scholarly journals Isolation and Activity Determination of Enzyme Phosphatase Secreted by Aspergillus niger

2019 ◽  
Vol 9 (1) ◽  
pp. 41-45
Author(s):  
Tafadzwa Zharare ◽  
Rumbidzai Mangoyi
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Recombinant Dna ◽  
Extracellular Enzyme ◽  
Enzyme Concentration ◽  
Recombinant Dna Technology ◽  
Activity Determination ◽  
Pure Cultures ◽  
Industrial Use

The use of enzymes on industrial scale saves a lot of energy and avoids pollution, thus holding a promise for green and economically sustainable alternative strategies in industrial transformations.  Generally, the fungi Aspergillus niger secretes enzymes which can be used in different industries. Thus, coming up with these enzymes in large amounts will definitely result in reduced costs encountered in importing them for industrial use.  This study focussed on isolation and activity determination of an enzyme phosphatase secreted by Aspergillus niger.  This enzyme can be of great importance in molecular biology industries, particularly for recombinant DNA technology.  For this study, pure cultures of Aspergillus niger were used.  Aspergillus niger was resuscitated on potato dextrose agar and then subcultured in Adam’s medium, a medium specific for the production of phosphatase.  Cells were centrifuged and the filtrate was collected whilst the residue was discarded. The filtrate was expected to contain the crude enzyme phosphatase since Aspergillus niger secretes the extracellular enzyme into the medium. Disodium phenyl phosphate was used as a substrate for the determination of the phosphatase activity. The enzyme activity was determined spectrophotometrically by reading absorbance of phenol formed in the presence of enzyme and the substrate. The concentration of phenol liberated was then used to calculate the enzyme activity expressed in King Armstrong Units (KAU).  Further work on enzyme activity determination was done by varying enzyme and substrate concentrations.  Results showed that the isolated alkaline phosphatase had activity of 4.0 KAU and 4.5 KAU at 25 ºC and 37 ºC respectively. Acidic phosphatase had activity of 5 KAU and 7 KAU at 25 ºC and 37 ºC respectively. Rate of activity increased upon increasing enzyme concentration and substrate.  Thus, Aspergillus niger produces the enzyme phosphatase, however, there is need to induce the production of these enzymes for industrial use.

scholarly journals The effects of Psidium guajava leaf extract on the production of cellulases and glucose oxidases by Aspergillus niger

2020 ◽  
Vol 5 (2) ◽  
pp. 118-122
Author(s):  
Isheanesu Dzambi ◽  
Rumbidzai Mangoyi
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Leaf Extract ◽  
Glucose Solution ◽  
Psidium Guajava ◽  
Absolute Methanol ◽  
Specific Substrate ◽  
Industrial Enzymes ◽  
Industrial Use

Aspergillus niger, is a filamentous fungus and producer of industrial enzymes such as glucose oxidases and cellulases. These enzymes are naturally produced by the fungus in order to digest and absorb nutrients from its environment. However, the enzyme quantities that are naturally produced are low. Thus, the aim of this study was to determine the effects of Psidum guajava leaf extract on the production of cellulases and glucose oxidases from Aspergillus niger. The leaves of Psidium guajava were extracted using absolute methanol. Aspergillus niger was then grown in the presence and absence of the extract in order to investigate the effects of extract on enzyme production by the fungus. The enzymes secreted into the broth medium were isolated by centrifugation. The supernatant which contained the secreted enzymes was used for the determination of enzyme activity. Enzyme activity was determined using the specific substrate for the specific enzyme, such as 2 % cellulose for cellulase and standard glucose solution for glucose oxidase. The results showed that the Psidium guajava leaf extract had an effect on production and activity of cellulases and glucose oxidases. From this study, it was noted that the Psidium guajava leaf extract may be used to induce the production of enzymes by Aspergillus niger and these enzymes may be of industrial use.

ISOLATION AND ACTIVITY DETERMINATION OF ENZYMES SECRETED BY ASPERGILLUS NIGER

2019 ◽  
Vol 2 (1) ◽  
pp. 67-74
Author(s):  
Oleen Machona ◽  
Ronald Mlambo ◽  
Tafadzwa Zharare ◽  
Rumbidzai Mangoyi
Keyword(s):  
Aspergillus Niger ◽  
Activity Determination

Isolation and identification of an extracellular enzyme from Aspergillus Niger with Deoxynivalenol biotransformation capability

