Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli

1993 ◽  
Vol 39 (1) ◽  
pp. 134-139 ◽  
Author(s):  
J. M. Tamblyn Lee ◽  
Y. Hu ◽  
H. Zhu ◽  
K. J. Cheng ◽  
P. J. Krell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Xylanase Activity ◽  
Hydrolytic Activity ◽  
Crystalline Cellulose ◽  
Xylanase Gene ◽  
Amorphous Cellulose ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Neocallimastix Patriciarum ◽  
Temperature Optima

An endo-β-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector λgtWESλB and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations. The xylanase was located in the periplasmic space of the host, Escherichia coli HB101. The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40 °C, respectively, and the Km for oat spelt xylan hydrolysis was 0.89 mg∙mL−1. It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan. It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.Key words: Neocallimastix patriciarum, xylanase, gene.

scholarly journals Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

2017 ◽  
Vol 3 (5) ◽  
pp. 139
Author(s):  
Mariana Wahjudi ◽  
Catherina . ◽  
Nita Marcelia Wangunhardjo ◽  
Ernest Suryadjaja ◽  
Xavier Daniel
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Sodium Dodecyl ◽  
Xylanase Activity ◽  
Cellular Protein ◽  
Host Cells ◽  
Chromosomal Dna ◽  
Clear Zone ◽  
Sds Page

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses.  The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>

Purification and properties of an extracellular xylanase from the thermophilic fungus Humicola grisea var. thermoidea

1991 ◽  
Vol 37 (9) ◽  
pp. 675-681 ◽  
Author(s):  
Rubens Monti ◽  
Héctor Francisco Terenzi ◽  
João Atílio Jorge
Keyword(s):  
Enzyme Purification ◽  
Xylanase Activity ◽  
Sole Source ◽  
Larch Wood ◽  
Purification And Properties ◽  
Sds Page ◽  
Component Form ◽  
Activity Form ◽  
Humicola Grisea ◽  
Temperature Optima

Humicola grisea var. thermoidea mycelium grown on xylan as the sole source of carbon produced at least two extracellular xylanases. The main xylanolytic component (form 2; 90% of recovered activity) was purified to homogeneity. The apparent molecular mass of the purified enzyme was estimated to be 23 000 and 25 550 Da by Bio-Gel P-60 filtration and urea–SDS–PAGE, respectively. The purified enzyme was a glycoprotein with 45% carbohydrate content and pH and temperature optima of 5.5 and 70 °C, respectively. The apparent Km and Vmax values determined with larch-wood xylan were 3.3 mg/mL and 229 μmol∙min−1∙mg protein−1, respectively. The enzyme was highly specific for xylan and degraded this substrate to produce xylo-oligosaccharides, suggesting that it is a β-1,4-endoxylanase (EC 3.2.1.8). The minor enzymatic component of H. grisea extracellular xylanase activity (form 1) was partially purified and some of its properties were studied for comparative purposes. The results obtained suggested that the mode of action of xylanases form 1 and 2 on xylan differs. Key words: xylanase, hemicellulase, enzyme purification, endoxylanase, Humicola grisea.

Fibrobacter succinogenes S85 possesses at least nine distinct glucanase genes

1993 ◽  
Vol 39 (9) ◽  
pp. 882-891 ◽  
Author(s):  
Laercio M. Malburg Jr. ◽  
Cecil W. Forsberg
Keyword(s):  
Escherichia Coli ◽  
Restriction Fragment ◽  
Carboxymethyl Cellulose ◽  
Southern Hybridization ◽  
Fibrobacter Succinogenes ◽  
Rumen Bacteria ◽  
Genomic Libraries ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Specific Activities

The construction of genomic libraries of Fibrobacter succinogenes S85 in λ-Dash, λ-DashII, and pUC19, and the screening of recombinant clones for carboxymethylcellulose hydrolysis yielded 38 glucanase clones. These clones along with a collection of 10 glucanase clones in pUC8, were compared by restriction fragment and Southern hybridization analyses. Seven distinct glucanase clones (pCe14, pCe15, and pCe17 in pUC8, pCe16 in pUC19, and LCe18, LCel10, and LCel12 in λ-Dash) were identified, which were nonhomologous to previously studied cel3, lichenase, and cello-dextrinase genes from F. succinogenes S85. Specific activities of the encoded enzymes expressed in Escherichia coli were higher for carboxymethyl cellulose, barley β-glucan, and lichenan, and lower for acid-swollen cellulose, laminarin, and xylan. The enzymes were predominantly acidic, with pIs between 3.5 and 5.2, with the exception of the pCe16 enzyme, which was basic with a pI of about 8.4. As determined by zymogram analysis after SDS-PAGE, the LCe18 enzyme was 97 kDa, the pCel10 enzyme exhibited components of 70 and 45 kDa, and the pCel12 enzyme components were 69 and 65 kDa. These seven new glucanase clones, along with the cel3 and lichenase genes, indicate that F. succinogenes S85 possesses at least nine distinct endoglucanase genes.Key words: Fibrobacter succinogenes, rumen bacteria, cellulolytic, endoglucanases, glucanases, genes.

