Journal of Life Sciences Volume 5 Number (4)
J LS
Journal of Life Sciences
Volume 5, Number 7, July 2011 (Serial Number 39)
Contents
Research Papers
483 Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal, Abnormal Karyotype hES Cells during Culture in vitro
Xueqin Zheng, Zhen Xiang, Xianlei Li, Wenling Lu, Li Tan, Qingguo Luo, Changqing He, Ye Yu, Yi Yao, Ying Li, Huaijiang Li and Yang Xiang
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA, an Endoglucanase with Cellulase and Mannanase Activity
Phillip J. Brumm, Spencer Hermanson, Joshua Luedtke and David A. Mead 497
Evaluation of Barley Genotypes for Resistance to Pyrenophora Teres Using Molecular Markers
Leona Leisova-Svobodova, Lenka Stemberková, Martina Hanusová and Ladislav Ku čera 503
Dynamics and Control of Infectious Diseases in Stochastic Metapopulation Models
Ariel Félix Gualtieri and Juan Pedro Hecht 509
The Research on Phosphate Mobilizing Bacteria from Soils of Southern Ukraine
Ludmila Chaikovska
513 Influence of NaCl and Na 2 SO 4 Treatments on Growth Development of Broad Bean (Vicia Faba, L.) Plant
Hameda El Sayed Ahmed El Sayed 524
Obsolete Pesticides and Phytoremediation of Polluted Soil in Kazakhstan
Asil Nurzhanova, Kabyl Zhambakin, Issbacar Rakhimbayev, Anatoly Sedlovskiy and Sergey Kalugin
536 Effect of Inorganic and Organic Based Fertilizers on Growth Performance of Tea and Cost
Implications in Kusuku, Nigeria
Rotimi Rufus Ipinmoroti, Gerald Oaikhena Iremiren, Olayiwola Olubamiwa, Akanbi Olutayo Fademi and Emmanuel Ogieriakhi Aigbekaen
The Effectiveness of Watermelon Endocarp Extract in Inhibiting Lipase Activity Relative to the Hypolipidemic Drugs
Subandi Hambali and Indah Langitasari
546
A New Record of Doria’s Comb Fingered Gecko, Stenodactylus Doriae, (Blanford, 1874), (Reptilia: Gekkonidae) from Southeastern of Iran, Sistan & Baluchistan Province
Nastaran Heidari, Nasrullah Rastegar-Pouyani and Hiva Faizi
549 Does Procambarus Clarkii (Girard, 1852) Represent a Threat for Estuarine Brackish Ecosystems of Northeastern Adriatic Coast (Italy)?
Sandra Casellato and Luciano Masiero 555
Metabolizable Energy and Amino Acid Bioavailability of Field Pea Seeds in Broilers Diets
Vassilios Dotas, Asterios Hatzipanagiotou and Konstantinos Papanikolaou 562
Chemical Composition of Meat in Castrated Male Brahman Cattle in Venezuela
José A. Miguel, Jesús Ciria, Begoña Asenjo, Hector Pargas and David Colmenarez
569 Alternative Distributions to Estimate Usual Intake of Nutrients for Groups
José Eduardo Corrente, Juliana M. Morimoto, Dirce Maria Lobo Marchioni and Regina Mara Fisberg
575
Computer Supported Sensory Profiling Analysis of Three Agaricus Cultivars
András Geösel, László Sipos, Brian McGuinness, Júlia Gy őrfi and Zoltán Kókai
Journal of Life Sciences 5 (2011) 483-487
Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal, Abnormal Karyotype hES Cells during Culture in vitro
Xueqin Zheng, Zhen Xiang, Xianlei Li, Wenling Lu, Li Tan, Qingguo Luo, Changqing He, Ye Yu, Yi Yao, Ying Li, Huaijiang Li and Yang Xiang Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414000, China
Received: March 07, 2011 / Accepted: March 24, 2011 / Published: July 30, 2011.
Abstract: Human embryonic fibroblasts (hEFs) can well maintain the pluripotency in human embryonic stem cells (hESs). However, recent research and reports indicated that a few of hES cell lines acquired genomic alteration during long-term culture of hES cells in vitro. This will directly restrict the therapy use of hES cells. Wnts are secreted lipid-modified signaling proteins that influence multiple processes ranging from cell proliferation to stem cell loss. Activation of Wnt signaling in many tissues has also been associated with cancer. Unchecked Wnt signaling and loss of cadherin expression can promote tumorigenesis. In this study, we found the caryotype of one hES cell line chHES-3 changed with duplication of 1p32-1p36 area after 34 passages. The results of RT-PCR indicated Wnt7a was expressed in hEFs after culture normal karyotype hES cells, but not expressed in control and abnormal karyotype hES cells. Wnt3 was expressed in hEFs after culture abnormal karyotype hES cells, not expressed in control and normal karyotype hES cells. Wnt3, Wnt9a and Wnt10b were detected weakly expression in normal hES cells, but higher in abnormal hES cells. At the same time, Wnt3a, Wnt4, Wnt5b, Wnt7a, Wnt8b and Wnt11 were expressed and E-cadherin was not tested in abnormal hES cells compared with normal hES cells. All that indicated Wnt7a was need for culture normal karyotype hES cells and Wnt3 was need for culture abnormal karyotype hES cells on hEFs. Wnt3, Wnt9a and Wnt10b high expression in hES cells and absence of E-cadherin may cause hES cells karyotype change.
Key words: hEFs, hES cell karyotype, differential expression, Wnts, β-catenin, E-cadherin.
