The production of extracellular amylase was investigated employing our laboratory isolate,
Microbial enzymes are widely used in several industries, notably in detergent, food processing, brewing, and pharmaceuticals [
Amylases are starch-degrading enzymes that catalyze the hydrolysis of internal alpha 1-4 glycosidic bonds in polysaccharides with the retention of alpha anomeric configuration in the products [
To overcome such limitations, great attention has been devoted for studies on amylases to tackle the problem [
The overall objective of the present study was to isolate a novel
Isolation of the
The soil suspension was diluted up to 10−3–10−6 times, and 0.5 mL of each diluted suspension was then transferred by spread plate method with a sterile glass spreader on petri plates containing potato dextrose starch agar medium (PDSA). These petri plates were incubated at 30°C for 4-5 days. Based on the zone of clearance on the starch agar plates, 13 colonies were picked up, and individual amylase activity of selected colonies was, investigated carried out and strain MK 07 showing good activity was used for further studies. The young colonies of fungal cultures were aseptically picked up and transferred to potato dextrose starch agar slants with 1% starch. These slants were then incubated at 30°C for 4 days, and after sufficient growth, they were stored at 4°C in the refrigerator till further use.
Actively growing and heavily speculating ten-day-old potato dextrose starch agar slant culture was added to 10 mL sterile 0.85% sodium chloride salt solution. The spores were gently scraped off with the help of a sterile needle, and contents were passed through glass wool so as to obtain spore inoculums free from mycelial bits. A volume of one mL of spore suspension contained more than 2.6 × 106 spores.
The activity of the alpha amylase was determined by the Bernfeld [
Maltose was used as standard reference for amylase activity. One mg/mL maltose solution was prepared and used as stock solution. Ten appropriate dilutions from 0.1 to 1.0 were made from standard stock solution. One mL of each dilution and 1 mL of DNS solution was added in each test tube, and blank was made with 1 mL of distilled water and 1 mL of DNS solution. These tubes were placed in boiling water bath for 5 mins and cooled to room temperature. The contents of the test tubes were diluted up to 10 mL with distilled water. All the tubes were mixed well, and optical density of the solution was measured at 540 nm. A standard curve was constructed taking concentration of maltose (mg/mL) on
The protein content of the enzyme preparations was estimated by the Lowry method using Bovine serum albumin as standard. One mg/mL stock solution is prepared, and from that stock solution various dilutions ranging from 0.1 to 1.0 mg/mL were prepared, and standard plot was performed. From each dilution, 0.2 mL of solution was taken in different test tubes, and to each tube 2 mL was added of alkaline copper sulphate reagent, and this mixture was mixed well and incubated at room temperature for 10 mins. Then 0.2 mL of the Folin-Ciocalteu reagent was added to each tube and incubated for 30 mins. After incubation, absorbance was read at 595 nm in an UV-Vis spectrophotometer. Then a standard graph was plotted with concentration on
After 48 hrs of incubation period, the culture broth from fermentor was collected in a sterile conical flask. To 250 mL of culture broth, 100 mL of phosphate buffer (0.02 M) and 0.02% tween 80 was added. The contents in the flasks were equally distributed into 250 mL centrifuge tubes, and these tubes were centrifuged in the centrifuge machine at 5000 ×g for 30 minutes. The substrate-free suspension was used for the estimation of alpha amylase [
The isolated
All enzyme purification steps were carried out at 0 to 4°C.
