Glucoamylase Lab Report

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Introduction
Starch-degrading enzymes like glucoamylase are gaining more importance among the industrial enzymes because of the importance of starch, sugars and other products in modern biotechnological era1. Amyloglucosidases also known as glucoamylase hydrolyzes α-1,4 and α-1,6 linkages and produce glucose as the sole end-product form starch related polymers. Glucoamylase have applications for dextrose production, confectionery, baking and in pharmaceuticals2-3. Due to the increasing demand for these enzymes in various industries, there is enormous interest in developing enzymes with better properties such as raw starch degrading amylase suitable for industrial applications and their cost effective production techniques4. Traditionally,
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The sterile medium was inoculated with known volume of inoculum and incubated at different temperatures (25, 30 and 350C) for different periods (48,72 and 96h) of fermentation. After fermentation the mouldy substrate was mixed with distilled water (1:4 w/v), agitated at through cheese cloth followed by centrifugation at 20,000 rpm for 20 minutes. The clear supernatant was used as crude enzyme.
Assay of glucoamylase The assay of glucoamylase was carried out according to the method of Shazia Malik, Tehreema Iftikhar and Ikram Ul Haq13. One unit (U) of glucoamylase is defined as the amount that liberates 1 µmol of reducing sugar as glucose/ml/min under the assay condition.
One ml of the diluted enzyme extract was added to 1.0 ml of 5% soluble starch solution prepared in acetate buffer (pH 4.8). The enzyme substrate mixture was incubated at 60 0 C for one hour. Then 2 ml of Dinitrosalicylic acid reagent (DNS) was added to each test tube. The test tubes were placed in boiling water for 5 minutes and cooled to room temperature. The transmittance of mixture was observed at 540 nm on spectrophotometer. The transmittance was converted to mg of glucose from standard
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Purification of glucoamylase The crude culture filtrate obtained after centrifugation was concentrated 5 times using rotary vacuum evaporator (EYELA Rotary Evaporator N 1000 Japan) followed by precipitation of enzyme at 40C using chilled acetone16. Precipitated protein was collected by centrifugation at 5,000 rpm for 15 mins at 40C. The precipitate was resuspended in 10 ml 0.1 M acetate buffer (pH 4.8) and enzyme activity was measured. DEAE-Cellulose chromatography
10 ml of the resuspended precipitate was applied to the previously equilibrated DEAE-Cellulose column 17. The enzyme was eluted with 0.1 M acetate buffer (pH 4.8) contaning a linear gradient of (0-0.1M) Nacl at a flow rate of 20 ml/h. Eluted fractions (5ml) were analyzed for protein content and enzyme activity. Active fractions were freeze dried and stored at 40C for further studies.
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