1. Introduction culture of any indefinite cell lines will lead to gene mutation and chromosome aberration which may
Human embryonic stem cells are derived from inner confer the cells a malignant phenotype [5]. More hES cell mass of human blastocyst and will be the unlimited cell lines could well maintain a normal karyotype cell source for future cell therapy, contributing to their during prolonged culture, but，a few of them did characteristics of pluripotency, capability of acquire chromosomal changes [6]. In our laboratory, differentiation to almost any type of cells [1]. At we found the caryotype of one hES cell line chHES-3 present a lot of inactive mouse embryonic fibroblasts was changed with duplication of 1p32-1p36 area after (mEFs) and human embryonic fibroblasts are required
34 passages.
to maintain and support the hES cell growth in an Wnts are secreted lipid-modified signaling proteins [7]. undifferentiated state [2-4]. hES cells have held great Wnts are powerful regulators of cell proliferation and promise for clinical use as a major resource in future differentiation, and their signaling pathway involves cell and tissue replacement therapy, but concerns are proteins that directly participate in both gene always kept on the safety of hES cells as long term transcription and cell adhesion [8]. The central player
Corresponding author: Yang Xiang, Prof., research field: is β-catenin, which is a transcription cofactor with T
molecular and cell biology. E-mail: xiangyangbio@126.com.
Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal,
Abnormal Karyotype hES Cells during Culture in vitro
cell factor/lymphoid enhancer factor TCF/LEF in the of the People’s Republic of China. The fibroblasts were Wnt pathway [9] and a structural adaptor protein
routinely expanded in DMEM (Gibco Invitrogen, USA) linking cadherins to the actin cytoskeleton in cell-cell
supplemented with 10% Fetal calf serum (Haita). adhesion [10]. Wnts influence multiple processes in
Seventy to eighty percent of confluent cells were animal development [8]. Nineteen Wnt genes exist in
rendered mitotically inactive by being resuspended in mammalian genomes, and the diversity of their
medium and treated them with 33G of gamma functions is exemplified by mutations that lead to
irradiation. Inactive cells were related with a dilution of developmental abnormalities ranging from stem cell
8×10 5 /mL into pre-gelled plates. The batch of hEFs loss to kidney and reproductive tract defects [9]. In
which could support the growth of hES cells were many tissues, activation of Wnt signaling has also been
subjected to proceed to next steps. hEFs in the control associated with cancer [11]. Recent studies have shown
group were handled in parallel not culture hES cells. that both Wnt signaling and cadherin-mediated
2.2 Culturing of Human Embryonic Stem Cell Line cell-cell adhesion are important in the organization and
chHES-3
maintenance of stem cells [12]. Alterations in cell fate, adhesion, and migration are characteristics of cancer in
chHES-3 was one of the several hES cell lines which cells ignore normal regulatory cues from their
established in our laboratory [1]. The hES cells were environment. Unchecked Wnt signaling [13] and/or the
grown on hEFs in a culture medium consisting of 85% loss of cell-cell adhesion [14-15] are involved in cancer Knock-out Dulbecco’s modified Eagle’s medium (KO-
induction and progression. Loss of cadherin expression DMEM), 15% Knock-out serum replacement, 1 mmol/L can also promote tumorigenesis [14-15].
L-glutamine, 0.1 mmol/L β-mercaptoethanol, 1% In our study, we used RT-PCR to analyze the
non-essential amino acids, and 4 ng/mL human basic differential expression of Wnts, β-catenin and fibroblast growth factor (hbFGF) (all Gibco Invitrogen
E-cadherin in hEFs and normal and abnormal products, USA). Cultures were passaged once a week caryotype hES cells during culture in vitro, so as to
by incubation in 200 U/mL collagenase IV (Gibco filter the candidate genes that may maintain hES cells
Invitrogen, USA) for 5-10 min at 37 ℃ and seeded on normal karyotype or urge hES cells karyotypic change.
freshly feeder layer. Re-feed was every other day. Our study suggested that Wnt7a was needed for culture
normal karyotype hES cells and Wnt3 was needed for
2.3 RT-PCR
culture abnormal karyotype hES cells on hEFs. High Total RNA was isolated from control hEFs (not expression of Wnt3, Wnt9a and Wnt10b in hES cells
culture hES cells), hEFs (culture hES cells), normal and absence expression of E-cadherin may cause hES
and abnormal hES cells, according to the cells karyotype change.
manufacturer’s recommended protocol. And then,
2. Materials and Methods
