scholarly journals Pereparation and characterization of two types of covalently immobilized amyloglucosidase

2005 ◽  
Vol 70 (5) ◽  
pp. 713-719 ◽  
Author(s):  
Nenad Milosavic ◽  
Radivoje Prodanovic ◽  
Slobodan Jovanovic ◽  
Irena Novakovic ◽  
Zoran Vujcic
Keyword(s):  
Specific Activity ◽  
Periodate Oxidation ◽  
Immobilized Enzymes ◽  
Soluble Starch ◽  
Soluble Enzyme ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Higher Temperature ◽  
Hydrolysis Of

Amyloglucosidase from A. niger was covalently immobilized onto poly( GMA-co-EGDMA) by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined. .

Purification of a third distinct xylanase from the xylanolytic system of Trichoderma harzianum

1986 ◽  
Vol 32 (7) ◽  
pp. 570-576 ◽  
Author(s):  
Ken K. Y. Wong ◽  
Larry U. L. Tan ◽  
John N. Saddler ◽  
Makoto Yaguchi
Keyword(s):  
Molecular Mass ◽  
Trichoderma Harzianum ◽  
Thermal Tolerance ◽  
Specific Activity ◽  
Gene Products ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Polysulfone Membrane ◽  
Xylan Hydrolysis ◽  
Hydrolysis Of

Three of the xylanases produced by Trichoderma harzianum E58 passed through a polysulfone membrane with molecular mass cut-off of 10 000 daltons, even though their molecular mass had been estimated to be 20 000, 22 000, and 29 000 daltons. The 22 000 dalton xylanase was purified to homogeneity from a preparation containing a mixture of 22 000 and 20 000 dalton xylanase using a combination of hydrophobic column chromatography and chromatofocusing. This enzyme has a pI of 8.5, a specific activity of 0.28 U/mg, a temperature optimum between 45 and 50 °C, a pH optimum between 4.5 and 5.0, and the ability to cleave xylotriose. It differs from the other two xylanases by having a lower pI, a lower specific activity, and a lower thermal tolerance. All three xylanases are highly specific for xylan hydrolysis and they do not cleave xylobiose or release arabinose substituents from arabinoxylan. Their amino acid compositions suggest that they are three distinct gene products. The three enzymes are major components of the xylanolytic system of T. harzianum, which consists of at least two other xylanases and two β-xylosidases which are responsible for the release of arabinose substituents and the hydrolysis of xylobiose.

scholarly journals Preparation and Characterization of 5′-Phosphodiesterase from Barley Malt Rootlets

2010 ◽  
Vol 5 (2) ◽  
pp. 1934578X1000500
Author(s):  
Jie Hua ◽  
Ke-long Huang
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Barley Malt

Two 5′- phosphodiesterases (5′-PDE-a and 5′-PDE-b) were isolated from barley malt rootlets, and further purified by gel filtration on Sephadex G-25 and Sephadex G-75. 5′-PDE-a had a pH optimum of 5.0, temperature optimum of 70oC, and specific activity of 0.0143 mM ·mg−1-min−1. 5′-PDE –b had a pH optimum of 6.0, temperature optimum of 65°C and specific activity of 0.0125 mM ·mg−1·min−1. Both enzymes can be used to hydrolyze RNA to form 5′-nucleotides. The enzymes were quite stable at 70oC for 420 minutes. The Km was 0.24 mM for 5′-PDE-a and 0.16 mM for 5′-PDE-b with t-RNA (yeast) as substrate.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

scholarly journals Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. ex4484_79

2021 ◽  
Vol 12 ◽  
Author(s):  
Anke Chen ◽  
Dan Wang ◽  
Rui Ji ◽  
Jixi Li ◽  
Shaohua Gu ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Maximum Activity ◽  
Homologous Proteins ◽  
Glycosidic Bonds ◽  
Catalytic Sites ◽  
Potential Applications ◽  
Beta Glucosidase ◽  
Hydrolysis Of

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

scholarly journals Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

1988 ◽  
Vol 250 (2) ◽  
pp. 453-458 ◽  
Author(s):  
H Sobek ◽  
H Görisch
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sulfolobus Acidocaldarius ◽  
Sedimentation Equilibrium ◽  
Prolonged Storage ◽  
Heat Stable ◽  
Equilibrium Data ◽  
Poly Ethylene ◽  
Hydrolysis Of