A truncated β-xylosidase from the anaerobic fungus Neocallimastix patriciarum 27

1994 ◽  
Vol 40 (6) ◽  
pp. 484-490 ◽  
Author(s):  
Hong Zhu ◽  
K.-J. Cheng ◽  
Cecil W. Forsberg
Keyword(s):  
Ion Exchange ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Barley Straw ◽  
Anaerobic Fungus ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Neocallimastix Patriciarum ◽  
Molecular Masses ◽  
Temperature Optima

Two extracellular β-xylosidases, xylosidase I and II, were isolated from the ruminal fungus Neocallimastix patriciarum 27 after growth in a barley straw medium. Xylosidase I was purified 88-fold to apparent homogeneity by ion-exchange, affinity, and gel filtration chromatography. The purified xylosidase I had an isoelectric point (pI) of 4.7 and was a monomelic protein with a molecular mass of 39.5 kDa as estimated by both SDS-PAGE and gel filtration. Xylosidase II was partially purified to approximately 95% purity. Xylosidase II had the same pI (4.7) as xylosidase I, and appeared to be a dimeric enzyme composed of two polypeptides with molecular masses of 85 and 45 kDa, respectively, on SDS-PAGE. Peptide mapping of the three proteins suggested that xylosidase I was a truncated product originating from xylosidase II. Xylosidases I and II had similar pH optima of 6.0, but different temperature optima of 50 and 40 °C, respectively. The Km and Vmax for xylosidase I were 0.59 mM of p-nitrophenyl-β-D-xylopyranoside and 38.04 U∙mg protein−1, respectively, and those for xylosidase II were 0.13 mM and 8.9 U∙mg protein−1, respectively. Both enzymes hydrolysed pNPX and xylobiose with the production of xylose, but only xylosidase I exhibited activity toward p-nitrophenyl-α-L-arabinofuranoside.Key words: xylosidase, Neocallimastix, patriciarum, glycosidase.

Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

1991 ◽  
Vol 37 (7) ◽  
pp. 554-561 ◽  
Author(s):  
Y. J. Hu ◽  
D. C. Smith ◽  
K. -J. Cheng ◽  
C. W. Forsberg
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Xylanase Activity ◽  
Ecori Fragment ◽  
Fibrobacter Succinogenes ◽  
Host Bacterium ◽  
Xylanase Gene ◽  
E Coli ◽  
Cloned Gene ◽  
Oat Spelt

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, λgtWESλB, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated λFSX101 and λFSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from λFSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-β-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the β-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of β-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan. Key words: rumen bacterium, xylanase gene, λgtWESλB, cellulolysis, Fibrobacter succinogenes.

scholarly journals Purification and characterization of an endoxylanase from Trichoderma koningii G-39

1991 ◽  
Vol 278 (2) ◽  
pp. 329-333 ◽  
Author(s):  
L Huang ◽  
T H Hseu ◽  
T T Wey
Keyword(s):  
Specific Activity ◽  
Xylanase Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Crystalline Cellulose ◽  
Trichoderma Koningii ◽  
Sds Page ◽  
Immunological Relationship ◽  
Oat Spelt

Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

Characterization of aNeocallimastixpatriciarumxylanase gene and its product

1999 ◽  
Vol 45 (11) ◽  
pp. 970-974 ◽  
Author(s):  
Jin-Hao Liu ◽  
Brent L Selinger ◽  
Cheng-Fang Tsai ◽  
Kuo-Jaon Cheng
Keyword(s):  
Catalytic Domain ◽  
Xylanase Activity ◽  
Binding Domain ◽  
Glycosyl Hydrolases ◽  
Cellulose Binding Domain ◽  
Xylanase Gene ◽  
Neocallimastix Patriciarum ◽  
Cellulose Binding ◽  
Linker Domain

A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.Key words: xylanase, cellulose binding domain, Neocallimastix patriciarum.

Expression of Trichoderma reesei β-Xylanase in Escherichia coli and its Function in Degradation of Juncao Holocellulose

2011 ◽  
Vol 236-238 ◽  
pp. 1058-1062
Author(s):  
Li Hua Liu ◽  
Zhi Wei Lin ◽  
Ling Lin ◽  
Yan Ling Yang ◽  
Zhan Xi Lin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Molecular Mass ◽  
Trichoderma Reesei ◽  
Optimum Activity ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Ph Range

In this study, the xyn2 gene, which encodes an endo-β-1,4-xylanase, was isolated with holocellulose extracted from Juncao Miscanthus floridulu as an inducer. The xyn2 gene expressed in Escherichia coli, with the estimated yield of 349 U·mL-1. Zymogram analysis showed that the purified Xyn2 had only one band on SDS-PAGE with an estimated molecular mass of 28 kDa. Enzymology analysis demonstrated that its optimum activity was at pH 6.0 and 60°C, with stability at pH range 4.0~7.0 and temperature up to 50°C. The metal ions Cu2+ and Mg2+ showed some inhibition effects, while Fe2+ and Fe3+ had small stimulating effects. Its values of Km and Vmax are 2.85 mM and 50.2 mM/min, respectively. Based on our results, we propose a novel way to convert Juncao biomass into energy and other useful products.

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

Biologia ◽  
2009 ◽  
Vol 64 (6) ◽  
Author(s):  
Yue-Hong Wang ◽  
Yu Jiang ◽  
Zuo-Ying Duan ◽  
Wei-Lan Shao ◽  
Hua-Zhong Li
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Optimal Condition ◽  
Industrial Application ◽  
Conversion Rate ◽  
Hydrolytic Activity ◽  
Thermoanaerobacter Ethanolicus ◽  
Transglycosylation Activity

AbstractIn this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K m and V max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).

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