Asparaginase production by Bacillus spp and its application in reducing acrylamide formation in fried food

Twenty three isolates of Bacillus spp were obtained from the National Commission for Biotechnology (NCBT), they were classified to the genus level, they were isolated from different local soils .Isolates were grown on M9 solid medium supplied with phenol red indicator, they were screened for their production of asparaginase. Liquid media were used to determine asparaginase activity. Enzymatic activity was measured by colorimetric method using Nesslers reagent spectrophotometerically at a wave length of 450 nm. The most efficient isolate number 3 was selected and its' species was classified according to Bergeys manual of classification in terms of morphological and biological characteristics. The isolate (3) was found to be Bacillus licheniforms.The conditions for producing asparaginase enzyme from Bacillus licheniformis were optimized using Asparagine as substrate in submerged fermentation by using the statistical program Minitab and the statistical design Response Surface Methodology (RSM) were applied. The highest enzyme activity was obtained at an incubation temperature of 30ºC, pH value of 9, aeration 150 rpm per minute , carbon source concentration (glucose) of 1.5% and nitrogen source concentration(yeast axtract) of 2.5%. Enzyme activity obtained under the above mentioned conditions was 1481.92 U/ml.Purification steps of enzyme started by precipitation with 80% acetone, then separation by column chromatography using Sephadex G-100 and Sephacryl DEAE-sepharose. The molecular weight was 34.4 kDa determined using SDS PAGE.The effects of temperature, pH, substrate concentration (asparagine), and some metallic ions (Cu+2, Na+, Hg+2, Mg+2, Mn+2, Ag+, Ca+2, Co+2, Fe+3, Zn+2)on purified asparaginase were determined. The enzyme was produced by submerged fermentation of Bacillus licheniformis isolated from local soils. Maximum enzyme activity was found at pH 9, temperature 60°C and 1 mole/ml of asparagine as a substrate. The enzyme remained stable at pH 8-9 and temperature at 30-60°C for 60 minutes. Asparaginase was activated by (Na+, Mg+2, Mn+2, Ag+, Co+2, Fe+3) ions at a concentration of 1mM/ml while other ions such as (Zn+2, Hg+2,Ca+2,Cu+2) reduced asparaginase activity at a concentration of 1mM/ml. The highest enzyme activity was observed with MgCl2 at a concentration of 1mM/ml , while EDTA at a concentration of 0.8mM/ml reduced the activity of the enzyme .The efficacy of purified asparaginase produced by Bacillus licheniformis in reducing the amount of acrylamide formed by Maillard reaction was tested in the frying of some food products including potatoes, chips, and donuts. The amount of acrylamide formed afterfrying was measured in the enzymatically treated samples and compared with those in the control (untreated samples). The results showed a reduction in the amount of acrylamide formed in the treated samples by 50.17%, 46.76%, and 40.63% in fried potatoes, chips and donuts, respectively, compared to the untreated control samples.