COMPARISON OF ACTIVITY OF PECTINASE USING AGRICULTURAL WASTE SUBSTRATES A STUDY M.P.KUSUMA* DR M.V.V.CHANDANA LAKSHMI** *Assistant Professor, RBVRR College of Pharmacy, Osmania University, India **Associate Professor, Department of Chemical Engineering, Andhra University, India ABSTRACT This paper deals with the estimation of pectinase activity using Saccharomyces cerevisiae inoculated on two agro-industrial residues, viz., black, and green gram husk alone and in combination with each other as substrates with the addition of nutrient substances to the media. The effect of inducers like Neurobion (a multivitamin solution of vitamins B 1, B 6, B 12 ; E-Merck India Limited) and citrus pectin on enzyme activity was also studied. Activity was estimated by both colorimetric assay and viscometric assay. It was found that pectinase activity was high when the two substrates were used in combination than when single substrate was used and maximum when Neurobion was added as inducer to the two substrates taken in combination. It can be inferred from the results that the husks, used as substrates, supply all the essential nutrients for the growth of the microorganism inoculated. Hence these substrates, which are agro-industrial wastes, can potentially be utilized for cheap and economic production of pectinase in a cheap and economic manner. KEYWORDS: Pectinase, Saccharomyces Cereviseae, Black Gram. INTRODUCTION Pectic substances and celluloses are the most abundant carbohydrates present in plants. Pectic substances like pectin, protopectin and pectic acids, present in cell wall and middle lamella, contribute firmness and structure to plant tissues. Pectins are the structural polysaccharides present in vegetable and fruit cells and are responsible for maintaining the plant tissues integrity. In pectic substances, d-galacturonicacid units are linked together by α- 1, 4-glycosidic linkages and the carbonyl side groups are esterified with methanol (Acuna- Arguelles,1995). This indicates that various forms of pectic substances are present in plant cells and for this reason pectinases exist in various forms. Pectic substances are widely distributed in fruits and vegetables (in turnips, peels of orange and in pulps of tomato, pineapple and lemon), hence they form important natural substrates for pectinases. The pectinases are further sub classified based on the nature of reaction. Endopectinases cleaves the substrate in random fashion while exopectinases cleaves in end-wise fashion 425
(Sathyanarayana et al., 2002). Pectinases are widely used in the food industry to clarify fruit juices and wine, to improve oil extraction, to remove the peel from citrus fruit, to increase the firmness of several fruits and to degum the fibers (Baker and Wicker, 1996; Chang et al., 1994). MATERIALS AND METHODS Microorganism: Saccharomyces cerevisiae isolated from the laboratory and identified by performing biochemical tests. Medium Preparation: The medium of composition given in the Table 1 is prepared in five different conical flasks. To the first flask, 8gm of wheat bran was added. To the second flask, 8gm of green gram were added. To the third flask 8gm each of wheat bran and green gram were added. To the fourth flask, 0.2gm of citrus pectin (0.5%) was added as inducer along with 8gm each of wheat bran and green gram, and To the fifth flask, 0.216 mg of neurobion (27micrograms for 1gram) was added along with 8gm each of wheat bran and green gram. All five flasks were sterilized and a loopful of Saccharomyces cerevisiae culture was inoculated aseptically and incubated at 30 o C for four days in an incubator (Des Raj Kashyap et al.,2003; Pandey et al.,2000). Table 1: Composition of the nutrient medium Materials gm/200ml Ammonium Sulfate 0.02 Magnesium Sulfate hepta hydrate 0.1 Potassium dihydrogen phosphate 0.1 Ferrous Sulfate hepta hydrate 0.0001 Yeast Extract 0.2 Enzyme Extraction To each of the flasks, 50ml of acetate buffer solution (ph4.4) was added. The flasks were then kept in a shaking incubator for 40min at 35 o C at 160rpm.The contents of all the five flasks were then filtered separately using What man filter paper. The filtrates were collected in five different conical flasks which were covered and labeled as M1, M2, M3, M4, M5 respectively corresponding to the order in which the substrates were taken. The residues collected after filtration was concentrated by repeated extraction. The conical flasks in which filtrates was collected were then labeled M1, M2, M3, M4 and M5 respectively as above. The conical flasks were stored in the refrigerator(kobayashi et al.,1996). 426
