymer hydrolyzing (C) activity. Experiments were conducted at 75 C for 12 h with various
maltooligosaccharides (G3-G7), amylose (AL), amylopectin (AP), soluble starch (SOL), pullulan (PUL), glycogen (GLY), and branched -CDs including G2--CD, G3--CD,
and G4--CD. The reaction mixture contained 10 mM maltooligosaccharides, branched CDs, and 1% oligomer was incubated with (+) or without () enzyme. Lane M,
maltooligosaccharide (G1G7) standards and Lane B, branched cyclodextrin standards (-CD, G1--CD, G2--CD).
Table 2
Kinetic analysis of TCMA. The reaction was performed with various concentrations
of substrates (G3, 110 mM; G4 and G5, 210 mM; and G2--CD, 0.0510 mM).
Substrates
Km (mM)
G2--CD
G3
G4
G5
0.70
4.25
18.2
16.2
0.16
0.68
3.82
3.02
kcat (min1 )
1806
687
237
231
206
48
34.7
29.2
14
Fig. 4. Effect of temperature and pH on the activity of TCMA. (A) Effect of temperature on the -1,4-hydrolyzing activity (closed circle, 100% activity is 8.29 U/mg) and
-1,6-hydrolyzing activity (open circle, 100% activity is 11.23 U/mg) of TCMA and (B) effect of pH on the -1,4-hydrolyzing activity (closed circle, 100% activity is 8.12 U/mg)
and -1,6-hydrolyzing activity (open circle, 100% activity is 11.11 U/mg) of TCMA.
Table 3
Comparison of maltose-forming -amylase enzymatic properties with other established GH57 enzymes.
4--Glucanotransferase
Hydrolysis
-1,4-Bond
Transglycosylation
Action mode
Localization
Reference
-1,4-Bond
Random (endo-type)
Intracellular
[32,33]
Amylopullulanase
-1,4-Bond
-1,6-Bond
Random (endo-type)
Extracellular
[12,29]
Branching enzyme
-1,4-Bond
-1,6-Bond
Random (endo-type)
Intracellular
[24]
Maltose-forming -amylase
-1,4-Bond
-1,6-Bond
Maltose (exo-type)
Intracellular
This study
Acknowledgment
A National Research Foundation of Korea (NRF) grant funded by
the Korean government (MEST) (No. 2013-031011) supported this
work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.enzmictec.
2014.03.009.
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