Amylase Production on Submerged Fermentation by Bacillus Spp

WHAT ARE AMYLASES ? 

Amylases are enzymes that break down starch or glycogen. ƒ Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extra-cellular digestion. When they have broken down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed into their cells. ƒƒ major advantage of using micro organisms major advantage of using micro organisms for the production of amylases is in for the production of amylases is in economical bulk production capacity and economical bulk production capacity and microbes are also easy to manipulate to microbes are also easy to manipulate to obtain enzymes of desired charecteristics. obtain enzymes of desired charecteristics.

ƒ The microbial amylases meet industrial demands a ƒ The microbial amylases meet industrial demands a large number of them are available commercially. large number of them are available commercially. ƒ Although many micro organisms produce this ƒ Although many micro organisms produce this enzymes the most commonly used for their industrial enzymes the most commonly used for their industrial application are application are ƒ “BACILLUS LECHENIFORMIS” ƒ “BACILLUS LECHENIFORMIS” ƒ “BACILLUS AMYLOLIQUIFACIENS” ƒ “BACILLUS AMYLOLIQUIFACIENS” ” ƒ “ASPERGILLUS NIGER ƒ “ASPERGILLUS NIGER ”

ƒ Amylases standout as a class of enzymes ,which are of ƒ Amylases standout as a class of enzymes ,which are of useful applications in food, brewing, textile,detergent and useful applications in food, brewing, textile,detergent and pharmaceutical industries. pharmaceutical industries. ƒ They are mainly employed for starch liquefaction(it is a process of dispersion of insoluble starch in aqueous solution) to reduce their viscosity, production of maltose, oligosaccharide mixtures, high fructose syrup and maltotetraose syrup. In detergents production, they are applied to improve cleaning effect and are also used for starch de-sizing in textile industry. ƒ The use of submerged culture is advantageous because of ƒ The use of submerged culture is advantageous because of ease of sterilization and process control is easier to ease of sterilization and process control is easier to engineer in these systems.Depending on strain and culture engineer in these systems.Depending on strain and culture conditions the enzyme can be constitutive or conditions the enzyme can be constitutive or inducible,showing different production patterns inducible,showing different production patterns

Classification of amylases: Classification of amylases: ƒ Amylases are classified based on how they break down starch molecules ƒ i. α-amylase (alpha-amylase) – Reduces the viscosity of starch by breaking down the bonds at random, therefore producing varied sized chains of glucose ƒ ii. ß-amylase (Beta-amylase) – Breaks the glucose-glucose bonds down by removing two glucose units at a time, thereby producing maltose ƒ iii. Amyloglucosidase (AMG) – Breaks successive bonds from the non-reducing end of the straight chain, producing glucose Many microbial amylases usually contain a mixture of these amylases

MICROBIAL AMYLASES:

The enzymes from microbial sources generally meet ƒ The enzymes from microbial sources generally meet industrial demands. Microbial amylases could be industrial demands. Microbial amylases could be potentially useful in the pharmaceutical and fine-chemical potentially useful in the pharmaceutical and fine-chemical industries if enzymes with suitable properties could be industries if enzymes with suitable properties could be prepared. With the advent of new frontiers in prepared. With the advent of new frontiers in biotechnology, the spectrum of amylase application has biotechnology, the spectrum of amylase application has widened in many other fields, such as clinical, medicinal widened in many other fields, such as clinical, medicinal and analytical chemistries, as well as their widespread and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, application in starch saccharification and in the textile, food, brewing and distilling industries food, brewing and distilling industries

MATERIALS AND METHODS FOR PRODUCTION MATERIALS AND METHODS FOR PRODUCTION ƒ MICROORGANISM:

Bacillus spp was isolated from environment ƒ MICROORGANISM: Bacillus spp was isolated from environment and maintained on nutrient agar slants and subcultured for every and maintained on nutrient agar slants and subcultured for every 10 days 10 days ƒ INOCULUM AND FERMENTATION MEDIUM: The inoculum was ƒ INOCULUM AND FERMENTATION MEDIUM: The inoculum was prepared by addition of sterile distilled water into the freshely prepared by addition of sterile distilled water into the freshely grown nutrient agar slants,from this 0.5ml of cell suspension was grown nutrient agar slants,from this 0.5ml of cell suspension was inoculated into 100ml of sterilized fermentation medium and inoculated into 100ml of sterilized fermentation medium and incubated at 35°C 10 hrs incubated at ƒ The composition of fermentation medium are: ƒ Bacteriological peptone – 6gm ƒ Magnesium sulfate – 0.5gm ƒ Potassium chlorate – 0.5gm ƒ Starch – 1gm (pH -7)