synthesis of first strand cDNA was performed as following: reaction mixture containing 10 µL
2.1 hEFs Isolation and Culture ribonuclease-free water, 1 µL total RNA (0.23-0.26
hEFs as feeder cells derived from two month µg/µL) and 1 µL oligo (dT) primer (0.5 µg/µL) was abortion fetus musculature were harvested hES cells in
incubated at 70 ℃ for 5min and chilled on ice, 4 µL our laboratory according to the method described in
5×reaction buffer, 1µL ribonuclease inhibitor (20 U/µL) Ref. [16]. The experiment was conducted according to
and 2 µL 10 mM dNTP were added. The mixture was the Guide for the Care and Use of Laboratory Animals,
incubated at 37 ℃ for 5 min, and then incubated at enunciated by the Ministry of Science and Technology
42 ℃ for 60 min after adding 1 µL RevertAid TM
Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal,
Abnormal Karyotype hES Cells during Culture in vitro
M-MuLV revers trancriptase (200 U/µL). The reaction Wnt16-R:5 ′-tagcagcaccagatgaacttaca-3′; was terminated by heating at 70 ℃ for 10 min and then
Beta-catenin-F:5 ′-atgctgaaacatgcagttgtaa-3′, kept at 4 ℃. The PCR primer pairs of 19 Wnts,
Beta-catenin-R:5 ′-ttgcattccaccagcttctaca-3′; β-catenin and E-cadherin were synthesized as following:
E-cadherin-F:5 ′-gtgaacacctacaatgccgcca-3′, Wnt1-F: 5 ′-tactacgttgctactggcactga-3′,
E-cadherin-R: 5 ′-ccaaatccgatatgttattttc-3′; Wnt1-R: 5 ′-cctctgttgccgtaaaggac-3′;
β-actin-F: 5′-ctcctgaagaaggggcgtctaaa-3′, Wnt2-F: 5 ′-ggctaacgagaggtttaagaagc-3′,
β-actin-R:5′-ggaagagcttcagggtagggaca-3′. Wnt2-R: 5 ′-ttgagaaagctcctttgagacac-3′;
At last PCR reaction condition is: the RNA isolated Wnt2b-F: 5 ′-tgtatatgccatctcatcagcag-3′,
from different hEFs and hESCs were reversely Wnt2b-R: 5 ′-tccacagtatttctgcattcctt-3′;
transcribed into cDNA. Then the amplification reaction Wnt3-F: 5 ′-ccaatctcaagtggactttgttc-3′,
was performed in a 20 µL reaction mixture containing Wnt3-R: 5 ′-gtgcatgtggtccaggatagt-3′;
13.55 µL deionized water, 2 µL 10 × PCR reaction Wnt3a-F: 5 ′-cccactcggatacttcttactcc-3′,
buffer, 2 µL 2.5 mM dNTP, 0.4 µL Taq DNA Wnt3a-R: 5 ′-tcgtacttgtccttgaggaagtc-3′;
polymerase, 0.45 µL primer pair mixture (0.5 µg/µL) Wnt4-F: 5 ′-cactgaaggagaagtttgatggt-3′,
and 1.6 µL cDNA, in a programmable thermal cycler, Wnt4-R: 5 ′-ttgttactccaccttaggtctgc-3′;
PE 9700: initial denaturation at 95 ℃ for 90 s, and then Wnt5a-F: 5 ′-agtgcaatgtcttccaagttct-3′,
35 cycles of 94 ℃ for 40 s, 60 ℃ for 40 s and 72 ℃ for Wnt5a-R: 5 ′-cagcatgtcttcaggctacat-3′;
58 s. The extension step in the last cycle was 72 ℃ for Wnt5b-F: 5 ′-cactctgcctcacaaaggtctat-3′,
5 min, and the mixture was finally kept at 4 ℃. The Wnt5b-R: 5 ′-attcagtttagggctttcctgac-3′;
PCR products were then separated on a 2.0% agarose Wnt6-F: 5 ′-agaagctgcctccatttcg-3′,
gel and analyzed using β-actin as contrast. The Wnt6-R: 5 ′-gtatccagaggcctttagactgg-3′;
products of PCR were sequenced. Wnt7a-F: 5 ′-actctcatgaacttgcacaacaa-3′,
3. Results
Wnt7a-R: 5 ′-agttgagggctctgagagatttt-3′; Wnt7b-F: 5
′-agagcaaagtgatgaggagactg-3′, 3.1 Analysis of Normal and Abnormal Karyotype of
Wnt7b-R: 5 ′-acgcccagctaatttttgtattt-3′; Wnt8a-F 5 ′-agaactgtagcatgggtgactt-3′,
hES Cells
The analysis of the caryotype of chHES-3 hES cell Wnt8a-R 5 ′-gcagtaatacttgctcaccaca-3′;
line showed that the caryotype of chHES-3 hES cells Wnt8b-F 5 ′-caattcctctgtgctctcctaga-3′,
was normal after 27 passages, but the caryotype of Wnt8b-R: 5 ′-agaggaacaaagatcctttggag-3′;
chHES-3 hES cells was abnormal after 34 passages Wnt9a-F: 5 ′-tcaaggagactgccttcctctat-3′,
with duplication of 1p32-1p36 area. Wnt9a-R: 5 ′-actccacatagcagcaccaac-3′;
3.2 Differential Expression of Wnts, β-catenin and Wnt9b-F: 5 ′-cctcaagtacagcaccaagtttc-3′, E-cadherin in hEFs after Culture Normal and Wnt9b-R: 5 ′-gcatgcatgtgatgacagtct-3′;
Wnt10a-F: 5 ′-cgagtcggagctgtgtgtc-3′, Wnt10a-R: 5 ′-gtaagcggtgcagcttccta-3′;
Abnormal Karyotype hES Cells
Wnts, β-catenin and E-cadherin gene expressions Wnt10b-F: 5 ′-caagagtttcccccactctct-3′,
were assessed in control hEFs after culture six days Wnt10b-R: 5 ′-cttacacacattcacccactctg-3′;
using CM (condition medium) (Fig. 1A), hEFs after Wnt11-F: 5 ′-ctatttgcttgacctggagagag-3′,
culture normal l hES cells six days using CM (Fig. 1B) Wnt11-R: 5 ′-cggtctgtgtaggggttgtag-3′;
and hEFs after culture abnormal hES cells 6 days using Wnt16-F: 5 ′-gcaccaaagagacagcatttatt-3′,
CM (Fig. 1C). Results showed that Wnt3 was detected
Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal,
Abnormal Karyotype hES Cells during Culture in vitro
Fig. 1 Wnts, β-catenin and E-cadherin expression in hEFs after culture normal and abnormal karyotype hES cells. A: control hEFs after culture hEFs six days using CM (condition medium); B: hEFs after culture normal hES cells six days using CM; C: hEFs after culture abnormal hES cells six days using CM.