2017 ◽  
pp. 742 ◽  
Author(s):  
Shaohua Yang ◽  
Yu Wu ◽  
Jun Yang ◽  
Rong Yan ◽  
Yinghui Bao ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Methyl Esters ◽  
Extracellular Enzyme ◽  
Transformation Product ◽  
Inhibitory Effect ◽  
Isolation And Identification ◽  
Enzyme Form ◽  
Optimized Protocol

In this study, the purification and characterization of an extracellular enzyme form Aspergillus niger was performed. With an optimized protocol, it was conducted a 42.6-fold purification with a yield of 26.2%. The purified lipase had a monomeric molecular weight of 40.5kDa and an isoelectric point of 6.01, and its maximum enzyme activity could be achieved at 40°C and pH 7.5-9.0. The enzyme could be activated by Ca2+, Mg2+ and Fe2+, while its activity could be inhibited by Zn2+ and Cu2+. Additionally, organic compounds exerted an inhibitory effect on the enzyme activity in a descending order of methanol, ethanol, DMSO, EDTA, acetone. Meanwhile, the specificity analysis of the enzyme indicated a preference to tributyrin and vegetable oils as well as long-chain fatty acid methyl esters (C12-C18). Most importantly, this enzyme could successfully transform deoxynivalenol (DON). Using HPLC analysis，it was detected a biotransformation rate of more than 70%.The liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular weight of the transformation product was 18.0 larger than that of DON, indicating that DON could be hydrolyzed by the enzyme. Overall, the proposed method here provides a new avenue for reducing the toxicity of DON, which appears to have a wide application outlook for DON biotransformation.

scholarly journals A RAPID METHOD FOR THE DETERMINATION OF PROTEOLYTIC ACTIVITIES OF ENZYME PREPARATIONS

1948 ◽  
Vol 32 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Bacon F. Chow ◽  
Mary-Ann Peticolas
Keyword(s):  
Enzyme Activity ◽  
Trichloroacetic Acid ◽  
Enzyme Concentration ◽  
Cent Solution ◽  
Enzyme Preparations ◽  
Proteolytic Activities ◽  
Activity Of Enzymes ◽  
Different Sources ◽  
Rate Of Activation

A method has been described for the determination of proteolytic activities of enzyme preparations using casein as substrate. The rate of digestion is proportional to the enzyme concentration used. This relationship is utilized as a measure of the enzyme activity. One unit of activity is defined as the amount which is required to digest casein in 15 minutes at 37.5°C. so that 50 per cent of the protein in 1 ml. of 0.25 per cent solution is not precipitable by trichloroacetic acid. This method has been used to determine the activity of enzymes from different sources and also used to follow the rate of activation of enzymes.

Purification and properties of endo-β-glucanase in the yeast Hanseniaspora valbyensis

1969 ◽  
Vol 15 (7) ◽  
pp. 697-701 ◽  
Author(s):  
Ahmed T. H. Abd-El-Al ◽  
H. J. Phaff
Keyword(s):  
Enzyme Activity ◽  
Cell Walls ◽  
Carbon Sources ◽  
Culture Fluid ◽  
Extracellular Enzyme ◽  
Enzyme Concentration ◽  
Random Pattern ◽  
Ph Optimum ◽  
Hanseniaspora Uvarum ◽  
Purification And Properties

Endo-β-glucanase was detected in Hanseniaspora valbyensis and Hanseniaspora uvarum. The extracellular enzyme activity of H. valbyensis was maximal with vigorous aeration at 30 C in a complete mineral medium with glucose and 0.2 M Na-succinate buffer. The enzyme concentration was lower when the buffer was 0.05 M Na-succinate. The use of a variety of carbon sources, including yeast cell walls, failed to induce higher enzyme activities. The enzyme was purified 34-fold from the culture fluid of H. valbyensis. The purified enzyme had a broad pH optimum between pH 3.5 and 4.5. Among the known β-glucan homopolymers, only laminarin (β-1 → 3 bonds) was hydrolyzed by the enzyme. The Km for laminarin was 0.11 mg/ml and the Vmax was 0.835 μmoles glucose equivalents/min mg protein. No action was detected with laminaribiose, laminaritriose, or oat glucan. Laminaritetraose, however, was hydrolyzed slowly in a random pattern. These results suggest the requirement for three consecutive β-1 → 3 bonds for activity.