The crude broth obtained after fermentation was centrifuged at 5000 ×g for 30 mins to remove the cell biomass. Solid ammonium sulphate was added slowly to the culture supernatant to get 60% saturation, stirred for 60 min, and left for overnight at 4°C. The precipitate was harvested by centrifugation at 10,000 ×g for 10 min, dissolved in 50 mM glycine-sodium hydroxide buffer and dialyzed against same buffer overnight (4°C). The dialyzed sample was then assayed for amylase activity and glucose content [
Dialyzed enzyme was loaded on to a column of sephadex G-100 (1.5 × 90 cm) previously equilibrated with 50 mM glycine-sodium hydroxide buffer (pH 11) and then eluted at a flow rate of 10 mL/h with the same buffer containing sodium chloride gradient from 0.1 to 1 M, and 1 mL volume fractions were collected. The absorbance of fractions was checked at 600 nm. Those fractions that showed absorbance were assayed for amylase activity with starch as substrate. Amylase-active fractions were pooled and concentrated for further characterization [
The pH optimum for purified amylase was assayed by analyzing its activity in the pH range of 5 to 12 using starch as a substrate and buffer systems of 0.05 moL L−1 phosphate buffer for pH 5.0 to 7.5, tris-HCl for pH 8.0 to 9.0, glycine-sodium hydroxide for pH 9.5 to 11.0, sodium phosphate for 11.5 to 12.0, and sodium carbonate for 12.5 to 13.0. pH stability study of the protein was analyzed by preincubating 5 mL of purified enzyme in 3.5 mL of selected pH buffer at 37°C for 1 to 48 h and subsequent analysis of residual activities under standard assay conditions.
To study the temperature optima of enzyme, the enzyme reaction mixture was incubated at different temperatures ranging from 35°C to 75°C in glycine-sodium hydroxide buffer (pH 11.0), and amylase activity was measured using starch (1%) solution as substrate. For determining thermal stability, the enzyme was preincubated for 1.0 h at different temperatures ranging from 35 to 90°C, and the relative activity was measured under standard assay conditions after incubating with starch as substrate [
Thermal inactivation assays were carried out by preheating 950
A clean and stiffed cloth with starch over that was used for the study. Equal sizes of pieces were used for the study, and they were weighed on an electric balance (5 × 5 inch). The cloth strip was then dipped in 100 mL of enzyme solution (pH 6.5) and then placed in the incubator at 70–80°C for 1.0 hr. After the time interval, the cloth strip was washed with tap water and then oven-dried. After drying, the cloth strip was again weighed.
The following parameters were studied for desizing of the cotton cloth.
The effect of enzyme concentration on the desizing of the cotton cloth by crude and partially purified enzyme was investigated. The concentration of the enzyme was varied from 100 to 500 U/mL/min.
The effect of temperature (25–50°C) on the desizing of cotton cloth by crude and partially purified enzyme was studied.
The effect of different pH (4.0–9.0) on the desizing of cotton cloth by crude and partially purified enzyme was studied.
The rate of desizing of cotton cloth at various time intervals by crude and partially purified alpha amylase was estimated. The cloth was treated with crude and partially purified enzymes at 80°C for 75 mins.
Kinetic study and volumetric rates of enzyme formation and biomass revealed that the mediums containing sucrose as carbon source gave better results compared to other medium tested. The medium used for amylase production consists of (g/L) sucrose 30; corn steep liquor 20; magnesium sulphate 0.5; potassium chloride 0.5; potassium phosphate 1; ferrous sulphate 0.01; peptone 5. Hence further fermentor studies were carried out with the above-mentioned medium for amylase production by the isolated
The following parameters were evaluated and optimized for amylase production by the isolated
The effect of fermentor volume on the production of alpha amylase by isolated MK 07 strain was evaluated. Maximum production of 1675 U/mL was achieved when the reactor volume was kept at 70%. As the volume of the fermentor media was increased, the enzyme production was decreased. The kinetic values of Y p/x, and Qp were also found to be significant when the volume of the fermentor was maintained at 70%. When the medium volume percentage in the fermentor was increased to 80%, a decrease in amylase production was obtained. Most of the fermentation studies for enhanced amylase production were carried out by synthetic media [
Effect of volume of media on amylase activity.
Effect of varying rate of agitation was investigated for alpha amylase production in fermentor by the isolated strain. The fermentation was carried out at 150, 200, 250, and 300 rpm. The production of enzyme following growth of the organism was found to be maximum of 1734 U/mL at an rpm of 250. When the agitation rate of the fermentation broth was increased above 250 rpm, a decrease in enzyme production was observed. Hence, for further studies, the rpm was maintained at 250 as shown in Figure
Effect of different RPM on amylase activity.
