scholarly journals An inducible hydrolase from Aspergillus niger, acting on carbon–carbon bonds, for phlorrhizin and other C-acylated phenols

1970 ◽  
Vol 116 (5) ◽  
pp. 889-897 ◽  
Author(s):  
T. Minamikawa ◽  
N. P. Jayasankar ◽  
B. A. Bohm ◽  
I. E. P. Taylor ◽  
G. H. N. Towers
Keyword(s):  
Aspergillus Niger ◽  
Metal Ion ◽  
Deae Cellulose ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Protamine Sulphate ◽  
Carbon Carbon Bonds ◽  
Alkaline Conditions ◽  
Km Value ◽  
Ammonium Sulphate Fractionation

1. An inducible enzyme catalysing the hydrolysis of phloretin to form phloroglucinol and phloretic acid has been extracted from the acetone-dried powders of the mycelial felts of an Aspergillus niger strain grown in the presence of phlorrhizin. The enzyme was partially purified by treatment with protamine sulphate, ammonium sulphate fractionation, negative adsorption on tricalcium phosphate gel, and DEAE-cellulose column chromatography. 2. The hydrolytic activity on phloretin appeared to be maximal at about pH9.6. However, the characteristics of the enzyme were studied at pH7.2, because of the lability of the product, phloroglucinol, under alkaline conditions. 3. The apparent Km value at pH7.2 was about 0.3–0.4mm for phloretin and 0.15mm for 3′-methylphloracetophenone. 4. Maximum activity of the enzyme was obtained without the addition of any cofactor or metal ion. The involvement of thiol groups in the reaction was demonstrated by the potent inhibitory action of both heavy-metal ions and p-chloromercuribenzoate. 5. The enzyme showed a rather broad substrate specificity, and some other C-acylated phenols related to phloretin were hydrolysed. It was found that 3′-methylphloracetophenone, phloracetophenone and 2′,4,4′-trihydroxydihydrochalcone were attacked more efficiently than phloretin. We propose the systematic name C-acylphenol acylhydrolase for the enzyme. This enzyme belongs to EC group 3.7.1.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals Free and Immobilised β-Glucosidases in Oenology: Biotechnological Characterisation and Its Effect on Enhancement of Wine Aroma

2021 ◽  
Vol 12 ◽  
Author(s):  
Pilar Fernández-Pacheco ◽  
Beatriz García-Béjar ◽  
Ana Briones Pérez ◽  
María Arévalo-Villena
Keyword(s):  
Volatile Compounds ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Maximum Temperature ◽  
Wine Aroma ◽  
High Concentration ◽  
Aroma Precursors ◽  
Km Value ◽  
Immobilised Enzymes ◽  
Optimal Ph

In grapes, monoterpenes and norisoprenoids are in the form of non-volatile compounds, flavourless glycosides which could enhance the aroma of wines after its hydrolysis using β- glucosidases enzymes. It is known that the use of immobilised enzymes offers advantages such as reusability and easy recuperation. In this study, a commercial β-glucosidase was immobilised by absorption in sodium alginate. Biotechnological characteristics and terpen hydrolysis (hydrolysis aroma precursors) in muscat wines were studied after treatment with both free and immobilised commercial β- glucosidase with two different concentrations. It was revealed that both forms shared an optimal pH (4.5) and a maximum temperature (64°C), even an increment on the activity between 40and 60°C. A similar Km value has been determined while Vmax from the immobilised enzyme was higher than the free (3.35 and 2.52 μmol min–1 mg–1, respectively). Additionally, the immobilised enzyme showed a better hydrolytic activity during 24 h, and its reusability has been proven. Regarding enzymatic hydrolysis in grape must, the best results were observed for the highest concentration of free β-glucosidase although glucose release was also determined for the immobilised enzyme along the days. In contrast, maximum activity was reached by the immobilised β-glucosidase in less time but in no case equalled the free ones. Finally, volatile compound liberation in wines treated with free or immobilised enzymes was analysed using HRGC-MS. Liberation for both enzymes and the greatest concentrations of some volatiles were detected when a double dose of the free β-glucosidase was used. Nevertheless, the wines treated with the immobilised β-glucosidase showed a high concentration of some volatile compounds such as nerol or geraniol.

