Enzymic hydrolysis of the potassium chloride soluble fraction of carrageenan: properties of "λ-carrageenases" from Pseudomonas carrageenovora

1973 ◽  
Vol 19 (7) ◽  
pp. 779-788 ◽  
Author(s):  
K. H. Johnston ◽  
E. L. McCandless
Keyword(s):  
Soluble Fraction ◽  
Reducing Sugar ◽  
Chondrus Crispus ◽  
Enzymic Hydrolysis ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Specific Viscosity ◽  
Chromatographic Resolution ◽  
Fraction I ◽  
Hydrolysis Of

An enzyme complex which hydrolyzed the KCl soluble carrageenan extracted from the red alga Chondrus crispus has been isolated from the cell-free medium of a culture of Pseudomonas carrageenovora grown on this polysaccharide. Three hydrolases could be separated. Fraction I, which caused a rapid decrease in the specific viscosity of the polysaccharide preparation with only minimal release of reducing sugar, could be distinguished from fraction II chromatographically on Sephadex G-100 and electrophoretically on agarose gel. Fraction IIa caused release of reducing sugar at pH 6.2, which activity was depressed at pH 7.5. Fraction IIb exhibited viscometric activity only at both pH 6.2 and pH 7.5. Fraction IIa had a sharp pH optimum at pH 6.2 and a temperature optimum at 28°. All hydrolases were inactivated by freezing, by dialysis against distilled water, by heating at 35° for 30 min, and by Hg2+ and 0.0001 mM EDTA. When fraction II (a and b) isolated after chromatographic resolution on Sephadex G-100 was incubated at pH 6.2 with KCl soluble carrageenan from C. crispus, products which had Rgal values of 0.74 and 0.17 were detected, were sulfated, and contained no 3,6-anhydrogalactose.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

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 The hydrolysis of monolayers of phosphatidyl-[Me−14C]choline by phospholipase D

1969 ◽  
Vol 113 (4) ◽  
pp. 697-705 ◽  
Author(s):  
R. H. Quarles ◽  
R. M. C. Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
High Pressures ◽  
Specific Radioactivity ◽  
Maximal Activity ◽  
Enzymic Hydrolysis ◽  
Ph Optimum ◽  
High Specific Radioactivity ◽  
Rapid Hydrolysis ◽  
Hydrolysis Of

1. The hydrolysis of monolayers of phosphatidyl[Me−14C]choline at the air/water interface by phospholipase D (phosphatidylcholine phosphatidohydrolase) was investigated by a surface-radioactivity technique by using a flow counter. 2. Phosphatidylcholine of high specific radioactivity was prepared biosynthetically in good yield from [Me−14C]choline by using Saccharomyces cerevisiae. 3. At initial monolayer pressures between 12 and 25 dynes/cm. the hydrolysis occurred in two stages, an initial slow hydrolysis followed by a rapid hydrolysis. Below 3dynes/cm. and above 28dynes/cm. no enzymic hydrolysis of pure phosphatidylcholine monolayers could be detected. 4. The rapid hydrolysis was proportional to the enzyme concentration in the subphase, its pH optimum was 6·6, and 0·2mm-Ca2+ was required for maximal activity. 5. Hydrolysis of the film was accompanied by a pronounced fall in the surface pressure even though the phosphatidic acid formed did not leave the film. When the pressure fell to low values the hydrolysis ceased even if the film was only partially hydrolysed. 6. Above monolayer pressures of 28dynes/cm. enzymic hydrolysis could be initiated by inclusion of phosphatidic acid (and less effectively stearyl hydrogen sulphate) in the film, although the rates were not appreciably higher than those observed at 25dynes/cm. with a pure phosphatidylcholine film. 7. The initiation of the hydrolysis by phosphatidic acid was facilitated by the inclusion of high Ca2+ concentrations and certain carboxylic acid buffer anions in the subphase, although these did not activate by themselves. 8. The initiation of the hydrolysis at high pressures could not be related to any change in the surface potential brought about by the addition of the long-chain anions to the film, nor could it be ascribed to a surface dilution effect. 9. The results are discussed in relation to previous studies on the hydrolysis of phosphatidylcholine particles by the enzyme and also similar investigations on phosphatidylcholine monolayers with other phospholipases.

THE ENZYMIC HYDROLYSIS OF CARRAGEENAN BY PSEUDOMONAS CARRAGEENOVORA: PURIFICATION OF A κ-CARRAGEENASE

1966 ◽  
Vol 12 (5) ◽  
pp. 939-947 ◽  
Author(s):  
J. Weigl ◽  
W. Yaphe
Keyword(s):  
Degradation Product ◽  
Culture Medium ◽  
Deae Cellulose ◽  
Reducing Power ◽  
Enzymic Hydrolysis ◽  
Specific Viscosity ◽  
Sulfated Oligosaccharides ◽  
Rapid Fall ◽  
Hydrolysis Of ◽  
Major Degradation Product

Enzymes specific for the κ and λ fractions of carrageenan were obtained from the cell-free culture medium of Pseudomonas carrageenovora. These enzymes were concentrated by precipitation with ammonium sulfate and separated by chromatography on hydroxylapatite.The κ-carrageenase was purified to electrophoretic homogeneity by incubation at 35 °C and chromatography on DEAE cellulose. The enzymic hydrolysis of κ-carrageenan was accompanied by a rapid fall in specific viscosity and increase in reducing power. The products were a homologous series of sulfated oligosaccharides with 3-O-(3,6-anhydro-α-D-galactopyranosyl)-D-galactopyranose-4-O-sulfate (neocarrabiose-4-O-sulfate) as the major degradation product, and an enzyme-resistant fraction. The alkali-modified enzyme-resistant fraction was degraded by the κ-carrageenase.