ƒ EXTRACTION OF AMYLASE FROM FERMENTATION MEDIUM: ƒ

After incubation the fermentation medium was harvested ƒ After incubation the fermentation medium was harvested by centrifugation at 5000 rpm for 20 min at 4°C .The by centrifugation at 5000 rpm for 20 min at supernatent was collected and subjected to estimate the amylase activity ƒ EFFECT OF TEMPERATURE: ƒ EFFECT OF TEMPERATURE: ƒ To study the effect of temperature on amylase ƒ To study the effect of temperature on amylase production,the submerged fermentation was carried out at production,the submerged fermentation was carried out at different temperatures (25°C, 30°C, 35°C and 40° C). different temperatures

ƒEFFECT OF PH: ƒ

The fermentation medium was prepared by varying pH values(5.0, 6.0, 7.0, 8.0) for the production of amylase. ƒ ASSAY OF AMYLASE: ƒ The amylase activity is determined following the method of Bernfeld.An assay mixture containing, enzyme extract,starch as a substrate and DNS as coupling reagent was used.one unit of amylase activity was defined as the number of micro moles of maltose liberated by 1ml of enzyme solution per minute

RESULTS AND DISCUSSION RESULTS AND DISCUSSION ƒ AMYLASE PRODUCTION IN SUBMERGED FERMENTATION: ƒ

AMYLASE PRODUCTION IN SUBMERGED FERMENTATION: ƒ In submerged fermentation the production of amylase was ƒ In submerged fermentation the production of amylase was reached maximum of 4u/ml at 10h of incubation reached maximum of 4u/ml at 10h of incubation period.further increase in the incubation period did not period.further increase in the incubation period did not show any significant increase in enzyme production rather show any significant increase in enzyme production rather it was decreased.Thus optimum time of enzyme synthesis it was decreased.Thus optimum time of enzyme synthesis was to be 10h after inoculation. was to be 10h after inoculation.

ƒ EFFECT OF TEMPERATURE: ƒ EFFECT OF TEMPERATURE: ƒ The maximum production of amylase was obtained at 35°C .The ƒ The maximum production of amylase was obtained at The optimum temperature was observed for the production of amylase from Banana stalk using B. subtilis was also 35°C. Increase in incubation temperature, decreased the production of enzyme. The production of the enzyme was greatly inhibited at 40°C. It might be due to that at high temperature, the growth of the bacteria was greatly inhibited and hence, enzyme formation was also prohibited

Effect of varying incubation temperature on amylase production ) 9 l m 7 / 8 7 U 6 ( 7 6 y t 5 5 i 6 v i t 4 c 5 4 a 3 4 3 e s 2 3 2 a l 1 y 2 1 m 0 A 1 0 0 25°C 30°C 35°C 40°C 45°C Incubation Temperature

ƒ EFFECT OF PH: ƒ EFFECT OF PH: ƒ The amylase production by bacillus spp was found maximum at pH ƒ The amylase production by bacillus spp was found maximum at pH 7.further increase in pH resulted in decrease in activity of 7.further increase in pH resulted in decrease in activity of amylase.However ,the pH of fermentation medium was found to be amylase.However ,the pH of fermentation medium was found to be optimum at pH 7.When pH is altered below or above the activity is optimum at pH 7.When pH is altered below or above the activity is decreased or becomes denatured.Different organisms have decreased or becomes denatured.Different organisms have different pH optima,and decrease or increase on either sides of different pH optima,and decrease or increase on either sides of optimum value results in poor microbial growth optimum value results in poor microbial growth

EFFECT OF VARYING pH on amylase production EFFECT OF VARYING pH on amylase production pH AMYLASE ACTIVITY pH AMYLASE ACTIVITY U/ml U/ml 5.5 3.2 6 4 4.1 6.5 4.9 7 11.0 7.5 9.0 8 7.0

APPLICATIONS OF INDUSTRIAL ENZYMES:

ENZYMES SOURCE APPLICATIONS ENZYMES SOURCE APPLICATIONS Alpha-amylase Bacterial a- Textiles, starch Alpha-amylase Bacterial a- Textiles, starch amylase (e.g., syrups, laundry and amylase (e.g., syrups, laundry and Bacillus subtilis), dishwashing Bacillus subtilis), dishwashing Fungal a-amylase detergents, paper Fungal a-amylase detergents, paper (e.g., Aspergillus desizing, (e.g., Aspergillus desizing, niger), Alkaline a- fermentation niger), Alkaline a- fermentation ethanol, animal ethanol, animal amylase amylase feed feed From a strain of Brewing, maltose From a strain of Brewing, maltose ß-amylase ß-amylase Bacillus Bacillus syrup syrup Glucoamylase Manufacture of Glucoamylase Manufacture of Aspergillus niger Aspergillus niger dextrose syrup and dextrose syrup and – -THANK YOU

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