Production of Microbial Amylase

Experiment 7 Production of microbial amylase Date: Enzymes are biological catalysts, i.e., the substances which accelerate the rate of a specific chemical reaction. They are either exoenzymes (extracellular) or endoenzymes (intracellular). Exoenzymes are released from the cell and act on the substrates. These are mainly hydrolytic enzymes that degrade, by the addition of water, high-molecular-weight substrates like polysaccharides, lipids, and proteins, into smaller components such as glucose that can enter into the cell and are later assimilated. For e.g. cellulase, amylase, pectinase, lipase, caseinase, gelatinase. Their production can be assayed in the laboratory by detecting a change in the substrate outside the microbial cell. Endoenzymes are utilized by the cell for further metabolic degradation of carbohydrates and are mainly responsible for the synthesis of new protoplasmic requirements and the production of cellular energy from assimilated materials. These enzymes function inside the cell include maltase, lactase, catalase, and urease. Amylase is an exoenzyme that hydrolyzes starch (polysaccharide) into maltose (disaccharide) and some monosaccharides such as glucose. These disaccharides and monosaccharides enter into the cytoplasm of the bacterial cell through the semi-permeable membrane and thereby used by the endoenzymes. All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. Amylases are classified into three groups viz., α-amylase, β-amylase, and -amylase. Alpha and beta amylases are essential in brewing beer and liquor made from sugars derived from starch. Amylases find use in bread making and to break down complex sugars, such as starch (found in flour), into simple sugars. Yeast then feeds on these simple sugars and converts it into alcohol and CO 2. This imparts flavor and causes the bread to rise. Amylase production is known in some bacteria while it is well-known in molds & yeasts. In yeast cells, it takes time to produce enough of these enzymes to break down significant quantities of starch in the bread. This is the reason for long-fermented doughs such as sourdough. Modern bread making techniques have included amylases (often in the form of malted barley) into bread improver, thereby making the process faster and more practical for commercial use. Amylases are used in the initial steps in several food fermentation processes to convert starch into fermentable sugars. They are also used to predigest foods for young children partially to clarify fruit juices and in the manufacture of corn and chocolate syrups. AGM 312 Microbial Biotechnology (1+1) Page 32 Principle The ability to degrade starch is used as a criterion for the determination of amylase production by a microbe. It is tested by determining the presence or absence of starch in the medium by using the iodine solution as an indicator. Starch in the presence of iodine produces a dark-blue coloration of the medium. The formation of the yellow zone around a colony indicates the amylolytic activity of the organism that has degraded the starch in the medium. This exercise deals with testing the hydrolysis of starch for the production of amylase by four test organisms viz., Aspergillus niger, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae by inoculating these on starch agar medium. Materials required:       Cultures of Aspergillusniger, Bacillus subtilis,and Escherichia coli Starch agar medium Starch (soluble) : 20.0g Peptone : 5.0g Beef extract : 3.0g Agar : 15.0g Distilled water : 1000 ml pH : 7.0 Gram‟s Iodine solution Sterile Petri plates Dropper Inoculation needle Procedure 1. 2. 3. 4. Melt the starch agar medium, cool to 45 ºC and pour into sterile Petri plates Allow for solidification Label each of the starch agar plates with the name of the organism to be inoculated Make a single streak of each organism into the center of the respective plate aseptically 5. Incubate the bacterial plates for 48 h and fungal plates for 72-96 h in an inverted position 6. Flood the surface of the plates with iodine solution with a dropper for 30 seconds 7. Pour off the excess iodine solution. AGM 312 Microbial Biotechnology (1+1) Page 33 Observations Examine the plates for the starch hydrolysis around the line of growth of each organism, i.e., the color change of the medium. Clear zones surrounding the microbial colonies indicate a positive reaction. An adverse reaction is indicated by a dark blue coloration of the medium. AGM 312 Microbial Biotechnology (1+1) Page 34 Review Questions 1. Differentiate Exo and Endo enzyme with suitable examples. 2. What is amylase and explain its role? 3. Name at least 10 microorganisms producing amylase enzymes. 4. List out the industrial applications of the amylase enzyme. 5. What is the reaction of amylase with starch? 6. What is the role of Gram‟s Iodine in the starch hydrolysis test? AGM 312 Microbial Biotechnology (1+1) Page 35 7. What is the significance of the brown color change in amylase assay? Reference Deepika, M. and S. Thulasi. 2016. Bacterial and Archaeal α-Amylases: Diversity and Amelioration of the Desirable Characteristics for Industrial Applications. Frontiers in Microbiol., (7): 1129. AGM 312 Microbial Biotechnology (1+1) Page 36