10-04-2017, 08:45 PM
Kinetics of production of alpha amylase using microorganisms
INTRODUCTION
Proteases and alpha amylases are the two most important industrial enzymes, together representing more than 70% of the total worldwide enzyme market (23). Proteases catalyze the cleavage of peptide bonds and cause the total hydrolysis of proteins while alpha amylases degrade 1 4 linkage of starch and other substrates in an endo fashion producing maltose and glucose (1, 22). Since both these enzymes are physiologically necessary for microorganisms they are widely distributed in a number of microorganisms where they are produced in large quantities by through the fermentation process.
Both fungi and bacteria are employed for the production of proteases and alpha amylases but the choice of organism mainly depends upon the application of the enzyme produced. For example, proteases to be used in the food industry are mainly produced by fungi, whereas the proteases used in leather or detergent industries are mainly produced by bacteria. Similarly, the alpha amylases produced by fungi are more stable than those from bacteria due to high thermostability and heat sensitivity (6).
Organism, Growth and Maintenance
A highly productive strain of Bacillus subtilis IIB-26 was taken from the culture bank of Institute of Industrial Biotechnology, GC University, Lahore, which was originally isolated from the field soil. The culture was grown and maintained on nutrient agar medium. The bacterial slopes were incubated at 37 C for 48 h and then stored at 4 C. However, the strain was maintained by weekly transfers onto fresh slopes.
Inoculum preparation
Inoculum of Bacillus subtilis was prepared in 500 mL Erlenmeyer flasks containing 100 mL of nutrient broth. The flasks were sterilized in an autoclave at 121 C (15 Ib/in2 pressure) for 15 min. After cooling, the medium was aseptically inoculated with bacteria from a 48 h old slope. The flasks after inoculation were incubated for 24 h on a rotary incubator shaker (Gallenkamp, UK) at 37 C and 200 rpm.
Fermentation Experiments
The fermentation experiments for the simultaneous production of alkaline protease and alpha amylase from Bacillus subtilis were carried out in a laboratory scale 7.5 L batch bioreactor (New Bruinswick Scientific, USA) with a working volume of 5.0 L. The bioreactor was equipped with monitors, which were used to measure and control foam, temperature, pH, stirring rate and dissolved oxygen. The vessel of the bioreactor was equipped with a four-blade turbine.
Peristaltic pumps were attached to control the foam and pH by automatic addition of an antifoam silicon agent or an acid/base respectively. Five liters of the fermentation medium containing (g/L) soybean meal, 20; starch, 10; glucose, 5.0; polypeptone, 10; KH2PO4, 1.0; (NH4)2SO4, 1.0 and Na2CO3, 5.0 (pH 8.0) was added to a fermentor vessel and sterilized in an autoclave. After cooling, the medium was inoculated with already-prepared 200 mL of the inoculum containing 3.5 108 CFU/mL. The fermentor was run for a batch operation for the said interval of time. After the fixed interval of incubation, the fermented broth was centrifuged at 3830 g for 10 min and the supernatant was assayed for protease and alpha amylase activities.
During all the experiments chemicals of analytical grade and calibrated labware were used.
Analytical Methods
Assay of Protease: The method of McDonald and Chen (16) was used for the assay of protease. Casein (4 mL of 1% solution in Phosphate buffer of pH 7.0 for the determination of neutral protease and pH 8.5 for the determination of alkaline protease) was incubated with 1 mL of enzyme sample at 30 C for one hour. The reaction was arrested by the addition of 5 mL of 5% trichloroacetic acid solution. The mixture was centrifuged at 2660 g for 10 min and 1 mL of supernatant was mixed with 5 mL of alkaline reagent (made by mixing 1 mL of 1% CuSO4, 1 mL of 2.7% Sodium potassium tartarate with 100 mL of 2% Na2CO3). To this mixture 1 mL of 1N NaOH was added to make the contents of the tube alkaline. After 10 min., 0.5 mL of Folin and Ciocalteau reagent was added to the test tubes and mixed. The optical density of the solution was measured with UV/VIS spectrophotometer (CECIL, CE 7200, Cambridge, England) at 700 nm after 30 min.
