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Enzyme and Microbial Technology 60 (2014) 915

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Enzyme and Microbial Technology


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Bioinformatic and biochemical analysis of a novel maltose-forming


-amylase of the GH57 family in the hyperthermophilic archaeon
Thermococcus sp. CL1
Eun-Jung Jeon a , Jong-Hyun Jung a,b , Dong-Ho Seo a , Dong-Hyun Jung a , James F. Holden c ,
Cheon-Seok Park a,
a

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

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E.-J. Jeon et al. / Enzyme and Microbial Technology 60 (2014) 915

such as high reaction temperatures. Also many hyperthermophilic


microorganisms such as Pyrococcus, Thermococcus, and Sulfolobus
species possess -amylases and related enzymes, such as glucosidases, pullulanases, and cyclodextrinases [1012]. Whole
genome sequence analysis has revealed that a few Pyrococcus and
Thermococcus strains only contain GH57 amylases and not GH13
amylases. For instance, Pyrococcus sp. ST04, whose whole genome
was recently determined and annotated, possesses three GH57
family enzymes including amylopullulanase (Py04 0209), branching enzyme (Py04 1358), and 4--glucanotransferase (Py04 0423),
and lacks homologs for GH13 enzymes [13,14]. Therefore, it is
reasonable that these GH57 amylases play an important role in
carbohydrate utilization in this hyperthermophilic archaeon [15].
Recently, a new type of GH57 amylase was identied in Pyrococcus sp. ST04 [16]. It is an exo-type -amylase acting only on
maltose (G2) from substrates such as starch, amylopectin, glycogen, and branched-cyclodextrin similar to the enzymatic activity
of -amylase. Normally, this enzyme hydrolyzes both -1,4- and
-1,6-glycosidic bonds of substrates akin to amylopullulanase [16].
Due to its activity prole, this enzyme was named Pyrococcus
sp. ST04 G2-forming amylase (PSMA). Remarkably, a comparative
genomic analysis revealed that PSMA homologs were exclusively
found in Pyrococcus and Thermococcus genera, but not in other
bacteria or archaea. Blesk et al. reported sequence features and
a ngerprint of well-established GH57 enzymes, but the report did
not include G2-forming -amylase, since its accurate biochemical
properties were not fully investigated at the time [7].
From the study of BLAST analyses and whole genome assignments, we observed that an homologs of PSMA was present in the
Thermococcus sp. CL1 (CL1 0868) genome. To determine that this
gene product (Thermococcus sp. CL1 G2-forming amylase, TCMA,
hereafter) has similar function to PSMA in Pyrococcus sp. ST04, we
conducted a detailed amino acid sequence analysis of TCMA and
determined the enzymatic characteristics of its recombinant gene
product in this study.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Thermococcus sp. CL1 was originated from Dr. James F. Holdens laboratory at University of Massachusetts, Amherst (Amherst, MA, USA) and its whole
genome sequence was recently determined, This hyperthermophilic strain was
cultivated using method described by Oslowski et al. [17]. Escherichia coli DH10B [FmcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 endA1 araD139(ara,
leu)7697 galU galK -rpsL deoRnupG] was used as the host for DNA cloning and
the E. coli BL21-CodonPlus(DE3)-RP strain [F ompT hsdS (rB ,mB ) dcm+ Tetr gal
(DE3) endA Hte (argU proL Camr )] was used as the heterologous host for protein
overproduction. E. coli transformants were grown in lysogeny broth (LB) [1% (w/v)
Bacto-tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl] containing chloramphenicol (34 g/mL) and ampicillin (100 g/mL) at 37 C.
2.2. Chemicals and enzymes
Amylose, amylopectin, soluble starch, and maltose (G2) were purchased from
Sigma Chemical Co. Maltooligosaccharides [from maltotriose (G3) to maltoheptaose
(G7)], short-branched cyclodextrin (6-O-maltosyl--CD; i.e., G2--CD), and pullulan
were purchased from Wako Pure Chemical Industries (Osaka, Japan). Long-branched
cyclodextrins [6-O-maltotriosyl--CD (G3--CD) and 6-O-maltotetraosyl--CD
(G4--CD)] were obtained from Dr. Kwan-Hwa Park at Sangmyung University
(Seoul, Korea). Silica gel K5F thin-layer chromatography (TLC) plates (Whatman,
Kent, UK) were used for analyzing the pattern of the hydrolysis. All restriction and
modifying enzymes were purchased from New England Biolabs (Ipswich, MA, USA).
2.3. Sequence analysis of putative G2-forming -amylases
Amino acid sequences of various amylases belonging to the GH57 family in
Thermococcus and Pyrococcus species were obtained from the National Center
for Biotechnology Information (NCBI) database. Sequence homology analysis was
performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence
alignments were performed using ClustalW2 [18]. The phylogenetic tree was

