scholarly journals Characterization of a Zn2+-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity

1992 ◽  
Vol 286 (2) ◽  
pp. 435-440 ◽  
Author(s):  
D E Sok ◽  
M R Kim
Keyword(s):  
Bacillus Cereus ◽  
Molecular Mass ◽  
Mouse Brain ◽  
Concanavalin A ◽  
Heat Denaturation ◽  
Phosphodiesterase Activity ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Optimal Ph

p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.

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.

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 Identification and Characterization of Bacterial Lipase from Plateu Soil in West Java

2017 ◽  
Vol 18 (02) ◽  
pp. 103-108
Author(s):  
Vivitri Dewi Prasasty ◽  
Vinella Winata ◽  
Muhammad Hanafi
Keyword(s):  
Heat Stability ◽  
Industrial Applications ◽  
West Java ◽  
Ph Optimum ◽  
Good Efficiency ◽  
Optimum Ph ◽  
Glycerol Ester ◽  
Lipase Screening ◽  
Hydrolysis Of

Lipases are known as glycerol ester hydrolases that catalyze the hydrolysis of triglycerides into free fatty acids and glycerol. Lipases are found in human, animal, plant, and microorganisms. The aim of this research is to identify lipase producers and characterize bacterial lipase from West Java plateau soil. Plateau soil bacteria samples were isolated on lipase screening medium containing Rhodamine B. Olive oil was used as a substrate in screening and production medium bacterial lipases. From 16 bacterial isolate of lipase producers, 14 were identified as Bacillus sp. and the others were identified as Pseudomonas alcaligenes. All isolates were taken into production step to determine their lipase activities. Moreover, top 3 lipase activities out of 16 lipase activities were chosen to find the optimum pH and temperature. Both characterizations showed pH optimum and temperature optimum from each lipase. These optimum condition were used in heat stability characterization for each lipase samples. The result showed that lipase from isolate COK 2 in optimum pH 4 and temperature 50oC was the most stable lipase due to this sample has good and stable activity for 1 to 5 hours incubation time. Lipase sample from isolate COK 2 has good efficiency for lipase productivity in acid condition and high temperature. Results of this investigation could encourage utilization of these activity enhancers for various industrial applications.

scholarly journals The biosynthesis of GDP-d-arabinopyranose in Crithidia fasciculata: characterization of a d-arabino-1-kinase activity and its use in the synthesis of GDP-[5-3H]d-arabinopyranose

1995 ◽  
Vol 311 (1) ◽  
pp. 307-315 ◽  
Author(s):  
P Schneider ◽  
A Nikolaev ◽  
M A Ferguson
Keyword(s):  
Molecular Mass ◽  
Kinase Activity ◽  
Substrate Inhibition ◽  
Size Exclusion ◽  
Crithidia Fasciculata ◽  
Optimum Ph ◽  
Exclusion Chromatography ◽  
Trypanosomatid Parasites

GDP-D-arabinopyranose (GDP-D-Ara) is the precursor of the uncommon D-arabinopyranose residues present in the glycoconjugates of a few trypanosomatid parasites. Biosynthetic labelling experiments with Crithidia fasciculata showed that GDP-D-Ara could be labelled with [3H]D-Ara, [2-3H]D-Glc and [6-3H]D-Glc, but not with [1-3H]D-Glc, suggesting that D-Ara can be either taken up directly by the parasite or derived from D-Glc through a pathway involving the loss of carbon C-1. In vivo pulse-chase experiments indicated that D-Ara was sequentially incorporated into D-Ara-1-PO4 and GDP-D-Ara prior to transfer to the acceptor glycoconjugate, lipoarabinogalactan. An MgATP-dependent D-arabino-1-kinase activity present in soluble extracts of C. fasciculata was purified away from phosphatase activities by size-exclusion chromatography. The D-arabino-1-kinase had an apparent molecular mass of 600 kDa, a neutral optimum pH, and displayed substrate inhibition at D-Ara concentrations above 100 microM. It had a KmATP of 1.7 mM and a KmAra of 24 microM. Competition studies indicated that the orientation of every single hydroxyl residue was important for D-Ara recognition by the enzyme, but that methyl or hydroxymethyl groups could be tolerated as equatorial substituents on C-5 of D-Ara. The partially purified D-arabino-1-kinase activity was used in the chemico-enzymic synthesis of GDP-[5-3H]D-Ara from [6-3H]D-GlcN.