Effect of different range of air supply (0.5–2.5 vvm) to the fermentation medium for the production of alpha amylase by the isolated strain MK 07 was studied. The production of alpha amylase enzyme following growth of the organism was optimum and maximum at 2.5 vvm (1576 U/mL), and further increase or decrease in air supply decreased the enzyme production. The production of amylase enzyme steadily increased with increase in air supply, indicating the isolated strain is an aerobic organism as shown in Figure
Effect of air supply on enzyme activity.
Effect of different sizes of inoculum on the production of alpha amylase by the isolated strain MK 07 was investigated. The production of enzyme following growth of the organism was increased with increase in inoculum volume up to 10%, and upon further increase in inoculum volume, enzyme production decreased, and the maximum amylase production obtained was 1691 U/mL as shown in Figure
Effect of different inoculum percentages on enzyme activity.
Effect of different pH on the production of alpha amylase by the isolated strain Mk 07 was investigated. The production of enzyme increased with increase in pH up to 5.0, and upon further increase in pH, the enzyme production decreased drastically, and the maximum amylase production obtained at a pH of 5.0 was 1723 U/mL, and the results are shown in Figure
Effect of different pH on the activity of amylase.
Under all optimized conditions a high enzyme production of 1723 U/mL was obtained in 48 hrs.
Different concentrations of calcium chloride like 0.2, 0.3, 0.4, and, 0.5 M were added to the fermentation medium and the enzyme produced by the
Effect of 0.2 M calcium chloride concentration on amylase activity.
Effect of 0.3 M Calcium chloride on amylase activity.
Effect of 0.4 M Calcium chloride on amylase activity.
Effect of 0.5 M Calcium chloride on amylase activity.
The alpha amylase was partially purified by ammonium sulphate precipitation and Sepahdex G 100 × chromatography. The specific activity of the enzyme was gradually increased after purifying the alpha amylase and found optimum after ammonium sulphate precipitation. The specific activity of the enzyme was found to increase after ammonium sulphate precipitation (see Table
Partial purification of alpha amylase from fermented broth.
Step | Amylase activity (U/mL) | Protein (mg/mL) |
---|---|---|
Fermentation broth | 327 | 3.75 |
Ammonium sulphate precipitation | 1427 | 1.63 |
Sephadex G 100 Chromatography | 3286 | 0.73 |
The residual activity of the partially purified alpha amylase enzyme was measured by incubating the enzyme at different temperatures. The results showed that the activity of the enzyme increased with increase in temperature. The partially purified enzyme activity was found to be highly active between 70–75°C. As the optimum activity was obtained at 75°C, thus this temperature was optimized for the conversion of starch to oligosaccharides, and the results of the following experiment are shown in Figure
Effect of temperature on enzyme activity.
The residual activity of the partially purified alpha amylase enzyme was measured by incubating the enzyme at different pH. The results showed that the activity of the enzyme increased with increase in pH. The partially purified enzyme activity was found to be highly active between pH 5.0 and 5.5. As the optimum activity was obtained at pH 5, thus this pH was optimized for the conversion of starch to oligosaccharides, and the results of the above study are shown in Figure
Effect of pH on enzyme activity interns of starch hydrolysis.
The effect of enzyme concentration on the desizing of the cotton cloth was studied by partially purified enzyme. The concentration of enzyme was varied from 50 to 500 U/mL/min. The desizing (separation of starch from the cloth) of the cloth was increased with increase in enzyme concentration and was found to be optimum at 300 U/mL/min. Further increase in the concentration had no significant effect on the desizing of the cloth. The results of the above experiment are shown in Figure
Effect of enzyme concentrations on the desizing of cotton cloth by partially purified alpha amylase.
The present investigation was aimed at achieving maximum enzyme production systematically by optimizing various process variables which have a significant role in the determination of growth of the strain as well as production of enzyme [