Fig. 2 Wnts, β-catenin and E-cadherin expression in normal and abnormal hES cells.
only in hEFs after culture abnormal karyotype hES
4. Discussion
cells six days (Fig. 1C). Wnt7a was detected only in hEFs after culture normal karyotype hES cells six days
So as to filter the canndidate Wnts, β-catenin and (Fig. 1B). The expression level of Wnt 9a was detected
E-cadherin genes that may maintain hES cells normal very high in control hEFs (Fig. 1A), but very low in
karyotype or urge hES cells karyotypic change. In this hEFs after culture normal and abnormal hES cells (Fig. 1B
study, RT-PCR results showed Oct 4 and Nanog were and Fig. 1C).
expressed in normal hES cells (data not shown), that indicated that normal and abnormal hES cells have hES
3.3 Differential Expression of Wnts, β-catenin and cells characterization. The analysis of the caryotype of E-cadherin in Normal and Abnormal hES Cells chHES-3 hES cell line was showed that the caryotype
Wnts, β-catenin and E-cadherin gene expressions of chHES-3 hES cells was normal after 27 passages, were assessed in normal hES cells (Fig. 2) and abnormal
but the caryotype of chHES-3 hES cells was abnormal hES cells (Fig. 2). Results showed that expression of Wnt3,
after 34 passages with duplication of 1p32-1p36 area. Wnt9a and Wnt10b were detected weakly in normal
The results of analysis of Wnts, β-catenin and hES cells, but higher in abnormal hES cells. At the
E-cadherin differential expression in hEFs after culture same, Wnt3a, Wnt4, Wnt5b, Wnt7a, Wnt8a and Wnt11
normal and abnormal karyotype hES cells showed were only expressed in abnormal hES cells. At the
Wnt3 was detected only in hEFs after culture abnormal same time, E-cadherin was expressed in normal hES
karyotype hES cells six days (Fig. 1C), which suggests cells and disappeared in abnormal karyotype hES cells.
Wnt3 was needed for culture abnormal karyotype hES
Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal,
Abnormal Karyotype hES Cells during Culture in vitro
cells and Wnt7a was detected only in hEFs after culture
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Journal of Life Sciences 5 (2011) 488-496
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA, an Endoglucanase with Cellulase and Mannanase Activity
Phillip J. Brumm, Spencer Hermanson, Joshua Luedtke and David A. Mead C5-6 Technologies, 2120 W. Greenview Drive, Middleton, WI 53562, USA
Received: September 28, 2010 / Accepted: March 01, 2011 / Published: July 30, 2011.
Abstract: The discovery of new, highly active, biomass-degrading enzymes is important to the development of a sustainable biofuels industry. Dictyoglomus turgidum, a thermophilic, anaerobic eubacterium that ferments cellulose and produces ethanol and hydrogen, was chosen as a candidate to screen for novel enzymes. A novel thermostable endoglucanase, CelA, was identified and purified during screening of a shotgun library of Dictyoglomus turgidum and subsequently subcloned and expressed in E. coli. The celA gene coding for a 312 amino acid protein showed low homology to proteins outside the genus Dictoglomi and lacked an apparent signal peptide. CelA had a broad substrate range, possessing both endo and exo activity on soluble and insoluble β-(1,4)-linked glucose-containing substrates as well as endo activity on soluble and insoluble β-(1,4)-linked mannose containing substrates. The specific activity of CelA was 226 U/mg using β-glucan, 66 U/mg using glucomannan, and 63 U/mg using CMC as substrates. The high temperature optimum of
70 ℃ to 80 ℃ and wide substrate range of the enzyme might make it an excellent tool for biomass degradation at high temperature.
Key words: Cellulase, mannanase, thermophilic, biomass degradation, Dictyoglomus turgidum.
1. Introduction contamination problems, and improved flow and loading of solid materials, all leading to improvement
Cellulose-containing plant cell walls provide an of the overall economy of the process. However, abundant and renewable source of glucose, pentose, growth on cellulosic substrates and production of and other small carbon compounds, many of which thermostable cellulases is rare among thermophilic have significant commercial value. Accordingly, there microorganisms. A review of the literature has been substantial interest in developing improved (summarized in Ref. [1]) revealed only a few truly techniques for enzymatic processing of cellulosic thermophilic organisms reported to produce cellulases. materials. Thermophilic cellulases are of considerable Dictyoglomus species represent a novel group of interest because of the potential benefits provided by thermophilic, anaerobic organisms with considerable the use of high temperature enzymes and organisms. biotechnological promise; these organisms are so Benefits of high temperature enzymes include higher unique that they have been given their own genus, specific activity that decreases the amount of enzyme Dictoglomi. Dictyoglomus turgidum (originally named needed, enhanced enzyme stability allows improved Dictyoglomus turgidus [2]) is one of only two accepted hydrolysis performance over time, high temperature members of the genus Dictoglomi; another being operation reduces or eliminates microbial Dictyoglomus thermophilum [3]. Dictyoglomus
turgidum is reported to be able to grow on cellulose, Corresponding author: Phillip J. Brumm, Ph.D., Chief
while Dictyoglomus thermophilum cannot [4]. The Scientific Officer, research fields: carbohydrate chemistry and
enzymology. E-mail: pbrumm@c56technologies.com. ability to ferment a variety of carbohydrates at high
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
temperatures makes the identification and characterization
2.2 Growth of Organisms
of the cellulytic enzymes from Dictyoglomus turgidum YT plate media was used in all molecular biology worthwhile. This could result in a better understanding screening experiments and Terrific Broth was used for of microbial strategies for decomposing biomass and
liquid cultures.
improved catalysts for conversion of carbohydrates to C5 and C6 sugars. In this report we describe unique
2.3 Enzyme Assays
properties of an unusual carbohydrase CelA. The endo-glucanase specificity of CelA was
2. Materials and Methods
determined in 0.50 mL of 50 mM acetate buffer, pH5.8, containing 0.2% azurine cross-linked-labeled (AZCL)
2.1 Materials insoluble substrate and 1.0 µg of enzyme protein.
Assays were performed at 70 ℃, with shaking at 1000 concentrate was a kind gift of Dr. Frank T. Robb,
Dictyoglomus turgidum
strain 6724 T bacterial cell
rpm, for 20 minutes in a Thermomixer R (Eppendorf, Center of Marine Biotechnology, University of Hamburg, Germany). Tubes were clarified by Maryland Biotechnology Institute. Dictyoglomus
centrifugation and absorbance values determined using turgidum strain 6724 T is on deposit at Deutsche
a Bio-Tek EL x 800 plate reader. The exo-glucanase Sammlung von Mikroorganismen und Zellkulturen
specificity of CelA was determined by spotting 1.0 µg GmbH. 10G electro-competent E. coli cells, BL21
of enzyme directly on agar plates containing 10 mM (DE3) chemically competent E. coli cells, pEZSeq (a
4-methyl umbelliferyl substrate. Plates were incubated lac promoter vector), Clostridium thermocellum CelA
in a 70 ℃ incubator for 60 minutes; after incubation, (CthCelA), CelG (CthCelG), CelI (CthCelI), CelO
the plates were examined using a hand-held UV lamp (CthCelO), and CAZyme β-Glucosidase 1 were
and compared to negative and positive controls. obtained from Lucigen, Middleton, WI. pET28a vector
Enzyme specific activity was measured using a was obtained from Merck Chemicals, San Diego, CA.
