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 a thermostable glucoamylase fromAspergillus fumigatus

1998 ◽  
Vol 44 (5) ◽  
pp. 493-497 ◽  
Author(s):  
William Brandani da Silva ◽  
Rosane Marina Peralta
Keyword(s):  
Aspergillus Fumigatus ◽  
Molecular Mass ◽  
Enzyme Purification ◽  
Soluble Starch ◽  
Carbohydrate Content ◽  
Apparent Molecular Mass ◽  
Starch Hydrolysis ◽  
Purification And Characterization ◽  
Original Activity

A thermostable glucoamylase from Aspergillus fumigatus was purified to homogeneity. It was a glycoprotein with 23% carbohydrate content and an apparent molecular mass of 42 kDa. The enzyme showed maximal activities at pH 4.5-5.5 and 65°C and preferentially attacked polysacharides, such as starch, glycogen, amylopectin, and amylose, rather than maltose and maltoriose. The Kmand Vmaxof soluble starch hydrolysis at 40°C and pH 5.0 were 0.1 mg ·mL-1and 161 µmol glucose equivalents liberated ·min-1·mg protein-1, respectively. The purified enzyme was remarkably insensitive to glucose. It was not affected by 500 mM D-glucose and retained about 80% of its original activity in the presence of 1000 mM of this sugar.Key words: amylase, Aspergillus fumigatus, enzyme purification, glucose insensitive, thermostableglucoamylase.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

2006 ◽  
Vol 52 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Subhas Das ◽  
Dileep Kumar Singh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Half Life ◽  
Indoleacetic Acid ◽  
Bacterial Strains ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Microbial Biodegradation ◽  
Membrane Bound

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34–41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%–16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.

scholarly journals Purification and characterization of beta-1, 3-glucanase from the secretion of Simira glaziovii colleters (Rubiaceae)

2006 ◽  
Vol 49 (6) ◽  
pp. 881-888 ◽  
Author(s):  
Felipe Almeida Vieira ◽  
Maura da Cunha ◽  
Denise Espellet Klein ◽  
André de Oliveira Carvalho ◽  
Valdirene Moreira Gomes
Keyword(s):  
Molecular Mass ◽  
Purification Process ◽  
Purification And Characterization ◽  
Chromatographic Methods ◽  
Sds Page ◽  
Optimum Ph

In this study, beta-1,3-glucanase was isolated from Simira glaziovii secretion. The purification process was achieved by a combination of chromatographic methods and was analyzed by SDS-PAGE. The purified enzyme presented an estimated molecular mass of 35 kDa. The optimum pH of enzyme was 5.2

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Purification and characterization of alpha-amylases from the intestine and muscle of ascaris suum (Nematoda).

2001 ◽  
Vol 48 (3) ◽  
pp. 763-774 ◽  
Author(s):  
K Zółtowska
Keyword(s):  
Molecular Mass ◽  
Isoelectric Focusing ◽  
Ascaris Suum ◽  
Iodoacetic Acid ◽  
Maximum Activity ◽  
Alpha Amylase ◽  
Purification And Characterization ◽  
Muscle Enzyme ◽  
Sds Page

Alpha-Amylase (EC 3.2.1.1) was purified from the muscle and intestine of the parasitic helminth of pigs Ascaris suum. The enzymes from the two sources differed in their properties. Isoelectric focusing revealed one form of a-amylase from muscles with pl of 5.0, and two forms of amylase from intestine with pI of 4.7 and 4.5. SDS/PAGE suggested a molecular mass of 83 kDa and 73 kDa for isoenzymes of a-amylases from intestine and 59 kDa for the muscle enzyme. Alpha-Amylase from intestine showed maximum activity at pH 7.4, and the enzyme from muscle at pH 8.2. The muscle enzyme was more thermostabile than the intestinal alpha-amylase. Both the muscle and intestine amylase lost half of its activity after 15 min at 70 degrees C and 50 degrees C, respectively. The Km values were: for muscle amylase 0.22 microg/ml glycogen and 3.33 microg/ml starch, and for intestine amylase 1.77 microg/ml glycogen and 0.48 microg/ml starch. Both amylases were activated by Ca2+ and inhibited by EDTA, iodoacetic acid, p-chloromercuribenzoate and the inhibitor of a-amylase from wheat. No significant differences were found between the properties of a-amylases from parasites and from their hosts.

