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.

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.

Purification and characterization of an extracellular glucoamylase from the thermophilic fungus Humicola grisea var. thermoidea

1993 ◽  
Vol 39 (9) ◽  
pp. 846-852 ◽  
Author(s):  
Luis Ricardo Orsini Tosi ◽  
Héctor Francisco Terenzi ◽  
Joāo Atílio Jorge
Keyword(s):  
Molecular Mass ◽  
Half Life ◽  
Carbohydrate Content ◽  
Thermophilic Fungus ◽  
Apparent Molecular Mass ◽  
Purification And Characterization ◽  
Sds Page ◽  
Humicola Grisea ◽  
Temperature Optima

Humicola grisea var. thermoidea mycelium grown on maltose as the main source of carbon produced at least two amylases. The major amylolytic component was purified to homogeneity and classified as a glucoamylase. The apparent molecular mass of the purified enzyme was estimated to be 63 000 Da by SDS-PAGE and 65 000 Da by Bio-Gel P-100 filtration. The purified enzyme was a glycoprotein with 1.8% carbohydrate content and pH and temperature optima of 5.0 and 55 °C, respectively. The purified glucoamylase was thermostable at 60 °C with a half-life of 16 min at 65 °C. In the presence of starch the purified enzyme retained 75% of its thermostability at 65 °C, while the addition of maltose failed to protect the activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch and amylopectin were the best substrates utilized and amylose was hydrolyzed faster than maltopentaose, maltotetraose, and maltotriose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.Key words: glucoamylase, amylase, Humicola grisea.

Purification and characterization of two xylanases from Chaetomium thermophile var. coprophile

1989 ◽  
Vol 35 (9) ◽  
pp. 836-842 ◽  
Author(s):  
Ramesh K. Ganju ◽  
Paul J. Vithayathil ◽  
S. K. Murthy
Keyword(s):  
Amino Acids ◽  
Enzyme Purification ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Larch Wood ◽  
Hydrolysis Products ◽  
Xylanase Enzyme ◽  
Temperature Optima

Two xylanases (I and II) out of several extracellular xylanases produced by the thermophilic fungus Chaetomium thermophile var. coprophile were purified to homogeneity by a combination of ion exchange and gel filtration chromatographic procedures. They had molecular weights of 26 000 (xylanase I) and 7000 (xylanase II). The temperature optima for xylanase I and II were 70 and 60 °C, and they were optimally active at pH 4.8–6.4 and 5.4–6.0, respectively. Xylanase I was found to be comparatively more stable than xylanase II at higher temperatures. Amino acid composition indicated that xylanase I contained high amounts of glycine, threonine, and low amounts of histidine and sulphur-containing amino acids. Each enzyme released different hydrolysis products from larch wood xylan. Xylanase I produced mainly xylobiose and xylotriose whereas xylanase II produced mainly xylobiose.Key words: Xylanase, enzyme purification, characterization, Chaetomium thermophile.

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 glutamate-phenylpyruvate aminotransferase from the ruminal protozoan Entodinium caudatum

2004 ◽  
Vol 55 (9) ◽  
pp. 991
Author(s):  
Md. Ruhul Amin ◽  
Ryoji Onodera ◽  
R. Islam Khan ◽  
R. John Wallace ◽  
C. Jamie Newbold
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Maximum Activity ◽  
Purification And Properties ◽  
Sds Page ◽  
Wide Range ◽  
S 100 ◽  
A Cell

Entodinium species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify, and investigate properties of one of the enzymes involved in amino acid metabolism by Entodinium caudatum, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 74-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with phenyl-superose, DEAE-Toyopearl 650M, Sephacryl S-100 HR, and Sephadex G-100. The molecular mass of GPA was estimated by SDS–PAGE to be 65.0 kDa. The optimum pH was 6.0 and it was found to be reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, and SDS, EDTA, and some heavy metal ions also inhibited activity. The purification and characterisation of the enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by Entodinium caudatum.

scholarly journals Coal Depolymerising Activity and Haloperoxidase Activity of Mn Peroxidase fromFomes durissimusMTCC-1173