INTRODUCTION
Proteases and alpha amylases are the two most important industrial enzymes, together representing more than 70% of the total worldwide enzyme market (23). Proteases catalyze the cleavage of peptide bonds and cause the total hydrolysis of proteins while alpha amylases degrade 1 4 linkage of starch and other substrates in an endo fashion producing maltose and glucose (1, 22). Since both these enzymes are physiologically necessary for microorganisms they are widely distributed in a number of microorganisms where they are produced in large quantities by through the fermentation process.
Both fungi and bacteria are employed for the production of proteases and alpha amylases but the choice of organism mainly depends upon the application of the enzyme produced. For example, proteases to be used in the food industry are mainly produced by fungi, whereas the proteases used in leather or detergent industries are mainly produced by bacteria. Similarly, the alpha amylases produced by fungi are more stable than those from bacteria due to high thermostability and heat sensitivity (6).
Organism, Growth and Maintenance
A highly productive strain of Bacillus subtilis IIB-26 was taken from the culture bank of Institute of Industrial Biotechnology, GC University, Lahore, which was originally isolated from the field soil. The culture was grown and maintained on nutrient agar medium. The bacterial slopes were incubated at 37 C for 48 h and then stored at 4 C. However, the strain was maintained by weekly transfers onto fresh slopes.
Inoculum preparation
Inoculum of Bacillus subtilis was prepared in 500 mL Erlenmeyer flasks containing 100 mL of nutrient broth. The flasks were sterilized in an autoclave at 121 C (15 Ib/in2 pressure) for 15 min. After cooling, the medium was aseptically inoculated with bacteria from a 48 h old slope. The flasks after inoculation were incubated for 24 h on a rotary incubator shaker (Gallenkamp, UK) at 37 C and 200 rpm.
Fermentation Experiments
The fermentation experiments for the simultaneous production of alkaline protease and alpha amylase from Bacillus subtilis were carried out in a laboratory scale 7.5 L batch bioreactor (New Bruinswick Scientific, USA) with a working volume of 5.0 L. The bioreactor was equipped with monitors, which were used to measure and control foam, temperature, pH, stirring rate and dissolved oxygen. The vessel of the bioreactor was equipped with a four-blade turbine.
Peristaltic pumps were attached to control the foam and pH by automatic addition of an antifoam silicon agent or an acid/base respectively. Five liters of the fermentation medium containing (g/L) soybean meal, 20; starch, 10; glucose, 5.0; polypeptone, 10; KH2PO4, 1.0; (NH4)2SO4, 1.0 and Na2CO3, 5.0 (pH 8.0) was added to a fermentor vessel and sterilized in an autoclave. After cooling, the medium was inoculated with already-prepared 200 mL of the inoculum containing 3.5 108 CFU/mL. The fermentor was run for a batch operation for the said interval of time. After the fixed interval of incubation, the fermented broth was centrifuged at 3830 g for 10 min and the supernatant was assayed for protease and alpha amylase activities.
During all the experiments chemicals of analytical grade and calibrated labware were used.
Analytical Methods
Assay of Protease: The method of McDonald and Chen (16) was used for the assay of protease. Casein (4 mL of 1% solution in Phosphate buffer of pH 7.0 for the determination of neutral protease and pH 8.5 for the determination of alkaline protease) was incubated with 1 mL of enzyme sample at 30 C for one hour. The reaction was arrested by the addition of 5 mL of 5% trichloroacetic acid solution. The mixture was centrifuged at 2660 g for 10 min and 1 mL of supernatant was mixed with 5 mL of alkaline reagent (made by mixing 1 mL of 1% CuSO4, 1 mL of 2.7% Sodium potassium tartarate with 100 mL of 2% Na2CO3). To this mixture 1 mL of 1N NaOH was added to make the contents of the tube alkaline. After 10 min., 0.5 mL of Folin and Ciocalteau reagent was added to the test tubes and mixed. The optical density of the solution was measured with UV/VIS spectrophotometer (CECIL, CE 7200, Cambridge, England) at 700 nm after 30 min.