Enzyme-Based Most Probable Number Method for the Enumeration of Bifidobacterium in Dairy Products

2001 ◽  
Vol 64 (12) ◽  
pp. 2001-2006 ◽  
Author(s):  
RODRIGO BIBILONI ◽  
ANDREA GÓMEZ ZAVAGLIA ◽  
GRACIELA DE ANTONI
Keyword(s):  
Dairy Products ◽  
Accurate Determination ◽  
Most Probable Number ◽  
Commercial Products ◽  
Probable Number ◽  
Selective Media ◽  
Activity Determination ◽  
Pure Cultures

An enzyme-based assay in combination with the most probable number (MPN) technique was developed for the enumeration of bifidobacteria. The assay employs the detection of fructose-6-phosphate phosphoketolase (F6PPK) activity as an indicator of the presence of bifidobacteria. The method was validated against viable counts and optimized with respect to selective media in order to quantitatively assess bifidobacteria in dairy products and other probiotic preparations. Several commercial products and homemade fermented milks were analyzed. Counts of bifidobacteria ranged from 107 to 108 MPN/ml in commercial products and homemade fermented milks. Commercial starters provided by Argentinean industries had between 107 and 1011 MPN/ml. The results obtained in this study suggest that the combination of F6PPK activity determination and the MPN methodology allows an accurate determination of Bifidobacterium in pure cultures, dairy products, and other probiotic preparations.

Case Applications

1993 ◽  
Keyword(s):  
Recombinant Dna ◽  
Family Relationship ◽  
Recombinant Dna Technology ◽  
Animal Science ◽  
Animal Remains ◽  
Service Load ◽  
Hard Evidence ◽  
Application Potential ◽  
Dna Profiles

A number of forensic and family relationship cases, as well as medical, animal science, wildlife poaching investigation, and plant science applications are presented in this chapter. As suggested by the titles and headlines from various journal and newspaper articles, the process of identification using recombinant DNA technology has proven to be very practical. Semen from a rape case, a hair follicle from a homicide, blood stains at a break-in, chorionic villi from a prenatal diagnosis, blood cells from a transplant patient, tumorous tissue, a big game animal gut pile, a freezer steak, rare condor blood, a whale skin biopsy, plant tissue, and ancient human and other animal remains are some of the sources of DNA used for typing. Perhaps the most apparent indicator of application potential can be deduced from the number of recent patent applications covering recombinant DNA processes and products. In addition, many new government and commercial ventures have been established to accommodate the anticipated service load. The analysis of DNA is providing hard evidence for the resolution of serious criminal acts and other difficult identification problems in homicide, rape, accident, missing persons, break-ins, and hit-and-run cases (Anderson 1989; Barinaga 1989; Conner 1988; Dodd 1985; Fowler 1988; Fox 1989; Fukushima 1988; Gill 1987; Giusti 1986; Hicks 1989; Higuchi 1988; Hewlett 1989; Jeffreys 1988; King 1989; Kobayashi 1988; Lander 1989a; Lewin 1989; McElfresh 1989; Marx 1988; Merz 1988; Newmark 1987, 1987a; Norman 1989; Ross 1989; Taylor 1989; Yokoi 1989). The determination of whether a series of crimes is serial or copycat, that is, committed by one or more than one perpetrator, is critical to the investigation of many cases. If DNA profiles match for specimens from different crime sites, this suggests that the same individual was involved and investigators can then concentrate their efforts on the hunt for one person. The forensic scientist first prepares a DNA identity profile of the crime (evidence), suspect, and victim specimens.

An Automated Method for Measuring Creatine Phosphokinase Activity in Serum

1970 ◽  
Vol 16 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Ronald K Wright ◽  
Roy L Alexander
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Creatine Phosphokinase ◽  
Creatine Phosphate ◽  
Enzyme Concentration ◽  
Magnesium Ion ◽  
Manual Method ◽  
Automated Method ◽  
Automated Procedures

Abstract We describe a procedure for automating the determination of creatine phosphokinase (CPK) activity in serum by use of the AutoAnalyzer. Enzyme activity is determined by measuring the creatine phosphate formed from the CPK-catalyzed reaction of creatine with adenosine triphosphate (ATP). The sensitivity of the automated procedure was comparable to that of the manual method. By use of the most favorable concentrations of creatine, ATP, and magnesium ion in the substrate, a linear relationship was obtained between enzyme concentration and enzyme activities up to 600 mU, representing a sixfold improvement over that obtained by the manual method. The degree of correlation between results obtained by the manual and automated procedures is shown.

scholarly journals A method for determining kinetic parameters at high enzyme concentrations

1982 ◽  
Vol 203 (1) ◽  
pp. 339-342 ◽  
Author(s):  
C J Halfman ◽  
F Marcus
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Graphical Method ◽  
Free Ligand ◽  
Enzyme Concentration ◽  
Fructose 1,6 Bisphosphatase ◽  
High Enzyme ◽  
Substrate Inhibitor ◽  
The Relationship

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.

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