Isolation and characterization of a novel thermostable neopullulanase-like enzyme from a hot spring in Thailand

Article abstract of DOI:10.1271/bbb.70754

A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60°C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and γ-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75°C with K_m and V_max toward pullulan of 1.22 ± 0.3% and 23.24 ± 1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources.

Full text of DOI:10.1271/bbb.70754

Biosci. Biotechnol. Biochem., 72 (6), 1448–1456, 2008
Isolation and Characterization of a Novel Thermostable
Neopullulanase-Like Enzyme from a Hot Spring in Thailand
Kittapong TANG, Rutchadaporn Sriprang KOBAYASHI, Verawat CHAMPREDA,
y
Lily EURWILAICHITR, and Sutipa TANAPONGPIPAT
BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology,
National Science and Technology Development Agency, Thailand Science Park,
113 Phaholyothin Rd., Klong 1, Klongluang, Pathumthani, 12120, Thailand
Received November 16, 2007; Accepted March 21, 2008; Online Publication, June 7, 2008
[doi:10.1271/bbb.70754]
A gene encoding a thermostable pullulan-hydrolyzing
enzyme was isolated from environmental genomic DNA
extracted from soil sediments of Bor Khleung hot spring
in Thailand. Sequence comparison with related enzymes
suggested that the isolated enzyme, designated Env
Npu193A, was most likely a neopullulanase-like enzyme.
Env Npu193A was expressed in Pichia pastoris as a
monomeric recombinant protein. The purified Env
Npu193A exhibited pH stability ranging from 3 to 9.
More than 60% of enzyme activity was retained after
incubation at 60 ꢀC for 1 h. Env Npu193A was found to
hydrolyze various substrates, including pullulan, starch,
and ꢀ-cyclodextrin. The optimal working condition for
Env Npu193A was at pH 7 at 75 ꢀC with Km and Vmax
toward pullulan of 1:22 Æ 0:3% and 23:24 Æ 1:7 U/mg
respectively. Env Npu193A exhibited distinct biochem-
ical characteristics as compared with the previously
isolated enzyme from the same source. Thus, a culture-
independent approach with sequence-basing was found
to be an effective way to discover novel enzymes
displaying unique substrate specificity and high thermo-
stability from natural bioresources.
enzyme discovery, but only a minor proportion of
them can be cultivated under standard conditions. It is
estimated that more than 95% of these microbes are
unculturable.2) With advances in biotechnology, the
unculturable microbes have become important targets
for the discovery of novel enzymes having specific
characteristics of academic and industrial interest. Mo-
lecular approaches searching for genes encoding target
enzymes based on activity-based screening and PCR-
based cloning have been exploited successfully in the
isolation of many genes encoding potent enzymes from
diverse environments, resulting in an increasing number
of potent industrial enzyme candidates obtained from
environmental DNA.3–8)
One enzyme property crucial in industrial applications
is thermostability. Extreme environments such as hot
springs are considered promising sources for the direct
isolation of thermostable enzymes. Our group has
reported the biodiversity and abundance of bacteria
and archaea from the Bor Khleung hot spring in
Thailand, and has found that the majority (up to 80%)
of the prokaryotes have not been reported elsewhere.9)
Recently, we isolated a novel cyclomaltodextrinase from
this hot spring using a PCR-based cloning approach.10)
This enzyme, Env Cda13A, exhibited activity against
cyclodextrins, starch, and pullulan to different degrees,
with a temperature optimum of 50 ꢀC. In this study,
we isolated another novel thermostable enzyme, Env
Npu193A, from the same hot spring. Although the
two enzymes were isolated from the same source, they
exhibited different biochemical characteristics, includ-
ing substrate specificity and thermostability. The struc-
ture-function relationship of Env Npu193A with respect
to other related enzymes in the neopullulanase subfam-
ily is further discussed, demonstrating the unique
distinct characteristics of this enzyme and thus the
potential of the molecular approach for the discovery
of novel enzymes of academic and industrial interest.
