Production of amylase

Introduction
Types of amylase
Production of amylase
Recovery and purification
Determination of enzyme activity
Industrial application

1. Don't blink now BY : ROHINI YADAV FERMENTATION TECHNOLOGY

2. Production of Amylase

3. Contents Introduction Types of amylase Production of amylase Recovery and purification Determination of enzyme activity Industrial application

4. Amylase Amylase is an enzyme that catalyses the hydrolysis of starch into sugars. Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion Thes e e zymes randomly cleave internal glycosidic linkages in starch molecules to hydrolyze them and yield dextrins and oligosaccharides

5. T ypes of Amylase: α-Amylase α-Amylase is a hydrolase enzyme that catalyses the hydrolysis of internal α-1, 4-glycosidic linkages in starch to yield products like glucose and maltose. It is a calcium metalloenzyme i.e. it depends on the presence of a metal co factor for its activity. There are 2 types of hydrolases: endo-hydrolase and exo-hydrolase. Endo- hydrolases act on the interior of the substrate molecule, whereas exo-hydrolases act on the terminal non reducing ends .

6. Amylase T he substrate that α-amylase acts upon is starch. Starch is a polysaccharide composed of two types of polymers – amylose and amylopectin. Amylose constitutes 20-25% of the starch molecule. It is a linear chain consisting of repetitive glucose units linked by α-1,4-glycosidic linkage. Amylopectin constitutes 75-80% of starch and is characterized by branched chains of glucose units. The optimum pH for activity is found to be 7. 0.

7. 2 β – Amylase β-Amylase is an exo-hydrolase enzyme that acts from the nonreducing end of a polysaccharide chain by hydrolysis of α-1, 4-glucan linkages to yield successive maltose units. It is unable to cleave branched linkages in branched polysaccharides such as glycogen or amylopectin, the hydrolysis is incomplete and dextrin units remain. During ripening of fruits, β-Amylase breaks down starch into maltose resulting in the sweetness of ripened fruit. The optimal pH of the enzyme ranges from 4.0 to 5.5.

8. 2 γ – Amylase γ-Amylase cleaves α(1-6)glycosidic linkages, in addition to cleaving the last α(1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, unlike the other forms of amylase, yielding glucose. γ- amylase is most efficient in acidic environments and has an optimum pH of 3.

9. 2 Production of α-Amylase

10. 2 S ources α-Amylase can be isolated from plants, animals or microorganisms. The enzyme has been isolated from barley and rice plants . I t has been found that cassava mash waste water is a source of α-Amylase which is active in wide range of pH and temperature . The most widely used source among the bacterial species is the Bacillus spp. B. amyloliquefaciens and B. licheniformis Fungal sources of α-Amylase are mostly Aspergillus species and only few species of Penicillium, P. brunneum , penicillium fellutanum

11. 2 Production Methods : There are mainly two methods which are used for production of α-Amylase on a commercial scale. These are : 1) Solid State fermentation. 2 ) Submerged fermentation

12. 1 M ethod used for microbes which require less moisture content for their growth. The solid substrates commonly used in this method are, bran, bagasse, and paper pulp. T he main advantage is that nutrient-rich waste materials can be easily recycled and used as substrates in this method. 1. Solid State Fermentation [SSF]

13. 1 employs free flowing liquid substrates, such as molasses and broths. The products yielded in fermentation are secreted into the fermentation broth. The substrates are utilized quite rapidly , hence the substrates need to be constantly replenished SmF is primarily used for the extraction of secondary metabolites that need to be used in liquid form . 2. Submerged Fermentation [SMF]

14. 1 Composition (g/l) Na2HPO4 . 6g KH2PO4 3g NH4Cl 1g NaCl 0.5g CaCl2 0.15g MgSO4.7H2O 0.25g Casein hydrolysate 0.20g Yeast extract . 0.10g Starch 20g Amylase Production Media

15. 1 1. Carbon Source: Common carbon sources used as substrates include maltose, sucrose and glucose. A. tamarii actively synthesizes α-Amylase when cultured on maltose, starch and glycogen under static conditions. . The aim is to use substrates that are waste or by products of other processes in order to make the process of enzyme production environment friendly. One such substrate is oil cake. Wheat bran has been used as substrate for α-Amylase production by B.licheniformis and A.niger. Substrate

16. Are you ready 2. Nitrogen Source The nitrogen source used for production of α-Amylase may be organic or inorganic. Few of the inorganic nitrogen sources include ammonium sulphate, ammonium chloride and ammonium hydrogen phosphate. Most commonly used organic sources of nitrogen include peptone, yeast extract and soyabean meal.