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

E.-J. Jeon et al. / Enzyme and Microbial Technology 60 (2014) 915


plate at 110 C for 30 min, 0.5 L aliquots of the reaction mixture were spotted onto
the plate and developed with a solvent system of isopropanol/ethyl acetate/water
(3:1:1, v/v/v). The developed TLC plate was dried in a hood and then visualized
by soaking quickly with 0.3% (w/v) N-(1-naphthyl)-ethylenediamine and 5% (v/v)
H2 SO4 in methanol. The plate was dried and baked in an oven for 10 min to observe
the reaction spots.
2.8. High performance anion exchange chromatography
High performance anion exchange chromatography (HPAEC) samples were prepared for analysis by ltering with a Nylon membrane lter (0.2 m pore diameter,
Whatman) after stopping the reaction by adding 10-fold 150 mM NaOH solution.
CarboPacTM PA-1 columns (0.4 cm 25 cm, Dionex, Sunnyvale, CA, USA) and an electrochemical detector (ED40, Dionex) were employed for detection. Two buffers,
A (150 mM NaOH) and B (150 mM NaOH and 500 mM Na-acetate), were used for
sample elution with a 0 to 100% gradient of buffer B for 60 min at a ow rate of
1.0 mL/min.
2.9. Gel permeation chromatography (GPC)
A protein sample (200 L of a 1.0 mg/mL solution) was applied to a Superdex
200HR column (10 mm 300 mm, GE Healthcare Bio-Sciences Ltd., Buckinghamshire, England) to estimate the apparent molecular weight of the recombinant
holoenzyme. The column was equilibrated with 20 mM sodium phosphate buffer
(pH 7.5) and the protein was eluted with the same buffer at a ow rate of 0.4 mL/min.
The standard proteins used were carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), -amylase (200 kDa), and thyroglobulin (669 kDa).

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

2.10. Circular dichroism spectroscopy


Circular dichroism measurements were carried out using a JASCO J-815 CD spectrometer (Jasco International, Tokyo, Japan) purged with N2 gas. Far-UV circular
dichroism spectra of PSMA and TCMA were recorded in the wavelength range of
190240 nm at 20 C. The parameters were as follows: bandwidth, 1.0 nm; scan
speed, 100 nm/min; data integration time, 1.0 s; and data pitch, 1.0 nm. Circular
dichroism studies were performed in the same buffer used to obtain a baseline.
Quantitative estimations of the secondary structure content were made with the
K2D3 sever.
2.11. Differential scanning uorimetry (DSF) analysis
DSF was performed as described previously [23]. A 7500 Fast Real Time PCR
System (Applied Biosystems, Carlsbad, CA, USA) was employed for the heating cycle.
Ten L each of enzyme (0.17 mg/mL) and a freshly prepared 10-fold water-based
dilution of SYPRO orange 5000 (Invitrogen, Grand Island, NY, USA) were added
to a Fast Optical 96 Well Reaction Plate (Applied Biosystems) and maintained on
ice. The plate was sealed with Optical Adhesive Film (Applied Biosystems) and then
gently vortexed for analysis in the real-time polymerase chain reaction (RT-PCR)
thermocycler. The heating cycle was comprised of a gradient between 30 C and
99 C in 70 steps of 1.0 min, each with a 1 C ramp. Data were collected based on the
calibration setting for TAMRA dye detection (ex 560 nm; em 582 nm) installed in
the instrument.
2.12. Kinetic analysis
The kinetic parameters of TCMA for various substrates were determined by
HPAEC analysis. All reaction conditions were the same as described above. The
amount of G2 was determined from various concentrations of substrates (for G3,
110 mM; for G4 and G5, 210 mM; and for G2--CD, 0.051 mM). Each kinetic
parameter such as Vmax , Km , and kcat were calculated using the Michaelis-Menten
equation of GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA).