Key words: pullulan-hydrolyzing enzyme; thermostable;
neopullulanase
The ꢀ-amylase family includes several starch-con-
verting enzymes exhibiting a diversity of substrates and
product specificities.1) Many enzymes in this family,
including ꢀ-amylases, isoamylases, pullulanases, cyclo-
maltodextrinases, and cyclodextrin glycosyltransferase
(CGTases), have been characterized and used widely
for industrial purposes such as starch production, brew-
ery, and baking. Industrial applications typically require
extreme conditions of pH and temperature, hence novel
amylolytic enzymes with compatible characteristics are
desirable.1)
Bacteria and fungi are the main sources for novel
y
To whom correspondence should be addressed. Tel: +66-2-5646665 ext 3472; Fax: +66-2-5646707; E-mail: sutipa@biotec.or.th

Novel Neopullulanase-Like Enzyme Isolated from Environmental DNA
1449
Materials and Methods
Molecular DNA cloning in P. pastoris. All PCR
products were cloned into pTZ57R/T vector (Fermentas)
and transformed into E. coli DH5ꢀF0. For expression
of Env Npu193A in P. pastoris, the corresponding gene
without its signal sequence was PCR amplified using
193picZXhoI (50-GTTGGGCTCGAGAAGAGGTCG-
CCAGTGGTACCTGC-30) and 193picZSacII (50-GC-
ACTGCCGCGGTCAGTTTTGCTTTGTAATTAGG-30)
primers (restriction sites for cloning underlined) and
cloned into pPICZꢀA vector. As a result, the gene was
fused in-frame with ꢀ-factor secretion signal of the
vector. The resulting recombinant plasmid, pPICZ-
BK193, was then integrated into P. pastoris KM71 by
electroporation, as described by the manufacturer (In-
vitrogen). DNA sequencing to verify integration was
performed by Bio Service Unit (National Center for
Genetic Engineering and Biotechnology, Bangkok,
Thailand) and Macrogen (Seoul, South Korea).
Strains, growth conditions, and plasmids. Escherichia
coli DH5ꢀF0 was used in the cloning all recombinant
plasmids. Bacterial cultures were grown in Luria-
Bertani broth (LB) at 37 ꢀC supplemented with ampi-
cillin (100 mg/ml) or zeocin (25 mg/ml) where appro-
priate. Plasmid pTZ57R/T (Fermentas, Vilnius, Lithua-
nia) was used for DNA cloning. Methylotrophic yeast,
Pichia pastoris KM71 (Invitrogen, CA), was used as
an expression host. Plasmid pPICZꢀA (Invitrogen)
was used as a yeast expression vector. Yeasts were
grown in YPD (Yeast Extract Peptone Dextrose me-
dium) supplemented with zeocin (100 mg/ml) where
appropriate.
Genomic DNA extraction from soil samples by
SDS-based DNA extraction and purification. Soil sedi-
ments were collected from Bor Khleung hot spring in
Ratchaburi Province, Thailand. Genomic DNA from
soil samples was extracted by the SDS-based DNA
extraction method established by Zhou et al.11) The
crude genomic DNA was purified with a QIAquick Gel
Extraction kit (Qiagen, Hilden, Germany). The quantity
and quality of purified DNA were analyzed by agarose
gel electrophoresis.
Expression of Env Npu193A in P. pastoris. A single
colony of recombinant P. pastoris was inoculated into
10 ml of YPD and incubated at 30 ꢀC overnight with
vigorous shaking. A 1-ml starter culture was transferred
to a 2-liter conical flask containing 400 ml of BMGY
(Buffered Glycerol-complex medium, Invitrogen), and
the culture was grown overnight under the same
conditions. After the culture reached an OD600 of 2–6,
cells were harvested by centrifugation at 3;000 ꢁ g for
5 min, and resuspended in 100 ml of BMMY (Buffered
Methanol-complex medium, Invitrogen) containing 3%
methanol as an inducer. Induction was continued at
30 ꢀC with vigorous shaking for 2 d with the addition
of methanol every 24 h to yield a final concentration
of 3%. Protein expression profiles were analyzed on
SDS–PAGE with Coomassie brilliant blue staining. The
culture medium containing secreted target enzyme was
collected by centrifugation at 3;000 ꢁ g for 5 min.