scholarly journals Purification and Characterization of Sepiapterin Deaminase from the Silkworm, Bombyx mori

Pteridines ◽  
1998 ◽  
Vol 9 (1) ◽  
pp. 18-21 ◽  
Author(s):  
Hiroshi Sawada ◽  
Motoki Kanekatsu ◽  
Motoko Nakagoshi ◽  
Kenjiro Dohke ◽  
Teruhiko Iino ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Bombyx Mori ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Native Form ◽  
Sds Page ◽  
Column Chromatographic ◽  
Silkworm Bombyx Mori

Summary Sepiapterin deaminase has been purified approximately 6,000-told from the larval integument of the lemon mutant of the silkworm by several column chromatographic procedures. Sepiapterin and isosepiapterin were active substrates among various pteridines tested. The molecular mass of this enzyme was estimated to be 74 kDa by SDS-PAGE and 70 kDa by gel filtration, suggesting that the native form of the enzyme is monomeric protein . All silkworm strains, to the best of our knowledge, had an activity of the enzyme and the enzyme was widely distributed in the larval tissues. Sepiapterin deaminase may have an important function on the silkworm.

Purification and characterization of a thermostable xylanase from a thermophilic fungus Thermoascus aurantiacus

1987 ◽  
Vol 33 (8) ◽  
pp. 689-692 ◽  
Author(s):  
Larry U. L. Tan ◽  
Paul Mayers ◽  
John N. Saddler
Keyword(s):  
Xylanase Activity ◽  
Barium Chloride ◽  
Thermophilic Fungus ◽  
Mercury Chloride ◽  
Thermoascus Aurantiacus ◽  
Purification And Characterization ◽  
Acting Mechanism ◽  
A Subunit ◽  
Temperature Optima

A thermostable endo-β-D-xylanase (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a thermophilic fungus Thermoascus aurantiacus C436, using a single chromatographic step on SP-Sephadex C50. The purified preparation was homogeneous based on denaturing polyacrylamide and isoelectric focusing gels. The xylanase had a subunit molecular mass of 32 000 daltons, isoelectric point at pH 7.1, apparent Km and Vmax of 0.17% (w/v) xylan and 61.3IU/mg protein, respectively, at 50 °C. The pH and temperature optima for xylan hydrolysis were pH 5.1 and 80 °C, respectively. The xylanase retained full activity following incubation at 60 °C for 97 h or 70 °C for 24 h. At 80 °C, the half-life of the enzyme was 54 min. The xylanase was not affected by copper sulfate, zinc sulfate, calcium chloride, cobalt chloride, barium chloride, magnesium sulfate, and EDTA at concentrations of 2 mM. Mercury chloride at 2 mM concentration abolished all xylanase activity, while lead acetate at the same concentration reduced xylanase activity by approximately 25%. From the initial hydrolysis products of xylan, the xylanase was deduced to hydrolyse xylan through an endo-acting mechanism.

scholarly journals Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

1999 ◽  
Vol 344 (2) ◽  
pp. 331-337 ◽  
Author(s):  
Lakhdar GASMI ◽  
Jared L. CARTWRIGHT ◽  
Alexander G. MCLENNAN
Keyword(s):  
Fusion Protein ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Apparent Molecular Mass ◽  
Protein Glycation ◽  
Human Homologue ◽  
Protein Fragment ◽  
Sds Page

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5′-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. Km and kcat values with ADP-ribose were 60 μM and 5.5 s-1 respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg2+ or 100-250 μM Mn2+ ions; fluoride was inhibitory, with an IC50 of 20 μM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD+ and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006

2021 ◽  
Vol 85 (3) ◽  
pp. 600-610
Author(s):  
Akihiro Fujita ◽  
Akira Kawashima ◽  
Yuuki Mitsukawa ◽  
Noriaki Kitagawa ◽  
Hikaru Watanabe ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Culture Supernatant ◽  
Coupling Reaction ◽  
The Other ◽  
Purification And Characterization ◽  
Other Hand ◽  
Sds Page ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.

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