micro version of the Modified Somogyi Method for Pure polysaccharides, azurine cross-linked-labeled,
reducing sugars as described in Ref. [6]. The reaction and D-Glucose (GOPOD Format) Assay Kit were
mixtures containing 200 µL of substrate (1% β-glucan obtained from Megazyme International (Wicklow,
or other carbohydrate in 50 mM acetate buffer, pH 5.8) Ireland). 4-methylumbelliferyl- β-D-cellobioside (MUC),
and 5 µL enzyme sample were incubated at 70 ℃ for 4-methylumbelliferyl- β-D-xylopyranoside (MUX), and
10 minutes. Micromoles of sugars formed were 4-methylumbelliferyl- β-D-glucoyranoside (MUG) were
determined using a glucose standard curve, and unit obtained from Research Products International Corp.
activity calculated as micromoles of reducing sugar per (Mt. Prospect, IL). Octyl Sepharose Fast Flow, Q
minute per milligram of protein.
Sepharose Fast Flow, and Sephacryl S-100 High Enzyme specific activity on cellulose (Avicel) and Resolution column media were purchased from GE
acid swollen cellulose (ASC) were determined using a Healthcare Life Sciences (Piscataway, NJ). CelLytic
coupled reaction. The reaction mixtures containing IIB reagent, pNP- β-glucoside, pNP-β-cellobioside,
1.0 mL of substrate (1% Avicel or 1% ASC plus 40 µL 4-methylumbelliferyl- β-D-lactopyranoside (MUL), of CAZyme β-Glucosidase 1 in 50 mM acetate buffer, carboxymethyl cellulose (CMC) and Avicel PH-101
pH 5.8) and 10 µL enzyme sample were incubated at cellulose powder were purchased from Sigma-Aldrich
70 ℃ for 30 minutes with shaking at 1000 rpm. The (St. Louis, MO). Acid swollen cellulose was reaction was terminated by centrifugation to remove prepared as described in Ref. [5]. All other chemicals
the insoluble substrate and glucose formation were of analytical grade.
determined using the Megazyme D-Glucose (GOPOD
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
Format) Assay Kit. The temperature optimum of CelA
2.5 Enzyme Purification
was determined using the reducing sugar assay with
E. coli cells containing the Dtur celA gene were 2% β-glucan as substrate at pH 5.8. The pH optimum of grown overnight at 37 ℃ in 2 L of TB containing 30 CelA was determined using the reducing sugar assay μg/mL kanomycin. Cells, 20.3 g, were resuspended in with 1% β-glucan as substrate at 70 ℃. 100 mL of 50 mM Tris-HCl, pH8.0, and lysed by HPLC analysis of hydrolysis products was carried sonication. The lysate was clarified by centrifugation out on a Shimadzu HPLC equipped with a Rezex ROA and E. coli proteins were precipitated by heat treatment organic acid column operating at 60 ℃. Elution was at 80 ℃ for 15 minutes. The heat-treated lysate was
achieved using a mobile phase of 0.005 N H 2 SO 4 at a
clarified by centrifugation. The clarified material (90 flow rate of 0.6 mL/min. Products were detected using mL) was diluted with an equal volume of 4 M
a refractive index detector and identified using a series (NH 4 ) 2 SO 4 and was then applied to a 30 mL Octyl of known cellodextrin standards. Sepharose Fast Flow column equilibrated with 2 M
2.4 Library Construction and Screening (NH 4 ) 2 SO 4 . The column was washed with 120 mL of
2 M (NH 4 ) 2 SO 4 in 100 mM Tris-HCl, pH7.5, and eluted
A cell concentrate of Dictyoglomus turgidum was sequentially with 200 mL total gradient of a) 2 M
lysed using a combination of SDS and proteinase K,
2 4 to 0 M (NH 4 ) 2 SO 4 in 100 mM Tris-HCl, pH extraction [7]. The genomic DNA was precipitated,
(NH 4 ) SO
and genomic DNA was purified using phenol/chloroform
7.5 and b) 0% to 60% propylene glycol in 100 mM Tris-HCl, pH7.5. Active fractions were identified by
treated with RNase to remove residual contaminating RNA, and fragmented by hydrodynamic shearing
hydrolysis of AZCL-HE-Cellulose, pooled, diluted 1:1 with deionized water, and applied to a 15 mL Q
(HydroShear apparatus, GeneMachines, San Carlos, CA) to generate fragments of 2-4 kb. The fragments
Sepharose Fast Flow column equilibrated with 50 mM were purified on an agarose gel, end-repaired, and
Tris-HCl, pH 8.0. The column was washed with 20 mL ligated into pEZSeq, a lac promoter vector. To identify
of 50 mM Tris-HCl, pH8.0, and the enzyme was eluted cellulases, the D. turgidum library was transformed into
with a 100 mL gradient of 0 to 1,000 mM NaCl in 50 10G electro-competent E. coli cells and screened on
mM Tris-HCl, pH8.0. Active fractions were pooled and YT plates containing 30 μg/mL kanomycin and 100
concentrated to 2.0 mL. The concentrate (0.8 mL) was μg/mL MUC. Positive (blue-fluorescing) cells were diluted with 0.2 mL of 80% glycerol and applied to a 150 mL Sephacryl S-100 High Resolution column
picked, re-streaked, and grown overnight at 37 ℃ in equilibrated with 50 mM Tris-HCl, pH 8.0. Active
2.0 mL of TB supplemented with 30 μg/mL kanomycin. fractions were pooled and concentrated to 1.0 mL for
The cultures were collected by centrifugation and the
characterization studies.