Although we proposed the fteen sequences in two subgroups


function as putative G2-forming -amylases, only one established
enzyme in this group was isolated and characterized from Pyrococcus sp. ST04 of subgroup A [16]. Therefore, it was worthwhile
to conrm how enzymes in subgroup B display their catalytic
properties. CL1 0868 in Thermococcus sp. CL1 (TCMA) is one of
the enzymes of subgroup B, which consists of 1728 nucleotides
corresponding to 575 amino acids with a theoretical molecular
mass of 66,999.24 Da. The gene is located between nucleotides
825,722827,449 and is surrounded by genes encoding members of
tRNA CCA-pyrophosphorylase (CL1 0867) and a hypothetical protein (CL1 0869). This gene organization is highly similar with other
Thermococcus species except for T. barophilus MP. Interestingly, the
gene organization in T. barophilus MP consists of two parts; the
upstream sequence shares similarity with that of Thermococcus
whereas the downstream sequence resembles the gene organization of Pyrococcus (data not shown).
The BLAST analysis showed that TCMA has 58% identity with
PSMA and shares high similarity (>80%) with other enzymes of
subgroup B. The ve conserved regions and catalytic residues
of amylolytic enzymes were identied in TCMA. GH57 family
enzymes employ an -retaining mechanism through two catalytic
residues. Using multiple sequence alignments, the nucleophile and
acidbase catalyst residues of TCMA were determined to be Glu150
and Asp244, respectively.
3.3. Cloning and overexpression of TCMA in E. coli

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

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E.-J. Jeon et al. / Enzyme and Microbial Technology 60 (2014) 915

Table 1
List of proposed maltose-forming -amylases in Thermococcales.
Group

Organism

Amino acid size

Accession number

Homologya (%)

Subgroup A

Pyrococcus sp. ST04 (PSMA)


Pyrococcus furiosus DSM 3638
Pyrococcus sp. NA2
Pyrococcus yayanosii CH1
Pyrococcus horikoshii OT3
Pyrococcus abyssi GE5
Thermococcus barophilus MP
Thermococcus litoralis
Thermococcus zilligii
Thermococcus sibiricus MM 739

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

Thermococcus gammatolerans EJ3


Thermococcus sp. CL1
Thermococcus onnurineus NA1
Thermococcus sp. 4557
Thermococcus kodakarensis KOD1
Thermococcus sp. AM4

575
575
578
575
578
575

YP
YP
YP
YP
YP
YP

002958784
006424867
002308198
004762801
184156
002581296

57
57
55
57
59
57

Homology was compared with PSMA.

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.

far-UV circular dichroism spectra (190240 nm), TCMA was found


to be comprised of 47.3% -helix and 12.7% of -sheet whereas
PSMA has 41.3% and 14% -helix and -sheet content, respectively.

3.4. Substrate specicity of TCMA

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).

naturally exists as a monomer. This is a unique property since


PSMA functions as a tetrameric enzyme (data not shown). However, the secondary structure of TCMA was similar to PSMA even
though their oligomeric states were distinct. From the result of

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).