Genome walking PCR. Genome walking PCR was
performed as described by the manufacturer (Clontech,
CA). Briefly, EcoRV-digested genomic DNA was
ligated with the genomewalker adaptor provided. To
obtain 50 and 30 flanking sequences of BK193, PCR was
performed on adaptor-ligated genomic DNA template
with BK193 gene-specific and adaptor primers. The
primary PCR products, of approximately 1.3 kb (50-
walking) and 2 kb (30-walking), were diluted and used
as templates for nested PCR using nested gene-specific
and adaptor primers, yielding nested PCR products of
approximately 1.2 kb (50-walking) and 1.9 kb (30-walk-
ing) respectively. The following primers pairs were
used: primary 50-walking primers, BK193-1UP (50-
CTTGGGATCTTTTCGGAAGGCAGGGAG-30) and
AP1 (50-GTAATACGACTCACTATAGGGC-30); nest-
ed 50-walking primers, BK193-2UP (50-TCACTCGT-
GTCTTTTGTGGCAGGGTCAT-30) and AP2 (50-AC-
TATAGGGCACGCGTGGT-30); primary 30-walking
primers, BK193-1DW (50-CGCGACACCGTGGCTTG-
AAGGAGATGA-30) and AP1; nested 30-walking pri-
mers, BK193-2DW (50-TCGCGCATGTTCGCAGAT-
CATATCCG-30) and AP2. To obtain the full-length
open-reading frame of the Env npu193A gene, primers
NdeI193 (50-ATCTGTCATATGAAGAGAACTCATT-
GCATAAGCAGAGCC-30) and EcoRI193 (50-ACTG-
CAGAATTCAGTTTTGCTTTGTAATTAGGACGC-30)
were used. The PCR condition to obtain the Env
npu193A gene was 1 cycle at 94 ꢀC for 1 min, 35 cycles
at 94 ꢀC for 30 s, 50 ꢀC for 30 s, 72 ꢀC for 2 min, and 1
cycle at 72 ꢀC for 5 min.
Purification of Env Npu193A. The culture medium
was concentrated by recirculating at a flow rate of
40 ml minꢂ1 in a Minimateꢀ Tangential Flow Filtration
(TFF) system (Pall, NY) with a 10 kDa-cut off Mini-
mateꢀ TFF capsule. The protein was then diluted in
potassium phosphate buffer, pH 6, to the initial volume,
and the concentration step repeated. The whole process
was repeated at least twice to replace the medium with
the buffer entirely. Size exclusion chromatography was
carried out on an AKTA Explorer system (GE Health-
care Biosciences, Uppsala, Sweden) to purify Env
Npu193A. Concentrated protein sample (300 ml) was
injected into a Superdex 200 10/300 GL column (GE
Healthcare) pre-equilibrated with 50 mM MOPS, pH 7,
and 50 mM NaCl and eluted in the same buffer at a flow
rate of 0.5 ml/min. Fractions exhibiting enzymatic
activity were collected and concentrated using an
Amicon Ultra-15 device (Millipore).
Enzyme assay. The enzyme activity was determined

1450
K. TANG et al.
quantitatively by the DNS method.12) Starch (0.5%)
(Carlo Erba, Rodano, Italy), 0.5% pullulan or 0.5%
cyclodextrin (Sigma, St. Louis, MO) substrates was
incubated with appropriate amounts of enzyme in 50 mM
MOPS buffer, pH 7.0, supplemented with 2 mM of
CaCl2. The reaction was incubated at 75 ꢀC for 45 min
and then 0.3 ml of DNS reagent was added. The reaction
was then boiled for 10 min. The amount of reducing
sugar was determined by measuring the absorbance
at 540 nm (OD540) and interpolating from a standard
curve of glucose or maltotriose (for pullulanase activity)
absorbance. The effect of metal ions on enzymatic
activity was examined after pre-incubation of the
alignments were performed using the program
Clustal X.