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Sunil Kumar Singh ◽  
Meera Yadav ◽  
Sudha Yadava ◽  
Kapil Deo Singh Yadav
Keyword(s):  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Sodium Lactate ◽  
Low Ph ◽  
Exchange Chromatography ◽  
Fungal Strain ◽  
Mn Peroxidase ◽  
Sds Page ◽  
Catalytic Rate ◽  
Temperature Optima

Mn peroxidase has been purified to homogeneity from the culture filtrate of a new fungal strainFomes durissimusMTCC-1173 using concentration by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The molecular mass of the purified enzyme has been found to be 42.0 kDa using SDS-PAGE analysis. The values using MnSO4and H2O2as the variable substrates in 50 mM lactic acid-sodium lactate buffer pH 4.5 at were 59 μM and 32 μM, respectively. The catalytic rate constants using MnSO4and H2O2were 22.4 s−1and 14.0 s−1, respectively, giving the values of 0.38 μM−1s−1and 0.44 μM−1s−1, respectively. The pH and temperature optima of the Mn peroxidase were 4 and , respectively. The purified MnP depolymerises humic acid in presence of H2O2. The purified Mn peroxidase exhibits haloperoxidase activity at low pH.

Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1

1999 ◽  
Vol 65 (2) ◽  
pp. 694-697 ◽  
Author(s):  
Khanok Ratanakhanokchai ◽  
Khin Lay Kyu ◽  
Morakot Tanticharoen
Keyword(s):  
Xylanase Activity ◽  
Crystalline Cellulose ◽  
Acetyl Esterase ◽  
Affinity Adsorption ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Alkaliphilic Bacillus ◽  
Alkaliphilic Bacterium ◽  
Adsorption Desorption ◽  
Xylanolytic Enzymes

ABSTRACT An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe2+, Ca2+, and Mg2+ greatly increased the xylanase activity, whereas Mn2+ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.

scholarly journals Activated Charcoal—A Potential Material in Glucoamylase Recovery

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
S. O. Kareem ◽  
I. Akpan ◽  
T. O. S. Popoola ◽  
L. O. Sanni
Keyword(s):  
Molecular Weight ◽  
Activated Charcoal ◽  
Contact Time ◽  
Enzyme Purification ◽  
Downstream Processing ◽  
Single Step ◽  
Major Protein ◽  
Rhizopus Oligosporus ◽  
Sds Page ◽  
Effects Of Ph

The potential of activated charcoal in the purification of fungal glucoamylase was investigated. Various concentrations of activated charcoal (1–4% w/v) were used to concentrate crude glucoamylase from Rhizopus oligosporus at different temperature values (30–50°C). Effects of pH (3.0–6.0) and contact time (0–60 min) on enzyme purification were also monitored. Activated charcoal (3% w/v) gave a 16-fold purification in a single-step purification at 50°C for 20 min and pH 5.5. The result of SDS-PAGE analysis of purified glucoamylase showed two major protein bands with corresponding molecular weight of 36 kDa and 50 kDa. The method is inexpensive, rapid, and simple which could facilitate downstream processing of industrial enzyme.

scholarly journals Effects of some antibiotics on glucose 6-phosphate dehydrogenase in sheep liver

2012 ◽  
Vol 47 (No. 10 - 11) ◽  
pp. 283-288 ◽  
Author(s):  
M. Çiftci ◽  
V. Turkoglu ◽  
S. Aldemir
Keyword(s):  
Gel Electrophoresis ◽  
Enzyme Purification ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sheep Liver ◽  
Sds Page ◽  
In Vitro Effects ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

In vitro effects of penicillin, sulbactam, cefazolin, and amikacine on the activity of the enzyme glucose-6-phosphate dehydrogenase in sheep liver were investigated. Glucose 6-phosphate dehydrogenase was purified from sheep liver, using a simple and rapid method. The purification consisted of two steps, preparation of homogenate and 2&rsquo;, 5&rsquo;-ADP Sepharose 4B affinity chromatography. As a result of the two consecutive procedures, the enzyme, having the specific activity of 11.76 EU/mg proteins, was purified with a yield of 35.72% and 1.913 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for the enzyme. In addition, I50 values of the antibiotics were determined by plotting activity % vs. antibiotic concentrations. I50 values were 17.71 mM for penicillin, 27.38 mM for sulbactam, 28.88 mM for cefazolin, and 30.59 mM for amikacine.

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