PURIFICATION AND PROPERTIES OF PHOSPHOGLUCOSE ISOMERASE FROM ASPERGILLUS NIGER

1959 ◽  
Vol 37 (8) ◽  
pp. 927-932 ◽  
Author(s):  
Kartar Singh
Keyword(s):  
Calcium Phosphate ◽  
Aspergillus Niger ◽  
Metal Ions ◽  
Maximum Activity ◽  
Phosphoglucose Isomerase ◽  
Muscle Enzyme ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Ammonium Sulphate Fractionation ◽  
Kinetics Of

Phosphoglucose isomerase from Aspergillus niger was purified about fiftyfold by ammonium sulphate fractionation and treatment with calcium phosphate gel. The properties and kinetics of the enzyme are described. The enzyme resembled the muscle phosphoglucose isomerase in not requiring metal ions for activation but differed in showing no inhibition with phosphate. The enzyme showed maximum activity between pH 7.8 to 8.0 as compared with the optimum pH 9.0 shown by the muscle enzyme.

scholarly journals Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic Endomycopsis fibuligera Using Sago Starch as a Substrate

2018 ◽  
Vol 16 (3) ◽  
pp. 302
Author(s):  
Ahyar Ahmad ◽  
Harningsih Karim
Keyword(s):  
Ammonium Sulphate ◽  
Deae Cellulose ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Degree Of Saturation ◽  
Sago Starch ◽  
Purification And Characterization ◽  
Ammonium Sulphate Fractionation ◽  
Crude Extract Enzyme

An investigation on purification and characterization of amyloglucosidase enzyme from Endomycopsis fibuligera by fermentation of sago starch has been carried out. This enzyme is inductive and can be produced by fermenting sago starch in a medium containing E. fibuligera. Crude enzyme was obtained by centrifuging the medium cultures containing E. fibuligera at 6,000 rpm for 20 min and then adding with 0.15 M acetate buffer (pH 5.0). Enzyme activity was determined using Somogyi-Nelson method by quantifying the released glucose from amyloglucosidase catalysis of starch (0.2%) as substrate. Prepurification process was conducted by ammonium sulphate fractionation and it showed that the ammonium sulphate fractionation with the degree of saturation of 40-60% produced a maximum activity of enzyme. Purification by DEAE-Cellulose and Sephadex G-75 column chromatography produced three and one fractions with purifity 17.4 and 22.5 times, respectively, compared to the crude extract enzyme. Characterization of this enzyme showed the optimum condition at pH 5.0 and 55 °C with 0.2% starch as substrate. The amyloglucosidase activities was strongly increased by addition of Co2+ and Mn2+ ions, whereas the activities were weakly decreased by addition of K+, Mg2+, and Fe3+ ions.

scholarly journals Salinity endurance of marine macro Rhodophycean algae with special emphasis on myo-inositol biosynthesis: An enzymological analysis from Halymenia venusta Børgesen

2021 ◽  
pp. 30-39
Author(s):  
Dibyendu Sekhar Mahanty ◽  
Sautrik Basu ◽  
Jukta Adhikari
Keyword(s):  
Divalent Cations ◽  
Deae Cellulose ◽  
Monovalent Cations ◽  
Present Communication ◽  
Inositol 1 ◽  
Marine Plants ◽  
Stress Relieving ◽  
Km Value ◽  
Ammonium Sulphate Fractionation ◽  
Intertidal Habitats