Results
Identification of a novel gene encoding a neopullula-
nase-like enzyme by genome-walking PCR
Previously, we reported the identification of partial
gene sequences with homology to the conserved region
of the starch-degrading enzyme family, and one of
these sequences, BK13, was further characterized.10)
The full-length gene of BK13, designated Env cda13A,
was identified by a genomic walking approach, and the
biochemical properties of the corresponding enzyme
were investigated.10) In this work, the same approach
was employed to obtain a full-length gene of another
partial sequence, BK193, previously isolated from the
same hot-spring environmental DNA sample. The full-
length gene, designated Env npu193A, was 1,815 bp in
length (GenBank accession EU022022) and coded for a
putative protein of 605 amino acid residues. BLAST
analysis showed that Env Npu193A shared the highest
amino acid sequence identity (48%) to that of Env
Cda13A, while it exhibited 45% identity with ꢀ-amylase
of Salinibacter rubber DSM13855 and 37% identity
with chain B of ꢀ-amylase II (TVAII) of Thermoacti-
nomyces vulgaris. The alignment of Env Npu193A with
related enzymes is shown in Fig. 1, and salient con-
served features are highlighted.1) The neopullulanase
subfamily conserved regions are shown as boxes A-C
in Fig. 1.14–16) Box A locates between the start of the
ðꢁ=ꢀÞ8-barrel and conserved region I, which is perhaps
a Ca2þ-binding site.17) The conserved sequences in
BoxA and BoxB (HKYXT and YQIFPER) are absent
in pullulan non-hydrolyzing amylases. Box C denotes
the fifth conserved region, used to distinguish between
oligo-1,6-glucosidase subfamily (QpDln) and neopullu-
lanase subfamily (MpKln), and is perhaps involved
in calcium binding.16) The amino acid sequences in
BoxC of Env Npu193A are different from both
subgroups, but are similar to that of cyclomaltodextri-
nase of Thermotoga maritima and Env Cda13A.10,15)
In addition, these latter three enzymes lack an extended
N-terminal domain, which is the unique characteristic
of true members of the neopullulanase subfamily.
Env Npu193A does not contain the consensus sequence
YNWGYDP, which is found in all type I pullulanases.18)
Therefore, Env Npu193A can be included in the group
of so-called neopullulanse-like enzymes.16)
enzyme solution with 2 mM of various cations (Mg2þ
,
Ca2þ, Sr2þ, Zn2þ, Cu2þ, Fe2þ, Ni2þ, Mn2þ) at optimal
temperature for 45 min, and then the remaining activity
was determined as described above. One unit of enzyme
activity was defined as the amount of the enzyme that
produces 1 mmole of reducing sugar at the optimal
temperature in 1 min. The enzyme activities measured at
different intervals were confirmed to be within the linear
range of the assay. The thermostability of the enzyme
was determined by pre-incubating the enzyme mixture
(appropriate amounts of Env Npu193A in 50 mM MOPS,
pH 7.0, and 2 mM CaCl2) at 45, 50, 60, 65, 70, and 75 ꢀC
for 30, 60, and 120 min. To determine pH stability, the
enzyme was incubated at 25 ꢀC for 4 h in 50 mM sodium
citrate (pH 3), 50 mM sodium acetate (pH 5), 50 mM
Tris–HCl (pH 7), or 50 mM glycine (pH 9). The residual
pullulanase activity was measured by the DNS method
using 4% (w/v) pullulan as substrate under optimum
conditions. The relative activity was calculated by
comparing the activity after each treatment to that of
the untreated enzyme, which was assigned to be 100%.
The kinetic parameters of the enzyme were calculated
by fitting initial velocities measured at various substrate
concentrations to the Michaelis-Menten equation using
Kaleida Graph version 3.51 data analysis software
(Synergy Software, PA).
Product specificities of Env Npu193A. Hydrolysis
products were analyzed by thin layer chromatography
(TLC). An appropriate amount of the substrate (cyclo-
dextrins, soluble starch, pullulan (0.5% each) and 2 mM
maltooligosaccharides) was incubated with the enzyme
at 75 ꢀC for 24 h. The reactions were heated at 100 ꢀC
for 10 min to stop the reaction, and spotted onto a silica
gel 60 aluminum plate (Merck, Darmstadt, Germany).
The plate was then placed in a TLC chamber containing
a solvent mixture of isopropanol-ethyl acetate-water
(3:1:1, v/v/v), and developed at room temperature.
After air-drying, the detection reagent (0.3% of N-(1-
naphthyl)-ethylenediamine and 5% concentrated H2SO4
in methanol) was evenly sprayed onto the plate. The
plate was heated at 110 ꢀC for 10 min for visualization.