pellets were lysed by incubation in 200 μL of CelLytic SDS denaturing gel electrophoresis was used to
IIB reagent for 30 minutes at 37 ℃. The lysates were verify the purity and size of the cellulase. clarified by centrifugation and 50 μL aliquots were
Electrophoresis was performed on a 4-20% gradient assayed at 70 ℃ in 0.5 mL of 50 mM acetate buffer,
acrylamide gel. The results (Fig. 1) show an pH 5.8 containing either 0.2% AZCL-HE-Cellulose for
approximately 37 kDa band in all fractions. cellulase activity or 0.2% AZCL-Arabinoxylan for
2.6 Subcloning and Expression
hemicellulase activity. The lysate of one MUC-positive culture, designated CelA, also released soluble dye
The DNA fragment containing the Dtur celA gene from AZCL-HE-Cellulose, indicating that CelA was sequenced by primer walking. Electronic translation
expressed an endo-cellulase. of the DNA sequence did not yield an apparent open
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
2.7 Bioinformatics InterProScan Family analysis (http://www.ebi.ac.uk/
Tools/InterProScan/), and BLASTP (Basic Local Alignment Search Tool [8]) (http://blast.ncbi.nlm. nih.gov/Blast.cgi) analysis tools were used to compare CelA with other proteins in the database. Phylogeny analysis was performed by using software at http://www.phylogeny.fr/version2_cgi/index.cgi. Multiple alignments were run using ClustalW [9] alignment with
A BB CC DD E E E
curation to remove positions with gaps [10]. Construction
of the phylogenetic tree was obtained using PhyML [11], Legend (from left to right): A: Molecular weight markers; B: E.
Fig. 1 SDS PAGE of Dtur CelA purification samples.
coli lysate, 1× and 3×; C: Heat-treated lysate, 1× and 3×; D: and graphically displayed using TreeDyn (http://www.
Concentrated Q sepharose active fractions, 1× and 3×; E: treedyn.org/). Operon predictions [12] were run using Concentrated S-100 active fractions, 1×, 3× and 5×.
http://www.microbesonline.org/operons/OperonList.html. Glycosyl hydrolase predictions were obtained from
reading frame of the correct size beginning with a http://www.cazy.org/geno/acc_geno.html. Signal
methionine codon. To determine the open reading sequence predictions were determined using
frame, the N-terminal sequence of the purified protein (http://www.cbs.dtu.dk/services/SignalP/) [13]. was determined by Edman degradation. The sequence The GenBank accession number of the sequence obtained was: MNNLPIKRGINFGDALEAPY.
reported in this paper is GeneID: 7083332. The Using 50 nanograms of template plasmid DNA, the
complete Dictyoglomus turgidum genome is available putative cellulase gene was amplified using the
at NCBI accession number NC_011661 and the following expression primers:
complete Dictyoglomus thermophilum genome is CelA Forward:
available at GenBank accession number CP001146. 5’-AACAATCTTATTAAGAGAGGAATTAATTTT-3’;
3. Results
CelA Reverse: 5’-TCATATATTCCTTTCAGGTATTAATGCCCT-3’.
Sequencing the cloned gene for celA in this work The amplified sequence corresponded to a 936 bp
gave the translated protein sequence that corresponds to Dtur_0670 (GenBank 7083332), the first 20 amino
open reading frame encoding a 37,002 Dalton protein. acids of which correspond exactly to the N-terminal
The amplified PCR product was cloned into the sequence obtained by Edman degradation of the pure pET28a vector. The ligated product was then protein. InterProScan Family analysis predicted the transformed into BL21 (DE3) chemically competent
enzyme was a Glycosyl Hydrolase, Family 5. The cells and the transformed clones were selected on
enzyme possessed no electronically detectable plates containing 30 μg/mL kanamycin and 200 μg/mL
cellulose binding module (CBM). BLASTP analysis 4-methylumbelliferyl- β-D-cellobioside. Eight fluorescent
against the non-redundant protein database revealed transformants were picked, grown in 50 mL cultures,
that CelA is most closely related to the electronically and induced with 1 mM IPTG. Lysates of the cultures
annotated Dictyoglomus thermophilum Endoglucanase were prepared and the expressed enzymes were shown
H (Dicth_0506) (85% amino acid identity and 94% to have properties identical to the original purified
amino acid similarity over 312 amino acids) and the sample of Dtur CelA.
electronically annotated Dictyoglomus turgidum
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
cellulase Dtur_0276 (68% amino acid identity and 82% to approximately 30% of this maximum value at 90 ℃ amino acid similarity over 312 amino acids).
(Fig. 4). CelA exhibited activity over the pH range of Multiple sequence alignment of the translated Dtur
4.0 to 6.8, with maximum activity between pH 5.6 and celA gene compared to cellulases from thermophilic
6.8. The activity of the enzyme was not stimulated by organisms shows that CelA is not closely related to
addition of either calcium or DTT, and was not other cellulases or endo-glucanases (Fig. 2).
inhibited by EDTA.
Outside the genus Dictoglomi, Dictyoglomus The endo-glucanase specificity of CelA was turgidum CelA is most homologous to an electronically
determined using insoluble AZCL substrates and annotated Family 5 glycoside hydrolase of Thermotoga
normalized to the highest activity (100%). CelA maritima RQ2 (UniProt Accession Number: MSB8
showed highest activity on AZCL-HE-Cellulose (100%), B1LAS2_THESQ); the homology is low, with 52%
followed by AZCL- β-glucan (55%). The enzyme had amino acid identity and 68% amino acid similarity over
low levels of activity on AZCL-arabinoxylan, 312 amino acids (Fig. 3).