THE WHEAT LEAF PHOSPHATASES: VI. SOME PROPERTIES OF THE ENZYME SYSTEM HYDROLYZING ADENOSINE-5′-PHOSPHATE AND PHENOLPHTHALEIN DIPHOSPHATE IN CRUDE JUICE PREPARATIONS

1963 ◽  
Vol 41 (5) ◽  
pp. 1275-1281 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Enzyme System ◽  
The Other ◽  
Enzymic Hydrolysis ◽  
Ph Optimum ◽  
Wheat Leaf ◽  
Hydrolysis Of ◽  
Leaf Juice

At least two enzymes are probably involved in the hydrolysis of mixtures of β-glycerophosphate, phenolphthalein diphosphate, and adenosine-5′-phosphate. One enzyme is primarily responsible for the hydrolysis of β-glycerophosphate whereas the other enzyme hydrolyzes adenosine-5′-phosphate and phenolphthalein diphosphate but has little activity on β-glycerophosphate.The liberation of orthophosphate from adenosine-5′-phosphate and phenolphthalein diphosphate by the enzyme in wheat leaf juice is inhibited by 0.005 M adenosine but not by 0.02 M phosphate. The inhibition of this enzyme by fluoride is markedly smaller than the inhibition of β-glycerophosphatase. The enzyme that hydrolyzes phenolphthalein diphosphate transfers phosphate from phenolphthalein diphosphate to adenosine to form adenosine-5′-phosphate.Experiments on the pH optimum for the enzymic hydrolysis of both adenosine-5′-phosphate and phenolphthalein diphosphate by undialyzed and dialyzed juice preparations with or without added Mg++ suggest that there may be more than one enzyme with different pH optima acting on both adenosine-5′-phosphate and phenolphthalein diphosphate.

THE WHEAT LEAF PHOSPHATASES: VI. SOME PROPERTIES OF THE ENZYME SYSTEM HYDROLYZING ADENOSINE-5′-PHOSPHATE AND PHENOLPHTHALEIN DIPHOSPHATE IN CRUDE JUICE PREPARATIONS

1963 ◽  
Vol 41 (1) ◽  
pp. 1275-1281 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Enzyme System ◽  
The Other ◽  
Enzymic Hydrolysis ◽  
Ph Optimum ◽  
Wheat Leaf ◽  
Hydrolysis Of ◽  
Leaf Juice

At least two enzymes are probably involved in the hydrolysis of mixtures of β-glycerophosphate, phenolphthalein diphosphate, and adenosine-5′-phosphate. One enzyme is primarily responsible for the hydrolysis of β-glycerophosphate whereas the other enzyme hydrolyzes adenosine-5′-phosphate and phenolphthalein diphosphate but has little activity on β-glycerophosphate.The liberation of orthophosphate from adenosine-5′-phosphate and phenolphthalein diphosphate by the enzyme in wheat leaf juice is inhibited by 0.005 M adenosine but not by 0.02 M phosphate. The inhibition of this enzyme by fluoride is markedly smaller than the inhibition of β-glycerophosphatase. The enzyme that hydrolyzes phenolphthalein diphosphate transfers phosphate from phenolphthalein diphosphate to adenosine to form adenosine-5′-phosphate.Experiments on the pH optimum for the enzymic hydrolysis of both adenosine-5′-phosphate and phenolphthalein diphosphate by undialyzed and dialyzed juice preparations with or without added Mg++ suggest that there may be more than one enzyme with different pH optima acting on both adenosine-5′-phosphate and phenolphthalein diphosphate.

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. .

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

Biochemical studies on the production of neuraminidase by environmental isolates of Vibrio cholerae non-O1 from Bulgaria

2011 ◽  
Vol 57 (7) ◽  
pp. 606-610 ◽  
Author(s):  
Rumyana Eneva ◽  
Stephan Engibarov ◽  
Tanya Strateva ◽  
Radoslav Abrashev ◽  
Ignat Abrashev
Keyword(s):  
Vibrio Cholerae ◽  
Pathogenic Bacteria ◽  
Environmental Isolates ◽  
Anaerobic Conditions ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Cholera Vibrio ◽  
Key Factor ◽  
Aeration Conditions ◽  
The One

Neuraminidase is a key factor in the infectious process of many viruses and pathogenic bacteria. The neuraminidase enzyme secreted by the etiological agent of cholera — Vibrio cholerae О1 — is well studied in contrast with the one produced by non-O1/non-O139 V. cholerae. Environmental non-O1/non-O139 V. cholerae isolates from Bulgaria were screened for production of neuraminidase. The presence of the neuraminidase gene nanH was detected in 18.5% of the strains. Тhe strain showing highest activity (30 U/mL), V. cholerae non-O1/13, was used to investigate the enzyme production in several media and at different aeration conditions. The highest production of extracellular neuraminidase was observed under microaerophilic conditions, which is possibly related to its role in the infection of intestine epithelium, where the oxygen content is low. On the other hand, this is another advantage of the microbe in such microaerophilic environments as sediments and lake mud. The highest production of intracellular neuraminidase was observed at anaerobic conditions. The ratio of extracellular to intracellular neuraminidase production in V. cholerae was investigated. The temperature optimum of the enzyme was determined to be 50 °C and the pH optimum to be 5.6–5.8.

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