Enzyme characterization
Env Npu193A contained a putative signal sequence of
27 amino acid residues at the N-terminus (M1-Q27)
(Fig. 1). This indicates that it is an extracellular protein.
It was expressed as a fusion protein (residues 28 to 605)
with the ꢀ-factor peptide, and secreted as an active
soluble protein in P. pastoris with an apparent MW of
approximately 66 kDa (Fig. 2). Purification of Env
Sequence analysis. The signal peptide cleavage
site was analyzed by SignalP 3.0.13) Multiple sequence

Novel Neopullulanase-Like Enzyme Isolated from Environmental DNA
1451
Fig. 1. Multiple Sequence Alignment of Env Npu193A Isolated from Hot Spring with Other Related Amylolytic Enzymes.
The four conserved regions commonly found among enzymes in glycosyl hydrolase family 13 are shown (labeled as I-IV). Conserved regions
located between the start of the ðꢁ=ꢀÞ8-barrel and conserved region I are indicated as BoxA and BoxB. The fifth conserved region, proposed to be
a selection marker for the neopullulanase subfamily, is indicated by BoxC.16) The putative signal sequence of Env Npu193A is underlined. The
Phe residue corresponding to Phe289 of Thermus sp. is indicated by an arrow. TVAII, chainB of ꢀ-amylase II of Thermoactinomyces vulgaris
(accession no. BAB40638); ThMA, maltogenic amylase of Thermus sp. IM6501 (accession no. AAC15072); TMG, maltogenic amylase of
Thermotoga maritima (accession no. AE001820); Cda13A, cyclomaltodextrinase Env Cda13A (accession no. DQ191050); Npu193A,
neopullulanase-like enzyme Env Npu193A (accession no. EU022022).
Npu193A by gel filtration revealed a monomeric protein
of similar size (data not shown). The purified enzyme
exhibited highest activity toward pullulan (23.24 U/mg),
and showed lower activity against starch (0.15 U/mg). It
was able to hydrolyze ꢂ-cyclodextrin, but with very poor
activity (data not shown). Ca2þ was essential for the
activity of the enzyme, since no activity was detected
when Ca2þ was not included in the reaction (data not
shown). The substrate specificity corresponded well with
the fact that Env Npu193A functioned extracellularly,
since it prefers large substrates that cannot enter the
cells. Since the enzyme showed highest activity toward
pullulan, it was chosen as a substrate to investigate
further the properties of the Env Npu193A enzyme. The

1452
K. TANG et al.
Discussion
kDa
116
Enzymes belonging to the neopullulanase subfamily
typically harbor the N-domain, which is involved in
dimer formation.14,19) In addition, amino acids at the
extended N-terminal domain of enzymes in the neo-
pullulanase subfamily have been found to play roles
in cyclodextrin binding, although those roles are not
essential.15,19) It was found that enzymes lacking this
N-domain were still able to hydrolyze cyclodextrins, but
with less activity than those harboring this region.20)
This lack of N-domain corresponded well with Env
Npu193A, which is monomeric, and the low activity
on cyclodextrins of both Env Cda13A and Env
Npu193A.10) The crystal structure of a maltogenic
amylase (ThMA) of Thermus sp. revealed three residues
in the N-terminal domain, Tyr45, Trp47, and Asp110,
which interacted with ꢁ-cyclodextrin at the upper part
of the active site.15) Another amino acid, Phe286, is
responsible for interaction with ꢁ-cyclodextrin. Replace-
ment of Phe286 by non-aromatic amino acid residues
causes a drastic decrease in the kcat=Km values, due to
the loss of interaction between the aromatic ring of
Phe and the ring structure of ꢁ-cyclodextrin.21) For both
Env Cda13A and Env Npu193A, this Phe is replaced
by Trp (Fig. 1). Although the two enzymes share the
same aromatic residue, which should make for similar
interactions with cyclodextrins, our result indicates that
the cyclodextrin substrates were converted more effi-
ciently by Env Cda13A than Env Npu193A. This clearly
indicates that other amino acid residues also contribute
to substrate recognition in the neopullulanase subfamily,
resulting in the clear difference in substrate specificity
of these two enzymes.
