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In this experiment, the focus is on isolation of pectinase producing fungi . For isolation purpose, nutrient agar and potato dextrose agar were prepared. Potato dextrose agar was used especially for isolation of fungi. This is because its pH of 3.5-4.0 does not permit normal bacterial growth. However, yeast and fungi are able to grow under such an acidic condition. In this experiment, three types of fungal isolates were obtained. These three isolates were subjected to pectinase -screening test. The purpose of screening is to detect the presence of pectinase -producing microorganism (fungi for this experiment). Two screening tests: Primary and secondary screenings were carried out over the isolates in order to test their ability to produce pectinase qualitatively and quantitatively. Note: The identities of the three isolates are unknown. However, they are differentiated based of the colonial morphology such appearance of colony, color of pigmentation, etc.
Primary screening is used to test the microorganisms¶ ability to produce pectinase qualitatively. This means the result of this screening will only show positive(capable of producing pectinase) or negative(incapable of producing pectinase). For the positive result, the amount of pectinase produced by a microorganism is indefinite. Differential solid growth medium (agar medium) was used for primary screening. In secondary screening, the quantity or amount of enzyme pectinase produced or amount of substrates formed by enzyme degradation were measured via special method such as DNS method/spectrophotometry. The quantity of enzyme production is very important as it allows the comparison between microorganisms of which one can produce the higher titer of enzyme p ectinase. In this experiment, the DNS method which incorporates spectrophotometric method and submerged fermentation was employed.
The medium used is considered to be differential as it distinguishes pectinase produce r from nonpectinase producer by formation of halo zone around the fungal colonies. Without centrimide application on the surface of agar medium. there is no observable change can be seen. CTAB) was used to observe the halo zone around the fungal colonies. This means the fungus is cultivated in the shake flask with appropriate liquid medium (mineral salt medium without agar powder). there is requirement of measuring the quantity/amount of enzyme produced in g/ml or U/ml. In primary screening. Only isolate «««. therefore no precipitation of pectin occurs.PRIMARY SCREENING In this experiment. presumptively. Nevertheless. However. . It needs the isolation of enzyme produced from submerged fermentation of the fungus. Thus. SECONDARY SCREENING In secondary screening. It is believed that enzyme pectinase will be released into the medium. From the result of primary screening. The purpose of centrimide application is to precipitate the pectin present in the agar medium. Note: Centrimide is cation surfactant that can form micelles in aqueous solution. only the fungus that formed the largest halo zone among the three isolates was chosen for further testing in secondary screening. The precipitation renders the formation of cloudiness (increas e of opacity) throughout the agar medium. However. This is because the degradation of pectin around the colony does not react with centrimide. It interacts with pectin to form a larger colloid in the aqueous solution. centrimide (cetyltrimethylammonium bromide. if there is pectinase production (or pectin degradation) around the fungal colonies.(forgot the code) was chosen due to the largest halo zone it formed. the size of the halo zones were not considered as quantitative measure because the re is no exact/ direct measurement of the enzyme concentration produced in g/ml. all the three fungal isolates were screened primary using mineral salt medium supplemented with pectin as carbon source for growth. clear halo zone is observed. This also means it can produce highest amount pectinase than the other two isolates. all three isolates were capable of producing pectinase.
(NOTE: The residue of the culture is used in biomass analysis) In testing enzyme activity. the concentration of enzyme produced is calculated by using formula as below: Note: enzyme concentration produced in a unit of time(min) and volume(ml) named as enzyme activity more accurately. This means DNS assay is used to measure only the amount of substrate (but not other substances produced in the liquid culture) . Enzyme activity = [C] x F V x Mg x t [C] = concentration of substrate/galacturonic acid(g/ml) F = dilution factor V = volume(ml) Mg = Molar mass of galacturonic acid (g/µmol) t = incubation time (min) Enzyme activity (U/ml) is defined as the amount of enzyme that releases 1 µmol of galacturonic acid in a unit time (min) and volume(ml). DNS (3. .5-dinitrosalicyclic acid) method was employed. However. This method indirectly measure amount of enzyme pect inase produced through assessing the quantity of substrate (degraded pectin/galacturonic acid formed). DNS versus GALACTURONIC ACID DNS is an aromatic organic compound that can react with any reducing sugar to form 3-amino-DNS(3-amino-5-nitrosalicyclic acid) that absorb light strongly at 540 nm. Since the substrate (galacturonic acid) isanreducing sugar. --------------. In another words.(1) is . we are correlating the substrate concentration to enzyme pectinase concentration.the liquid of the culture is collected via filtration for testing the enzyme activity. it can react with the DNS to produce product that can be measured spectrophotometrically at 540 -570 nm.
the OD . b. a slight different in the substrate concentration can change the OD value greatly.STANDARD GRAPH OF OD (nm) VS GALACTURONIC ACID (g/ml) DNS assay only gives result with OD(nm) value that is correlated to concentration of galacturonic acid.(2) y = [OD] = spectrophotometric value at wavelength 570 nm m = gradient of the straight line x = [C] = concentration of galacturonic acid c = value of y at x equals to zero (line that cuts y -axis) With this equation. This is because the medium may consist of traces of pectin which is not used in the fungal growth. [C] produced on different day can be calculated by substituting the value of y or [OD] into the equation. The graph of straight line is obtainable with equation [OD] = m[C] + c (y = mx + c) with:---------. The OD values may not be the exactly represent the substrate conce ntration from the enzymatic reaction. Since the spectrophotometer is a very sensitive probe. a standard graph of OD versus galacturonic concentration is plotted. Therefore. In order to get any value for galacturonic acid concentration. the concentration of galacturonic acid. ANALYSIS OF GROWTH PROFILE (BIOMASS) AND ENZYME ACTIVITY POSSIBLE ERRORS The possible errors exist in this experiment that can reduce the accuracy of the result/reading:a. The volume of substrate or enzyme pippetted may vary.
. Thus. the more substrate will be produced from pectin degradation.reading may be resulted from degradation of pectin remains in the medium and pectin that we added in latter. There are other errors may occur throughout the experiment but of those that ca n affect enzyme activity (secondary screening ) are stated above. The incubation time may be delayed slightly but it is not taken into consideration. The longer of incubation time. this can affect the OD value as well (see equation 1 above). The incubation time is somewhat very important as it can affect the enzymatic activity. c.
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