A Samrot, A Vijay
A Samrot, A Vijay. Α-Amylase Activity Of Wild And Mutant Strains Of Bacillus Sp. The Internet Journal of Microbiology. 2008 Volume 6 Number 2.
α-Amylase is an enzyme capable of hydrolyzing starch. Alternate names of this enzymes are 1,4-α-D-glucan glucanohydrolase and glycogenase. Amylase enzymes are used extensively in bread making to break down complex sugars such as starch (found in flour) into simple sugars. Recently, various strategies in the pharmaceutical and chemical industries have used amylases in the synthesis of optically pure drugs and agrochemicals. The production of amylase by bacteria and fungi has been studied in terms of enzyme production, protein properties and purification. The aim of this work was to describe the production of an extracellular amylase by wild and UV mutated
Enzymes are the proteins capable of catalyzing biochemical processes. Some of these are capable of catalyzing hydrolytic cleavage (digestion) of such biological polymers as proteins, carbohydrates and fats. These are known as hydrolases. Amylase is the name given to glycoside hydrolase enzymes that break down starch into glucose molecules. Although the amylases are designated by different Greek letters, they all act on α-1,4-glycosidic bonds. Under the original name of
Among all the amylolytic enzymes, the genetics of alpha-amylase in
Bacterial amylases have been extensively researched and are prolifically described in the patent literature. Unfortunately, only two
Having known the importance of amylase enzyme this work was aimed to isolate amylase from soil bacteria and to mutate the bacteria to overproduce the same.
Materials And Methods
Collection of sample
Soil samples from various places were collected in sterile container. Samples were serially diluted and subjected for total colony count. Colonies isolated were subjected for the production of amylase.
Assay for amylase production
Soluble starch-1g, Yeast extract - 0.2g, Peptone - 0.5g, MgSO4 - 0.05g, NaCl -0.05g, CaCl2 - 015g, Agar - 2g,, Distilled water - 100mL, pH 7.0
Starch agar plates were prepared. Isolated colonies were inoculated unto the medium. Plates were incubated at 370C for 24 hours. Iodine solution was added and checked for the production of amylase enzyme in the starch agar plate. Formation of zone after iodine solution confirms the presence of amylase. All the starch hydrolyzing organisms were identified using bergey’s manual (Claus and Berkeley, 1986).
Identification of bacteria
The isolated bacteria were identified according to Gram characteristics, spore morphology and motility. In addition, the following identified tests were carried out: utilization of citrate, VP test, casein hydrolysis, gelatin liquefaction starch hydrolysis and production of acid from D-glucose, L-arabinose and D-xylose. The production of gas from glucose, catalase production and nitrate reduction were also tested (Claus and Berkeley, 1986).
Media Composition(in gms)
NaH2PO4.2H2O - 1.56, NH4Cl - 5.35, KCl - 0.745, Na2SO4.10H2O -0.644, Citric acid -0.42, MgCl2.6H2O -0.25, CaCl2 - 2.2x10-3, FeCl3.6H2O -2.7x10-2 , MnCl2.4H2O-1.0x10-2, CuCl2.2H2O -8.5x10-4, H3BO3 - 3.0x10-4, Na2MoO4 -1.0x10-3, Bactotryptone - 10.0, Yeast extract - 2.5, Soluble starch –5, pH - 6.9-7.0, pH was adjusted with 1.0 M NaOH and this basal medium was sterilised by autoclaving at 121ºC for 15min used in this work for α-amylase production contained (g/L): Yeast extract, Bacto-tryptone and Soluble starch were sterilised separately and aseptically added to the flasks containing the liquid medium, after cooling. The above medium (50 mL in 250 mL Erlenmeyer flasks) was inoculated with 1 mL of an overnight culture and incubated at 37ºC with vigorous aeration in a rotary shaker at 150 rpm for 144h. At time intervals, the turbidity of the cultures was determined by measuring the optical density at 470 nm in a Systronics spectrophotometer (India). Before assay, the cells were separated by centrifugation at 13.000 rpm for 15 min and the clear supernatant was used as crude enzyme preparation (Cordeiro
Amylase Assay (Miller, 1959)
The activity of α-amylase was assayed by incubating 0.3 mL enzyme (crude enzyme preparation) with 0.5 mL Soluble starch (1%, w/v) prepared in 0.05M Phosphate buffer, pH 6.5. After incubation at 37ºC for 10 min the reaction was stopped The reducing sugars released were assayed colorimetrically by the addition of 1 mL of 3-5-dinitrosalicylic acid reagent. An enzyme unit is defined as the amount of enzyme releasing 1 mM of glucose from the substrate in 1 min at 90ºC.
Effect of pH on activity and stability of α -amylase
Effect of pH on the activity of α -amylase was measured by incubating 0.3 mL of enzyme and 0.5 mL of buffers, adjusted to pH of 5.5 to 8.5, containing Soluble starch (0.5%). The buffers used were: sodium acetate pH 5.5; phosphate pH 6.0 – 8.0; Tris-HCl pH 8.5. Stability of the enzyme at different pH values was also studied by incubating the enzyme at various pH values ranging from 5.5 – 8.5 for 24h and then estimating the residual activity.
Effect of temperature on activity and stability of α -amylase
The effect of temperature on the enzyme activity was determined by performing the standard assay procedure as mentioned earlier for 10 min at pH 6.5 within a temperature range of 40 –100ºC. Thermostability was determined by incubation of crude enzyme at temperatures ranging from 40-100ºC for 2hr in a constant-temperature water bath. After treatment the residual enzyme activities were assayed.
Salt tolerance test
Enzyme was incubated in 10 mM Phosphate buffer (pH 7.0) containing various NaCl concentrations (0.05 to 5M) for 24h at 4ºC and in each case activity of the enzyme was measured in the same way as mentioned earlier.
Induction of mutation
Wild species was exposed to UV radiation by following method. Wild
Results And Discussion
Totally 180 samples were isolated from the soil samples. About 28 samples showed α-amylase production (Table 1). All the 28 organisms were subjected for the identification of
B10 was further explored for the UV mutagenesis study. Efficient α –amylase producing B10 was exposed to UV irradiation for various time intervals viz. 30, 60, 90, 120 and 150 sec. 7, 6, 4, 3 and 1 colonies were observed in the 30, 60, 90, 120 and 150 sec UV exposed plates respectively. Reduction in colony count was due to effect of UV. All these 21 colonies were subjected for the α –amylase activity. One from colony each 90, 120 and 150 sec exposed groups found to produce more α –amylase than the wild strain B10 (Table 3). Among these three M3 (i.e. 150 sec UV exposed strain) produced 35U/mL of α-amylase.
When studying the time course production of α-amylase, B10
B10 and M3 were also subjected to produce α-amylase in various concentration of NaCl. At the concentration 0.75%, B10 (wild strain) showed α –amylase activity of 29U/mL and M3 showed α –amylase activity as 37U/mL (Fig.4). The overall results clearly said us that mutant strains are more capable of producing more α–amylase than wild strain.
Mutants form of