Recently, there was a study of mutagenesis of a
conserved residue Ala290 of a maltogenic amylase,
ThMA, from Thermus sp. IM6501.22) Replacement of
this amino acid residue with one with a bulky side
chain resulted in a change in substrate specificity of
the enzyme. The mutant, ThMA-A290I, produced
more maltose than glucose, while the wild-type enzyme
produced both sugars in comparable amounts. It was
found that the mutation at this position increased the Km
value for maltose, resulting in an accumulation of this
sugar.22) Concomitantly, the presence of Trp residue at
the corresponding position (W251) of Env Npu193A is
likely to be the reason maltose was the main product
obtained from starch and cyclodextrin hydrolysis by the
enzyme. The bulky side chain of Trp residue might affect
the substrate binding affinity in such a way that maltose
was not further hydrolyzed to glucose. Env Cda13A, in
contrast, hydrolyzed various substrates, yielding glucose
as the main product.10) Since Env Cda13A harbored Phe
at the corresponding position, it is possible that the less
bulky side chain of Phe residue allows higher affinity for
maltose binding, leading to more glucose as the product
as compared with Env Npu193A.
66 kDa
66
45
35
25
Fig. 2. Expression of Env Npu193A in P. pastoris.
Culture was induced for 2 d with methanol, as described in
‘‘Materials and Methods,’’ and the supernatant from day 2 was
collected and subjected to SDS–PAGE.
recombinant enzyme showed optimal temperature and
pH of 75 ꢀC and 7 (Fig. 3A and B). The Km and Vmax
values for pullulan were 1:22 ꢃ 0:3% and 23:24 ꢃ
1:7 U/mg respectively. The high enzymatic activity
toward pullulan of Env Npu193A was different from
that of the previous isolate, Env Cda13A, from the
same environment, which preferred cyclodextrins as
substrates (23.24 U/mg for Env Npu193A as compared
with 0.3 U/mg for Env Cda13A when pullulan was used
as a substrate). Env Npu193A was stable across broad
pH range, from 3 to 9, with at least 50% remaining
activity (Fig. 4A). In addition to its ability to function
at high temperatures, Env Npu193A exhibited high
thermostability as compared with Env Cda13A. After
incubation at 60 ꢀC for 60 min, it retained more
than 80% of maximal activity (Fig. 4B) whereas Env
Cda13A activity was completely abolished under these
conditions (data not shown).
Hydrolysis products of Env Npu193A
Products obtained from Env Npu193A-catalysed
hydrolysis were analyzed by TLC (Fig. 5). The results
showed that this novel enzyme hydrolyzed large
molecules of pullulan, starch, and ꢂ-cyclodextrin, but
not ꢀ or ꢁ-cyclodextrins (Fig. 5A). The enzyme also
hydrolyzed linear maltooligosaccharides (G4-G6)
(Fig. 5B). The final products obtained from complete
Env Npu193A hydrolysis of starch, ꢂ-cyclodextrin,
and malto-oligosaccharides were maltose, but not
glucose. Pullulan degradation by Env Npu193A yielded
only maltotriose, not panose. This suggests that this
enzyme exhibited activity only on ꢀ-1,6-glucosidic
linkages of pullulan, not on ꢀ-1,4-glucosidic bonds.
This supports the assumption that Env Npu193A is
not a true member of the neopullualnase subfamily,
which, by definition, hydrolyses ꢀ-1,4-glucosidic link-
ages in pullulan yielding panose. Some are also able
to hydrolyze ꢀ-1,4-glucosidic and ꢀ-1,6-glucosidic
linkages in pullulan.18)
In this study, the preferred substrate for Env Npu193A

Novel Neopullulanase-Like Enzyme Isolated from Environmental DNA
1453
A
% Relative activity
100
80
60
40
20
0
0
2
4
6
8
10
12 pH
B
% Relative activity
100
80
60
40
20
0
30
40
50
60
70
80
90
Temp (°C)
Fig. 3. Activity Profiles of the Purified Env Npu193A.
The enzyme activities were measured at various pH (A) and various temperatures (B). Optimal pH was determined by measuring the enzyme
activity in buffer ranging from 4–11 (100 mM): sodium citrate (pH 4), acetic acid (pH 5–6), MOPS (pH 6.5–7), Tris–HCl (pH 7–9), and Glycine
(pH 9–11). The optimal temperature was determined at selected temperatures from 30 to 90 ꢀC.
was pullulan, which was hydrolyzed only to maltotriose.