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

scholarly journals Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

1982 ◽  
Vol 205 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Toru Momoi ◽  
Yoav Ben-Yoseph ◽  
Henry L. Nadler
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Significant Activity ◽  
Acid Ceramidase ◽  
Ph Optimum ◽  
Farber Disease ◽  
Normal Controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C12:0), N-myristoyl- (C14:0) and N-palmitoyl- (C16:0) sphingosine (C18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C18:0) substrate. The activity towards N-linolenoylsphingosine (C18:3/C18:1), N-linoleoylsphingosine (C18:2/C18:1) and N-oleoylsphingosine (C18:1/C18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C18:0/C18:1). The activity towards N-stearoyldihydrosphingosine (C18:0/C18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C6:0/C18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher Km values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.

scholarly journals Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate

1992 ◽  
Vol 287 (3) ◽  
pp. 685-690 ◽  
Author(s):  
O Ploux ◽  
P Soularue ◽  
A Marquet ◽  
R Gloeckler ◽  
Y Lemoine
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Bacillus Sphaericus ◽  
Careful Study ◽  
Metal Chelators ◽  
Ph Profile ◽  
E Coli ◽  
Coa Ligase ◽  
Hydrolysis Of

The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP. Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.

AN ISOPHENOXAZINE SYNTHASE FROM PYGNOPORUS COCCINEUS (FR.) BOND. AND SING.

1964 ◽  
Vol 42 (11) ◽  
pp. 1515-1526 ◽  
Author(s):  
P. M. Nair ◽  
L. C. Vining
Keyword(s):  
Electron Acceptor ◽  
Specific Activity ◽  
Fold Increase ◽  
Maximum Activity ◽  
Flavin Mononucleotide ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Hydroxyanthranilic Acid ◽  
Pycnoporus Coccineus ◽  
Citrate Phosphate

Mycelium from cultures of the red polypore Pycnoporus coccineus (Fr.) Bond. and Sing, contains an enzyme which catalyzes the oxidative condensation of 2 molecules of 2-aminophenol to yield 2-amino-3H-isophenoxazin-3-one. Fractionation of the crude extract has given a preparation with an 893-fold increase in specific activity. The purified enzyme has a pH optimum in citrate-phosphate buffer of 5.0, and a temperature optimum of 55°. The Km value is 4.35 × 10−4 M. FMN and Mn++ ions were required for maximum activity. FAD also served as an electron acceptor. Of the metal ions tested only Mn++ activated the reaction. Hg++ and Fe++ inhibited strongly. The course of the reaction when cofactors were added separately suggested that flavin mononucleotide is the initial electron acceptor and that Mn++ ions are required for reoxidation of the flavin. The enzyme has narrow specificity, and does not catalyze the oxidation of 3-hydroxyanthranilic acid, 3-hydroxykynurenine, or pyrocatechol.

scholarly journals INVESTIGATION OF ENZYMATIC HYDROLYSIS OF FATTY WASTE PROCESSING OF SUNFLOWER SEEDS BY IMMOBILIZED LIPASE

2020 ◽  
Vol 20 (1) ◽  
pp. 5-11
Author(s):  
V. Skliar ◽  
G. Krusir ◽  
V. Zakharchuk
Keyword(s):  
Crop Production ◽  
Agricultural Sector ◽  
Lipolytic Activity ◽  
Immobilized Lipase ◽  
Immobilized Enzymes ◽  
Lipase Immobilization ◽  
Ph Optimum ◽  
Prolonged Incubation ◽  
Physical And Chemical ◽  
Hydrolysis Of

Ukraine has a developed agricultural sector, in particular crop production, which is a source of large quantities of production residues and waste. One of the most promising areas for solving environmental problems in the production of grain products is the processing of industrial waste by enzymes and the use of processing products in other industries. The current needs of sustainable environmental practices have increased the use of enzymatic technologies in production processes. Lipases be used in the processing of waste from oil and fat enterprises, namely, waste from the stage of demetallization of hydrogenated fat from vegetable oils by enzymatic hydrolysis.The work is devoted to the study of conditions of enzyme Rhizopus japonicus lipase immobilization and its physical and chemical characteristics. Factors for obtaining immobilized biocatalysts, methods and conditions for determining the activity and stability of immobilized enzymes are highlighted. Lipolytic activity of the enzyme immobilized under these conditions remains more than 30% compared to native, which is a high indicator of activity retention. It has been shown that immobilization promotes the expansion of the pH- and thermo-optimum of the lipase. It was determined that for the Rhizopus japonicus immobilized lipase, the pH optimum increased with a shift from 7.0 to 6.5, and there was an increase in pH stability during prolonged incubation of the immobilized enzyme for alkaline and acidic pH values. It has been established that lipase immobilization leads to expansion of the thermo-optimum, as well as stabilization of the enzyme during prolonged incubation at 40 ° C and at higher temperatures (+60-80 ° C), which are possible when drying the final product. The high activity and stability of the immobilized lipase make it possible to recommend it for biotechnological processing ofoil-fat waste.

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