Altered salinity is one the most important perils encountered by marine plants inclusive of algae. Under hyper saline condition plants accumulate several stress relieving osmolytes including myo-inositol, the most widespread cyclitol in plants. The present communication reports the occurrence of myo-inositol biosynthesis in six different Rhodophycean seaweeds growing under stressful intertidal habitats of the Okha coast (Gujarat, India), on the basis of a study conducted on two marker enzymes of myo-inositol biosysnthesis [L-myo-inositol-1-phosphate synthase and D/L-myo-inositol-1-phosphate phosphatise]. Both enzymes were partially purified from Halymenia venusta to about 27 and 39 folds respectively over the homogenate following low-speed centrifugation, 30-75% ammonium sulphate fractionation, successive chromatography through DEAE-cellulose / CM-Cellulose, Sephadex G-200 and BioGel 0.5m / UltrogelAcA 34 columns. The temperature and pH optima for both the enzymes were similar and were recorded to be 350C and 7.5 respectively. For MIPS, D-glucose-6-phosphate and NAD were the exclusive substrate and coenzyme respectively and D/L-MIP was the sole substrate for MIPP. The Km values for D-glucsoe-6-phosphate and β-NAD were recorded to be 3.599 mM and 0.2366 mM respectively, while the Km value for D-MIP was found to be 0.4070 mM. Monovalent cations K+ had slight stimulatory, Li+ was strong inhibitory for both the enzymes. Divalent cations Ca2+ exhibited slight stimulatory and Cd2+ reduced MIPS and MIPP activities. MIPP was stimulated by Mg2+. Cu2+ and Hg2+ were strong inhibitors of both the enzymes. A steady and proportionate increase in the content of free myo-inositol was observed along with elevated levels of recorded salinity.

1-Methyladenosine Hydrolase of Starfish (Pisaster ochraceous)

1973 ◽  
Vol 30 (12) ◽  
pp. 1861-1866 ◽  
Author(s):  
H. L. A. Tarr
Keyword(s):  
Deae Cellulose ◽  
Maximum Activity ◽  
Half Maximum ◽  
Enzyme Protein ◽  
Freezing And Thawing ◽  
Phosphorylase Activity ◽  
Ph Optimum ◽  
Ammonium Sulphate Fractionation ◽  
Noncompetitive Inhibitors

A nucleosidase specific for 1-methyladenosine was purified up to 42-fold from mature testes of starfish, and less extensively from mature ovaries, stomach tissue, and pyloric caeca, by extraction, ammonium sulphate fractionation, and DEAE-cellulose chromatography. The enzyme, the products of which were 1-methyladenine and D-ribose, was free from nucleoside phosphorylase activity and had the following properties. The pH optimum was 7.8–8.0 with half maximum activity at pH 6.2 and 9.5; the enzyme was rapidly inactivated at 40 C and by freezing and thawing; of 10 purine and pyrimidine ribonucleosides and deoxyribonucleosides studied, only 1-methyladenosine was hydrolyzed; the Km was 6.65–7.15 × 10−4 M; 1-methylinosine and 1-methylguanosine were noncompetitive inhibitors; the Ki for these were 1.25 × 10−4 M and 1.4 × 10−4 M, respectively. Vmax values of from 6.3–10.4 μmoles of 1-methyladenosine hydrolyzed per mg of enzyme protein per hr at 25 C were calculated. It is not known if this enzyme plays a role in the in vivo formation of 1-methyladenine, which induces maturation of starfish oocytes.

PURIFICATION AND PROPERTIES OF PHOSPHOGLUCOSE ISOMERASE FROM ASPERGILLUS NIGER

1959 ◽  
Vol 37 (1) ◽  
pp. 927-932 ◽  
Author(s):  
Kartar Singh
Keyword(s):  
Calcium Phosphate ◽  
Aspergillus Niger ◽  
Metal Ions ◽  
Maximum Activity ◽  
Phosphoglucose Isomerase ◽  
Muscle Enzyme ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Ammonium Sulphate Fractionation ◽  
Kinetics Of

Phosphoglucose isomerase from Aspergillus niger was purified about fiftyfold by ammonium sulphate fractionation and treatment with calcium phosphate gel. The properties and kinetics of the enzyme are described. The enzyme resembled the muscle phosphoglucose isomerase in not requiring metal ions for activation but differed in showing no inhibition with phosphate. The enzyme showed maximum activity between pH 7.8 to 8.0 as compared with the optimum pH 9.0 shown by the muscle enzyme.