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.

Characterization of 2β(R)-17-O-AcetylajmaIan: Acetylesterase — a Specific Enzyme Involved in the Biosynthesis of the Rauwolfia Alkaloid Ajmaline

1987 ◽  
Vol 42 (4) ◽  
pp. 333-342 ◽  
Author(s):  
Leo Polz ◽  
Helmut Schübel ◽  
Joachim Stoekigt
Keyword(s):  
Substrate Selectivity ◽  
Specific Enzyme ◽  
Naturally Occurring ◽  
Optimum Ph ◽  
Relative Molecular Weight ◽  
Inhibition Studies ◽  
Hydrolysis Of ◽  
Activity Point ◽  
Rauwolfia Alkaloid

A novel enzyme was isolated, partially purified (217-fold) and characterized from cell suspen­sion cultures of Rauwolfia serpentina Benth. The enzyme catalyzes one of the late biochemical reactions in the biosynthesis of ajmaline by hydrolysis of 17-O-acetylated alkaloids of the ajmalan group forming the appropriate deacetylated compounds. This esterase exhibits an unusually high substrate selectivity and exclusively accepts acetylated ajmaline derivatives with the naturally occurring 2β(R)-configuration. The properties of the enzyme were determined showing an optimum pH at 7.5, an isoelectric point of pH 4.9 and a relative molecular weight of 33 ± 2 kDa. Inhibition studies of enzyme activity point to the necessity of SH-groups. The esterase seems not to be inhibited by ajmaline. the end product of the pathway. The highest enzyme activities were observed in leaves and cell suspension tissues of the tribe Rauwolfieae which are known to synthe­size ajmaline and its congeners. The specific function of the esterase in the biosynthesis of the later alkaloids was established.

Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell

1994 ◽  
Vol 72 (3-4) ◽  
pp. 157-162 ◽  
Author(s):  
Makoto Arao ◽  
Toru Yamaguchi ◽  
Toshitsugu Sugimoto ◽  
Masaaki Fukase ◽  
Kazuo Chihara
Keyword(s):  
Parathyroid Hormone ◽  
Molecular Mass ◽  
Hydrolytic Activity ◽  
High Molecular Mass ◽  
Phenylmethylsulfonyl Fluoride ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cell ◽  
Hydrolysis Of

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Sue, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from α-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250–300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1–84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.Key words: opossum kidney, parathyroid hormone, chymotrypsin, endopeptidase.

scholarly journals Purification and characterization of a new endoglucanase from Clostridium thermocellum

1992 ◽  
Vol 283 (1) ◽  
pp. 69-73 ◽  
Author(s):  
M P M Romaniec ◽  
U Fauth ◽  
T Kobayashi ◽  
N S Huskisson ◽  
P J Barker ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Reducing Sugars ◽  
Rapid Decrease ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Hydrolysis Of ◽  
Optimal Ph

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Isolation, Purification, and Characterization of the PR Oxidase from Penicillium roqueforti

1998 ◽  
Vol 64 (12) ◽  
pp. 5012-5015 ◽  
Author(s):  
Shenq-Chyi Chang ◽  
Wen-Yee Lei ◽  
Ying-Chieh Tsai ◽  
Yau-Huei Wei
Keyword(s):  
Molecular Mass ◽  
Extracellular Enzyme ◽  
Culture Broth ◽  
Penicillium Roqueforti ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Optimum Ph

ABSTRACT The PR oxidase, an extracellular enzyme, involved in the conversion of PR toxin into PR acid, was purified from the culture broth ofPenicillium roqueforti ATCC 48936. The enzyme has a pI of 4.5 and a molecular mass of approximately 88 kDa, and it is a monomer. The optimum pH for this enzyme is ca. 4.0, and the optimum temperature is 50°C.

Sign in / Sign up

Export Citation Format

Share Document