The involvement of conserved region II for substrate
specificity among starch-hydrolyzing enzymes has been
demonstrated with TVAI ꢀ-amylase from T. vulgaris.23)
Substitution of amino acid sequences, AAQY, at the
C-terminal of conserved region II in this enzyme into
VANE resulted in the alteration of substrate specificity.
The mutant TVAI enzyme hydrolyzed mainly the ꢀ-1,6-
glucosidic bond of pullulan, yielding maltotriose, where-
as the action patterns for the other substrates remained
the same as the wild type.23) Mutation in conserved
region II caused the alteration of the kcat=Km value of
the enzyme on ꢀ-1,4-glucosidic linkages.23) This might
explain the preference for pullulan of Env Npu193A,
which contains the sequence VPNE in conserved
region II (Fig. 1). On the other hand, Env Cda13A with
VADM sequences and the cyclodextrinase of T. mar-
itima harboring four completely different amino acid
residues (CGHD) in this conserved region degraded
pullulan poorly (Fig. 1).15) A pullulan hydrolase from
Thermococcus aggregans that attacks both the ꢀ-1,4-
glucosidic and the ꢀ-1,6-glucosidic bonds in pullulan
has a hybrid sequence in this region (APQE).24) This
enzyme has been proposed to be a pullulan hydrolase
type III.18) This indicates the important role of amino
acid sequences in conserved region II for interaction and

1454
K. TANG et al.
A
% Residual activity
120
100
80
60
40
20
0
3
5
7
9
pH
t0
t4
B
% Residual activity
100
80
60
40
20
0
0
min
30
50°C
60
90
120
70°C
45°C
60°C
65°C
75°C
Fig. 4. Effect of pH (A) and Temperature (B) on the Stability of Env Npu193A.
To assess the pH stability of Env Npu193A, the enzyme was pre-incubated at 25 ꢀC for 4 h in 50 mM sodium citrate (pH 3), 50 mM sodium
acetate (pH 5), 50 mM Tris–HCl (pH 7), and 50 mM glycine (pH 9) before it was mixed with the reaction mixture, as described in ‘‘Materials and
Methods.’’ , Enzyme activity from Env Npu193A that was not pre-incubated; , Enzyme activity from Env Npu193A after 4 h of pre-
incubation. The thermostability of the enzyme was determined by pre-incubating Env Npu193A for 30, 60, and 120 min at various temperatures
before it was mixed with the reaction mixture. Residual pullulanase activity was measured by the DNS method. Values are shown as percentages
of the original activity, which was taken to be 100%. Each data plot is the average of two.
reaction specificity on pullulan. In conserved region III
(EIWD), the Ile residue has been found to play an
important role in the substrate specificity of neopullu-
lanase.25) Replacement of this amino acid residue with
Trp (I358W) caused the neopullulanase of B. stearo-
thermophilus to hydrolyze starch rather than pullulan.
On the other hand, the I358V mutation increased
the specificity of neopullulanase for ꢀ-(1,6)-branched
oligosaccharides. Therefore, it is proposed that this Ile
residue in conserved region III affected the substrate
preference of the enzyme.
In summary, the neopullulanase-like enzyme Env
Npu193A is capable of functioning at high temperatures
and of exhibiting high thermostability. It displays
different biochemical characteristics to Env Cda13A,
previously isolated from the same source, and many
related enzyme previously reported. The unique proper-
ties of Env Npu193A indicate the potential of culture-
independent methods to retrieve such novel enzymes
from natural bioresources. The sequence-based screen-
ing approach can be used to discover new genes that
might go undetected using expression-based screening.

Novel Neopullulanase-Like Enzyme Isolated from Environmental DNA
1455
National Science, and Technology Development Agen-
cy of Thailand, is greatly appreciated.
A
G1
G2
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Acknowledgments
We are grateful to Dr. Phil Shaw and Dr. Piyanun
Harnpicharnchai for critical editing of the manuscript.
We thank Dr. Sittiruk Roytrakul for useful discussion.
Financial support (B21 FJ0010) from the National
Center for Genetic Engineering and Biotechnology,

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