E.-J. Jeon et al. / Enzyme and Microbial Technology 60 (2014) 915

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

kcat /Km (mM1 min1 )


2566
161.8
13.01
14.25

3.5. Kinetic analysis


In order to understand the enzymatic action of TCMA, the kinetic
parameters of the recombinant protein were determined with G3,
G4, G5, and G2--CD substrates using a Michaelis-Menten equation (Table 2). Analysis of the Km value of TCMA for representative
substrates G3 and G2--CD revealed that the Km value on -1,4linkages was higher than that of -1,6-linkages. Also, the Km value
of substrates that consisted of an -1,4-linkage decreased as the
length of substrate glucose units increased (Table 2). These data
suggest that TCMA easily bound small substrates and had lower Km
value toward -1,6-glycosidic linkages than -1,4-linkages. This
was also observed in the catalytic efciencies acquired from kcat /Km
values. The catalytic efciency of G2--CD was 16-fold higher than
that of G3 (Table 2). Compared to the kcat /Km value toward G3, the
values for longer substrates such as G4 and G5 were negligible.
3.6. TCMA has different optimal conditions depending on
glycosidic bond
As described above, TCMA hydrolyzes both -1,4- and -1,6glycosidic bonds, and reactions with G3 and G2--CD as the
substrate showed the highest activity. We determined the optimal
reaction conditions for TCMA to release G2 from these substrates. In
G3 hydrolysis, the highest activity of TCMA was displayed at 85 C,
whereas the optimum temperature for G2--CD hydrolysis activity increased above 98 C (Fig. 4A). Moreover, the optimum pH of
TCMA toward G3 and G2--CD were determined as pH 5.0 and pH
6.0, respectively (Fig. 4B). When the G3 hydrolysis reaction was performed at pH 7.0 and 8.0, the relative activity decreased to below
20%. This was distinct from G2--CD hydrolysis, which showed
bell-shaped activity over a broad pH range (Fig. 4B). These results
suggest that the interaction between the enzyme and two different substrates might be highly dependent upon the environmental
conditions such as temperature and pH.
TCMA originates from the hyperthermophilic archaeon Thermococcus sp. CL1, which grows optimally at 85 C. Therefore, the

thermostability of TCMA is an essential property to establish its


physiological role in vivo. DSF analysis showed that melting temperatures of TCMA were 67 C, 91 C, and 105 C at pH 4.0, pH 5.0,
and pH 6.0, respectively (data not shown). These data indicate that
thermostability of TCMA is signicantly affected by pH condition.
We also checked the effect of pH on TCMA activity. The half-life
of TCMA at 85 C was 69 min, 225 min, and 255 min at pH 5.0, 6.0,
and 7.0, respectively (data not shown). These results are signicantly consistence with each other, indicating that thermostability
is substantially correlated with pH in TCMA.
4. Discussion
GH57 amylolytic enzymes are well-known to play an essential
role in Thermococcus and Pyrococcus genera [15,24,25]. Based on
the evolutionary relationships, G2-forming -amylase homologs
are strictly separated from the four distinguished amylolytic
enzymes of the GH57 family in Thermococcus genera, including
-galactosidases, amylopullulanases, branching enzymes, and 4-glucanotransferases (supplemental Fig. S2). The sequence logo
of G2-forming -amylases in Thermococcales showed that the conserved regions, in particular IV and V, are clearly different from
those of other GH57 enzymes (Fig. 1B). Blesk et al. suggested
that the arrangement of domain and sequence features of GH57
enzymes were distinct from each other depending on their substrate specicities [7]. Among the conventional GH57 enzymes,
a group of -galactosidases was located near the G2-forming amylase homologs rather than other groups (supplemental Fig.
S2). Interestingly, -galactosidase and G2-forming -amylase have
greater specicities to the shorter substrates than longer ones.
Recently revealed PSMA displayed a distinctive hydrolysis of
G2 connected by either -1,4- or -1,6-glycosidic bonds [16]. This
activity is a unique characteristic which has not been observed
in GH57 family and other amylolytic enzymes to date (Table 3).
In addition, it was realized that PSMA homologs are exclusively
present in Thermococcales from bioinformatics analysis. They share
a wide range of homology from 55% to 87%, and divided into two
groups by their sequence arrangement. Subgroup A consists of
PSMA-like G2-forming -amylases that shows above 70% homology with PSMA, whereas subgroup B has low amino acid identity
with PSMA. TCMA is one of the G2-forming -amylases that was
classied in subgroup B. The enzymatic properties of TCMA were
similar with those of PSMA. However, there are some distinct
properties between two enzymes. From GPC analysis, we identied that TCMA and PSMA show different oligomeric states. TCMA
acts as a monomer while PSMA functions as a tetrameric enzyme.