AZCL-xyloglucan and AZCL-galactaomannan Dtur CelA had a temperature optimum between
(approximately 4% each). The enzyme had no activity
70 ℃ and 80 ℃; the activity of the enzyme dropped on the following substrates: AZCL-rhamnoglacturanon,
Fig. 2 Phylogenetic comparison of thermostable cellulases analyzed by ClustalW.
Dtur_0276, Dictyoglomus turgidum GeneID: 7083099; Dtur CelA, Dictyoglomus turgidum GeneID: 7083332; Dicth_0506, Dictyoglomus thermophilum GeneID: 6946331; Cth_CelC, Clostridium thermocellum GeneID: 12584559; Cth_CelG, Clostridium thermocellum GeneID: 462211; Cth_CelO, Clostridium thermocellum GeneID: 7208816; Cth_CelK, Clostridium thermocellum GeneID: 2978565; Cth_CelD, Clostridium thermocellum GeneID: 40671; Cth_CelA, Clostridium thermocellum GeneID: 144752; Cth_CelI, Clostridium thermocellum GeneID: 7208809; Thema_celB, Gene Thermatoga maratima ID: 1297062; Thermotoga_RQ_GH5, Thermatoga RQ2 Gene ID: 6092506; Acel_E1, Acidothermus cellulolyticus Gene ID: 1708075; Acel_0135_Cel, Acidothermus cellulolyticus Gene ID: 4485572; Rhomr_celA, Rhodothermus marinus Gene ID: 2304961; Pyrfu_EglA, Pyrococcus furiosus Gene ID: 5870829; Calsa, Caldicellulosiruptor saccharolyticus Gene ID: 1708078.
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
Fig. 3 ClustalW alignment of Dictyoglomus turgidum CelA with Tma, the electronically annotated Family 5 glycoside hydrolase of Thermotoga sp. RQ2.
Table 1 Specific activity of Dtur CelA.
Substrate
Specific activity (U/mg) β-glucan 226
ASC 0.59 Avicel 0.01
Xylan n.d.
pNP- β-cellobioside
Fig. 4 Temperature-activity curve for Dtur CelA.
pNP- β-glucoside
Activity of Dictyoglomus turgidum cellulase as a function of
n.d.
temperature at pH5.8 using 1% beta-glucan as substrate as n.d.: not detectable, less than 0.01 U/mg of activity. described in Materials and Methods.
ASC are estimates based on low dosages of enzyme, AZCL-curdlan, AZCL-galactan, or AZCL-arabinan.
the enzyme did not show a linear response between CelA hydrolyzed MUC, MUL and MUG substrates,
dosage and product formation with these two substrates. indicating that the enzyme possessed at least a minimal
ASC hydrolysis was carried out for 65 hr at 60 ℃. At exo-activity on glucans. No activity was detectable on
10% conversion of ASC to reducing sugars, the major MUX.
products formed were glucose (1.5%) and cellobiose Dtur CelA showed significant activity on five
(8.5%); no cellotriose or cellotetraose was detectable. soluble and three insoluble polymeric substrates (Table 1).
4. Discussion
The highest specific activity, 226 U/mg was obtained using β-glucan as substrate. Enzymatic activity was
Dictyoglomus turgidum is an anaerobic, thermophile observed with both Avicel and ASC using a coupled
able to degrade a wide range of biomass components [2] assay (CelA plus b-glucosidase) to remove any product
including starch, cellulose, pectin and lignin. The broad inhibition by cellobiose. The values for cellulose and
range of substrate utilization is reflected in the high
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
percentage of CAZymes present in the genome, 2.35% (887 A.A.) or CelO (660 A.A.). The observation that of its total genes, higher than the percentage present in
Dictyoglomus turgidum CelA is able to hydrolyze the cellulose-degrading, thermophilc anaerobe, insoluble cellulose substrates without the presence of Clostridium thermocellum , with 2.2% of its total genes
either an identifiable carbohydrate binding domain (Garret Suen, University of Wisconsin, Madison,
(CBD) or cellulosomal structure suggests that the private communication). This apparent strong enzyme possesses an alternative mechanism for the degradation capacity, coupled with the uniqueness of
recognition and binding to these substrates. the organism, suggested that Dictyoglomus turgidum
Alternatively, the enzyme could be an intracellular would be an excellent source of new and novel
protein that breaks down oligosaccharides imported biomass-degrading enzymes.
into the cell.
Dictyoglomus turgidum CelA was isolated from Functionally, Dictyoglomus turgidum CelA is a plate screening of a shotgun library in E. coli. The
novel cellulase, active on a much wider range of enzyme was purified, its gene sequenced and substrates than other cellulases. The enzyme has broad subcloned into pET28a for high level expression.
substrate specificity, being able to hydrolyze both Subsequent sequencing of the Dictyoglomus turgidum
native and dye-linked substrates containing either genome indicated the CelA protein corresponded to the
β-(1,4)-linked glucose and β-(1,4)-linked mannose predicted Dtur_0670 gene product. Amplification and
residues. The specific activity of the enzyme for cloning of the Dtur_0670 gene product directly from
β-(1,4)-linked glucose-containing substrates is similar genomic DNA and cloning into pET28a gave a protein
to that reported for cloned Clostridium thermocellum with molecular weight and properties identical to CelA,
cellulases (Table 2).