scholarly journals Pemurnian dan Karakterisasi Enzim Lipase dari Aspergillus oryzae pada Kopra Berjamur

2012 ◽  
Vol 14 (1) ◽  
pp. 26
Author(s):  
Seniwati Dali Dali ◽  
Abdul Rauf Patong ◽  
Muhammad Noor Jalaluddin ◽  
Pirman Andi Parenrengi
Keyword(s):  
Ammonium Sulphate ◽  
Aspergillus Oryzae ◽  
Enzyme Purification ◽  
Maximum Activity ◽  
Crude Enzyme ◽  
Lipase Catalysis ◽  
Lipase Enzyme ◽  
Km Value ◽  
Ammonium Sulphate Fractionation

An investigation on purification and characterization of lipase enzyme production Aspergillus oryzae from copra by fermentation of oliveoil has been carried out. This enzyme can be produced by fermenting olive oil in a medium containing Aspergillus oryzae. Crude enzyme isobtained by centrifuging the medium cultures containing Aspergillus oryzae at 3500 rpm for 30 minutes and than adding 0.2 M borat buffer(pH 8.2). Enzyme activity was determined from paranitrophenol as product of lipase catalysis of paranitrophenylbutirat (0.2 M) assubstrate measured by the Vorderwulbecke method. Prepurification process was by ammonium sulphate fractionation. Precipitation60-80% ammonium sulphate produced maximum activity of enzyme. Purification by Q sepharosa FF and sephadex G-75 collumchromatography produced four and three fractions with purifity of 12.85 and 20.25 times than crude enzyme respectively. Characterizationof this enzyme showed optimum condition at pH 8.2, temperature at 350C, the Km value at 0.046 M, and Vmaks is 1.926 μmol/menit and themolecular weight at 40.7 kDa.

Kinetic Behaviour of Amylase According to pH: A New Perspective for Starch Hydrolysis Process

2020 ◽  
Vol 16 (2) ◽  
pp. 135-144
Author(s):  
Ravneet K. Grewal ◽  
Baldeep Kaur ◽  
Gagandeep Kaur
Keyword(s):  
Metal Ions ◽  
Competitive Inhibition ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Cost Effective ◽  
Starch Hydrolysis ◽  
Divalent Metal Ions ◽  
Activity Data ◽  
Time Saving ◽  
Alkaline Conditions

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency. Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH. Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot. Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5. Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

scholarly journals Characterization of a Solvent-Tolerant Amidohydrolase Involved in Natural Product Heterocycle Formation

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 892
Author(s):  
Lea Winand ◽  
Dustin Joshua Vollmann ◽  
Jacqueline Hentschel ◽  
Markus Nett
Keyword(s):  
Natural Product ◽  
Metal Ion ◽  
Building Blocks ◽  
Maximal Activity ◽  
Kinetic Properties ◽  
Pharmaceutical Drugs ◽  
Highly Active ◽  
Km Value ◽  
Amidohydrolase Superfamily ◽  
Solvent Tolerant

Heterocycles are important building blocks in pharmaceutical drugs and their enzymatic synthesis is attracting increasing interest. In recent years, various enzymes of the amidohydrolase superfamily were reported to catalyze heterocycle-forming condensation reactions. One of these enzymes, MxcM, is biochemically and kinetically characterized in this study. MxcM generates an imidazoline moiety in the biosynthesis of the natural product pseudochelin A, which features potent anti-inflammatory properties. The enzyme shows maximal activity at 50 °C and pH 10 as well as a kcat/Km value of 22,932 s−1 M−1 at its temperature optimum. Experimental data suggest that the activity of MxcM does not depend on a catalytic metal ion, which is uncommon among amidohydrolases. MxcM is highly active in diverse organic solvents and concentrated salt solutions. Furthermore, we show that MxcM is also capable to introduce imidazoline rings into derivatives of its natural substrate myxochelin B. Overall, MxcM is a solvent-stable, halotolerant enzyme with promising biochemical and kinetic properties and, in future, might become a valuable biocatalyst for the manufacturing of pharmaceutical drugs.

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