Estimation of enzyme activity Colorimetric assay Standard curve using d-galacturonic acid: 2ml of d-galacturonic acid solution of 10mg/ml strength was diluted to 100ml to obtain d- galacturonic acid of 0.2mg/ml. This was then confected with the DNS solution prepared earlier and distilled water as shown in table 3. The test tubes were then boiled for 10min, cooled and 10ml distilled water added to each tube.the OD of the contents of each test tube was then measured at 540nm using colorimeter and the standard curve of absorbance at 540nm against concentration d-galacturonic acid was plotted as shown in fig 2 (Des Raj Kashyap,2003). Determination of enzyme activity: 2.5ml of each of the five enzymes stored in the conical flasks labeled M1, M2, M3, M4 and M5 were taken in five different test tubes and enzyme activity was determined. The values noted (table 4). One unit of enzyme activity (U) was defined as the amount of enzyme which releases 1µmol of d-galacturonic acid per minute. Viscometric assay Viscometric assay for determination of activity of enzymes obtained is done by first measuring and noting the time required in the viscometer for each of the enzymes. Then the enzymes obtained are heat-inactivated by taking them in separate test tubes and boiling them in a water bath boiling at 90 degrees centigrade. The values are tabulated in the table 5 The activity is determined using the formula below: A = T a T (1) T a - T o Where A is the activity of the enzyme as already defined, T a is the time for heat-inactivated enzyme in the viscometer (sec), T o is the time for water in the viscometer (sec), T = {Blank Original} in seconds. The results are represented graphically in fig 3. 427
RESULTS AND DISCUSSION The selection of an ideal agro-biotech waste for enzyme production in a solid-state fermentation process depends upon several factors, mainly related with cost and availability of the substrate material. COLORIMETRIC ASSAY Five samples each containing 2.5ml of the extract was taken and enzyme activity was determined using DNS method. It can be seen that the activity of the enzyme labeled M5 (the medium containing wheat bran and green gram as substrate with neurobion as the inducer) is the greatest of all the enzymes obtained. The media M3 and M4 were showing equal and comparable activity. Fig 2: Standard Curve of galacturonic acid Table 4: Table of absorbance of the enzymes at 540nm and concentration of d-galacturonic acid obtained from standard curve: Flask Number Substrate Inducer Absorbance at 540nm M1 Black -- 0.57 0.095 M2 Green -- 0.74 0.023 M3 Black and green -- 0.81 0.135 M4 Black and green Citrus 0.82 0.138 Pectin M5 Black and green Neurobion 0.86 0.143 Concentration of d-galacturonic acid, mg 428
1 0.8 1 Absorbance at 540nm 0.6 0.4 0.2 0 M1' M2' M3' M4' M5' Type of medium used Fig 3: Graphical Representation of Results of Colorimetric Assay: ii) VISCOMETRIC ASSAY: Table 5: Results of viscometric assay Flask Substrate Inducer Time for Time for Activity Number ordinary enzyme, sec heatinactivated enzyme, sec M1 Black -- 46.06 36.59-10.16 M2 Green -- 43.58 46.15 6.381 M3 Black and green -- 40.60 38.38-31.9205 M4 Black and green Citrus 48.59 45.61 6.034 Pectin M5 Black and green Neurobion 47.70 45.60 6.19 CONCLUSION The present investigation relates to a simple, novel, low-cost process for the high-level production of alkaline pectinase from Saccharomyces cereviseae by using agricultural cheap substrate. Although a number of solid substrates have been used for the production of bacterial pectinases, combination of Black with inducer have never been used for pectinase production either in SSF or SmF systems. REFERENCES 1. Sathyanarayana. N.Gummadi, T.Panda, Process Biochemistry 38, 2003, 987-996. 2. Baker R.A., Wicker, L, Trends Food Science and Technology, 7, 1996, 279-284. 3. Chang, T.S., Siddiq, M., Sinha, N.K., Cash, J.N., Journal of Food Science 59, 1994, 1065-1069. 429
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