14

E.-J. Jeon et al. / Enzyme and Microbial Technology 60 (2014) 915

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]

The aforementioned multiple sequence alignment results showed


that there was a region that was distinctly different between subgroup A and B. It is interesting that this region is located at the
surface where each monomer interacts in PSMA. A computer simulation showed that there was a different level of -helix and
-sheet content in this region between PSMA and TCMA. Previously, there was an interesting report regarding the oligomeric
state of hyperthermophilic branching enzyme. A branching enzyme
from T. kodakarensis and AmyC from Thermotoga maritima (RMSD
shared a highly conserved fold organization and arrangeof 1.90 A)
ment [26]. However, they display a different oligomeric state. The
branching enzyme is a monomeric enzyme due to a -hairpin
insertion at the 185204 amino acid segment whereas AmyC is a
tetrameric enzyme [26]. Hence, there is a possibility that different regions between subgroup A and B in PSMA homologs (6874,
89112, 181186, 197203, 227235, 304308, 379393, 424431,
and 480493 PSMA numbering) might cause a different oligomeric
state of these enzymes (supplemental Fig. S1).
TCMA is an intracellular enzyme displaying hydrolysis activity
toward -1,4- and -1,6-glucosidic linkages as PSMA. However, Km
values of TCMA for substrates were different from those of PSMA.
The catalytic efciency of TCMA for -1,6-glucosidic linkage is
lower than that of PSMA while it is higher toward -1,4-glucosidic
linkage [16]. Although the preferences between two enzymes were
different, their roles in cytoplasm are supposed to be comparable. TCMA most likely hydrolyzes the isomaltooligosaccharides
or phosphorylase-limited dextrin generated from glycogen storage polymer, because it is only intracellular amylase hydrolyzing
-1,6-glucosidic linkage. Thermococcus genera produce diverse
amylolytic enzymes classied as GH13 and GH57. However, there
are no debranching enzymes found in bioinformatical studies
except for G2-forming -amylase. Although amylopullulanase is
displaying the strong -1,6-hydrolysis activity, its role is to degrade
polymeric substrates outside of the cells, not in cytoplasm [27].

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

Interestingly, TCMA showed different optimum conditions


depending on the type of substrate. It thought that the binding between TCMA and G3 or G2--CD might cause different
binding afnities resulting in a somewhat different enzyme thermostability. Protein-ligand complex is more stable than sole
protein [23,28], which properties are commonly used in inhibitor
screening. Also amylopullulanase from P. furiosus was stabilized by
addition of Ca2+ ion, which reduced the Km value and increase the
optimum temperature [29]. And Thermoactinomyces vulgaris R-47
-amylase I showed a shift of optimum temperature by truncating
the N-terminal region because truncation reduced enzyme stability [30]. The different optimum pH of TCMA was also explained
by substrate binding sites. Keating et al. explained that pH displaying activity was determined by residues interacting with substrates
[31]. Therefore, we concluded that amino acid residues in the
substrate-binding site of TCMA have dissimilar afnity toward G3
or G2--CD.
5. Conclusions
G2-forming -amylase is a new member of the GH57 family that
has distinct enzymatic properties from other GH57 enzymes. To
date, only one enzyme has been isolated from Pyrococcus sp. ST04
(PSMA). In silico study showed that in Thermococcus and Pyrococcus species, 15 proteins divided into two phylogenetic subgroups
that were identied as homologs of PSMA. One of the far distinct
homologs, CL1 0868 in Thermococcus sp. CL1 (57% homology with
PSMA), had G2-forming -amylase activity but displayed a different oligomeric state and afnity toward substrates. In particular,
it differentially exhibited optimum conditions based on substrate
linkage. From the results, we propose that the 15 proteins are new
members of the G2-forming -amylase GH57 family based on their
specic sequence logos, which can be informatively used to predict
the specicity of hypothetical GH57 enzymes.

E.-J. Jeon et al. / Enzyme and Microbial Technology 60 (2014) 915

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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