confirming the identification (data not shown). The enzyme does not cleave AZCL-curdlan, Structurally, Dictyoglomus turgidum CelA is a novel
indicating it does not recognize β-(1,3)-linked glucose cellulase, showing little structural similarity to known
residues. The high activity on β-glucan, CMC, and cellulases or other proteins outside the genus glucomannan (which contains a backbone of Dictoglomi . Noticeably absent from the enzyme is a
β-(1,4)-linked glucose and mannose residues in a 5:8 signal peptide sequence, suggesting either an ratio) indicates cleavage between β-(1,4)-linked intracellular location for the enzyme or an alternate
glucose residues appears to be preferred. Steric route for secretion of the protein. Unlike some
hindrance by the high degree of xylose substitution on cellulases active on cellulose, CelA does not contain
the glucose backbone of xyloglucan may explain the any dockerin domains, and there is no evidence from
relatively low rate of hydrolysis of this substrate. the genome of Dictyoglomus turgidum for the
Hydrolysis of mannan and galactomannan indicates existence of other cellulosomal components such as
the ability of CelA to recognize and cleave both soluble scaffoldins. Therefore, it is unlikely that the
Table 2 Comparison of Dtur CelA activities to various Cth
Dictyoglomus turgidum CelA is a component of a
cellulytic enzymes.
larger cellulytic structure. In contrast to many CMC β-glucan ASC Avicel Source noncomplexed cellulases, Dictyoglomus turgidum
0.59 0.01 This work CelA does not contain an identifiable carbohydrate
Dtur CelA 63 226
9.7 48 3.5 0.08 Schwarz [14] binding domain separate from the hydrolytic domain of
Cth CelA
0.94 0.17 Gilad [15] the enzyme. This is further supported by the small size
Cth CelI
12.2 n.d.
Cth CelQ 159
1.4 0.40 Arai [16]
74 49 0.092 0.055 of the enzyme, 312 A.A., significantly smaller than C. Arai [17]
Cth CelJ
2.1 0.002 Zverlov [18] thermocellum CBM-containing cellulases such as CelI
Cth CelO
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
(galactomannan) and insoluble (mannan) substrates possess a novel growth condition where large spherical between β-(1,4)-linked mannose residues. This wide
aggregates containing over 100 cells and surrounded range of substrate utilization is not common among
by a common outer membrane can be seen [2, 3]. This thermophilic cellulases; pure Clostridum thermocellum
observation and the apparent lack of signal sequences cellulases CthCelA (Glycosyl Hydrolase, Family 8),
on many secreted proteins suggest the presence of CthCelC (Glycosyl Hydrolase, Family 5), CthCelG
some unknown mechanism of protein secretion in this (Glycosyl Hydrolase, Family 5), CthCelI (Glycosyl
gram-negative thermophile.
Hydrolase, Family 9), CthCelK (Glycosyl Hydrolase, The high temperature optimum and wide substrate Family 9), CthCelL (Glycosyl Hydrolase, Family 5),
range of Dictyoglomus turgidum CelA may make it an and CthCelO (Glycosyl Hydrolase, Family 5) showed
excellent tool for biomass degradation at high no activity on either AZCL-galacomannan or temperature as well as an excellent model for studies AZCL-xyloglucan, even at high enzyme dosing (data
on cellulase structure and function. The discovery of not shown).
this novel cellulase suggests that Dictyoglomus The enzyme possesses significant exoglucanase
turgidum may be an exciting source of new and novel activity along with its endoglucanase activities. The
enzymes for biomass conversion and other applications. high specific activity on pNP- β-cellobioside, coupled
The recent completion of the genome sequence of with the product distribution observed with ASC,
Dictyoglomus turgidum , with 54 potential CAZyme suggests that Dictyoglomus turgidum CelA can be
genes, should provide a foundation for the functional classified as a processive endo-cellulase.
annotation of the many other enzymes this bacterium Only a handful of genes have been expressed and
utilizes to degrade plant polysaccharides. characterized from Dictyoglomi, including: xylanase
5. Conclusions
xyn
B [4] beta-mannanase manA [19], xylanase xynA [20], and amylases amyA, B and C [21, 22]. In D.
Screening a genomic library of Dictyoglomus thermophilum , almost all amylase activity is found in
turgidum resulted in the discovery of a novel the media, although AmyA and AmyB lack a typical
thermostable endoglucanase enzyme, CelA. The signal sequence at their amino terminal end [22].
enzyme shows low homology to other cellulases and ManA [4] does not have an apparent signal peptide.
endoglucanases previously described. The enzyme Xylanase XynA [20] and xylanase xynB [4] from D.
utilize a broader range of substrates than other thermophilum possess signal peptides that appear to be
thermophilic cellulases and endoglucanases, suggesting functional in E. coli.
the enzyme evolved to degrade a broad range of Dictyoglomus thermophilum , the type species of this
polymeric substrates found in low concentrations in the genus, was described as growing only on soluble
hot spring environment. This broad range of substrate substrates [3], while Dictyoglomus turgidus, obtained
utilization, combined with the high temperature originally from Uzon Caldera, was found to grow
optimum of 70 ℃ to 80 ℃, makes this enzyme a weakly on solid polysaccharides, including useful tool for degrading a range of biomass polymers microcrystalline cellulose [2]. A preliminary analysis
at elevated temperature. The unusual structure of the from the recent whole genome sequencing of these two
molecules makes it an excellent candidate for further species reveals that both have a similarly large number
structural characterizations and comparisons. of glycosyl hydrolases (Dtu, 54; Dth, 55
Acknowledgments
[www.cazy.org]), few of which have a recognizable signal peptide (data not shown). Both strains also
This work was funded in part by DOE grant DOE
Identification, Cloning and Characterization of Dictyoglomus Turgidum CelA,
an Endoglucanase with Cellulase and Mannanase Activity
DE-FG36-06GO16106, “Novel enzyme products for analysis for the non-specialist, Nucleic Acids Res. 36 (2) (2008) 465-469.
the conversion of defatted soybean meal to ethanol” [12] M.N. Price, K.H. Huang, E.J. Alm, A.P. Arkin, A novel
and funded in part by the DOE Great Lakes Bioenergy method for accurate operon predictions in all sequenced Research Center (DOE Office of Science BER
prokaryotes, Nucleic Acids Research 33 (2005) 880-892. DE-FC02-07ER64494).
[13] O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen, Locating proteins in the cell using TargetP, SignalP, and
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