17. The optimum process control parameters vary depending on the microbial source, desired end product, method of fermentation employed and many other such factors. P rocess Parameters: 1.Temperature There are two temperatures that need to be in optimum range during production. They are temperature for the growth of the microbial source and optimum temperature at which maximum production of enzyme takes place.

18. The optimum temperatures for growth and α-Amylase production were found to be 45°C to 46 °C and 50 °C, respectively. 2. pH Optimum pH is a critical factor for the stability of enzyme produced. Enzymes are pH sensitive and hence care must be taken to control the pH of the production process. Pyrococcus furiosus produces α-Amylase which shows activity at an optimum pH of 6.5–7.5

19. go . Duration of fermentation This is a crucial factor in fermentation process. If the process is carried out for a time period shorter than the optimum duration the maximum yield cannot be obtained. The enzyme activity increases with increase in incubation time till it reaches the optimum duration. In most cases, the production of enzyme begins to decline if the incubation time is further increased. This could be due to the depletion of nutrients in the medium or release of toxic substances. Bacillus subtilis, a well known producer of alpha amylase was studied for comparison between different fermentation hours and the study revealed a high yield of alpha amylase after 48 hours of fermentation Different incubation time durations were compared for yield of amylase from Penicillium fellutanum isolated from mangrove rhizosphere soil. The culture when incubated at 96 h, showed the maximum activity. Optimum levels of initial moisture content may vary depending on the microbial source used. For fungal sources the moisture content required is less whereas bacterial sources need more moisture content for high yield of the enzyme. production of α-Amylase by Penicillium janthinellum the moisture content was varied within a range of 20- 80% by varying the amount of salt solution used in moistening the substrate particles. Moisture

20. WPS WPS This is a crucial factor in fermentation process. The enzyme activity increases with increase in incubation time till it reaches the optimum duration. In most cases, the production of enzyme begins to decline if the incubation time is further increased. This could be due to the depletion of nutrients in the medium or release of toxic substances. Bacillus subtilis, show high yield of alpha amylase after 48 hours of fermentation Penicillium fellutanum isolated from mangrove rhizosphere soil. The culture when incubated at 96 h, showed the maximum activity. Duration of fermentation

21. is The desired enzyme produced may be excreted into the culture medium (extracellular enzymes) or may be present within the cells (intracellular enzymes). D epending on the requirement, the commercial enzyme may be crude or highly purified. Further, it may be in the solid or liquid form. The steps involved in downstream processing i.e. recovery and purification steps employed will depend on the nature of the enzyme and the degree of purity desired. Recovery and purification of enzymes

22. office In general, recovery of an extracellular enzyme which is present in the b roth is relatively simpler compared to an intracellular enzyme. For the release of intracellular enzymes, special techniques are needed for cell di sruption. Microbial cells can be broken down by physical means ( sonication, high pressure, glass beads) The recovery and purification steps will be the same for both intracellular and extracellular enzymes, once the cells are disrupted and intracellular enzymes are released. The most important consideration is to minimise the loss of desired enzyme activity. Removal of cell debris: Filtration or centrifugation can be used to remove cell debris.

23. software Removal of nucleic acids : Nucleic acids interfere with the recovery and purification of enzymes. They can be precipitated and removed by adding poly-cations such as polyamines, streptomycin and polyethyleneimine.  Enzyme precipitation: En ymes can be precipitated by using salts (ammonium sulfate) organic solvents (isopropanol, ethanol, and acetone). Precipitation is advantageous since the precipitated enzyme can be dissolved in a minimal volume to concentrate the enzyme. Liquid-liquid partition : Further concentration of desired enzymes can be achieved by liquid-liquid extraction using polyethylene glycol or polyamines.

24. company Separation by chromatography: t here are several chromatographic techniques for separation and purification of enzymes. These include ion-exchange, size exclusion, a ffinity, hydrophobic interaction and dye ligand chromatography. A mong these, ion- exchange chromatography is the most commonly used for enzyme purification. Drying and packing: The concentrated form of the enzyme can be obtained by drying. This c an be done by film evaporators or freeze dryers (lyophilizers). The dried enzyme can be packed and marketed.

25. It 1. Dinitrosalicylic Acid Method (DNA) 2. Nelson-Somogyi (NS) method 3. Iodine Activity Determination of enzyme activity

26. established Hydrolysis of starch into the extreme forming a less viscous starch suspension. In bakers industry, converting starch in two smaller fermentable sugars . In textile industry, used to removal of starch sizing agent for woven fabric . Fuel alcohol production, converting starch into smaller fermentable sugars which are acted upon by East to produce alcohol. Application of amylase enzyme

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