Amylase enzyme:
- Starch, a glucose polymer is one of the most widely available plant polysaccharides which is hydrolysed by an enzyme called amylase.
- One of the main uses of amylases is in the production of sweetener for the food industry.
- The hydrolysis of starch with amylase result first in the production of short chain polymers called dextrins then disaccharide maltose and finally glucose.
- The most important enzyme in the starch saccharification process are
- α-amylases,
-amylases, glucoamylases, glucose isomerases, pullulanases, and isoamylases.
- α-amylase: (1-4 α
-Dglucan-glucanohydrolases)
- α-amylases are extracellular enzymes which hydrolyse
α-1,4-glycosidic bond.
- These enzymes are endoenzyme splitting the substrate in the interior of the molecule.
- Bacteria which produces α
-amylases are Bacillus subtilis, B. cereus, B. amyloliquefaciens, B. coagulens, B. polymyxa, B. acidocaldarins, B. lichiniformis, Lactobacillus micrococcus, Pseudomonas, Arthrobacter, Escherichia, Proteus, Thermomonospora, Serretia
- Fungi- Aspergillus, Penicillium, Cephalosporium, Neurospora and Rhizopus.
- The most important
-amylase produced are Bacillus amyloliquefaciens, B. lichiniformis, and Aspergillus oryzae.
- α-amylases are extracellular enzymes which hydrolyse
- β-amylase: (α
-1,4-D glucan-maltohydrolases)
- β
-amylases are usually of plant origin but some micro-organisms also produce.
- This enzyme catalyzes the hydrolysis of the second
α-1,4-glycosidic bond from non-reducing end.
- E.g. of β
-amylase production micro-organisms are B. polymyxa, B. cereus, B. megaterium, Streptomyces spp., Pseudomonas spp., Rhizopus spp. etc.
- β
- Gluco-amylase: (
α-1,4-D glucan-glucohydrolase)
- This enzyme acts on starch by splitting glucose unit from the non-reducing end.
- Micro-organism involved are Aspergillus niger, A. oryzae, Rhizopus spp.
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Bacterial α-amylase production:
- Bacterial
α-amylase production involves the function of the normal cell machinery for protein synthesis.
- The use of different type of antibiotic affects the enzyme production capacity. E.g. when actinomycetes D is added to amylase producing culture to inhibit RNA synthesis.
- Both RNA synthesis and growth ceases after 30 minutes.
- But the production of amylase continues.
- The enzyme formation rate is very low during exponential growth in many strains but just before the growth rate decreases and spore formation begins amylase production increases.
- The production of α
-amylase is regulated by several genes which have been only partially characterized whereas single step mutation increase yield by a factor of 2 to 7 so mutants have been selected after 5 steps which produce yields 250 times greater than the wild strain.
Production process:
- Preparation of pre-inoculum:
Bacillus spp. (B. lichiniformis, B. amyloliquefaciens, B. subtilis) grown on NA slant for24hrs.
- Transfer some colony with sterile needle into the flask containing sterile NB.
- Incubate at 37oC for 24hrs.
- Fermentation:
- Solid or submerged fermentation.
- Batch or Fed-batch culture with inoculum of 0.5ml in 100ml of sterilized fermentation medium.
Medium composition:
| 5% starch | 0.5%peptone |
| 0.56% NH4NO3 | 0.2%yeast extract |
| 0.28% sodium citrate | pH=6.8 |
| 0.13% KH2PO4 | Temp- 27-30oC |
| 0.05% MgSO4.7H2O | Time- 48hrs |
| 0.01%CaCl2.2H2O |
Extraction of α-amylase:
- Centrifugation at 3000 rpm for 30 mins or 5000 rpm for 20mins at 4oC and collect the supernatant for crude enzyme.
Fungal α-amylase production:
- Inoculum- spores of Aspergillus oryzae obtained from DDA or SDA plate at 28oC for 5-6 days.
- Medium
| 1-2% NaNO3 | 0.05% Mg(H2PO4)2 |
| 0.1% K2HPO4 | 2% mast extract |
| 0.1% MgSO4 | Temp- 28-30oC |
| 0.05% KCl | Time – 3-4 days |
| 0.003% FeSO4 | pH: 3-4 |
| 0.08% Mg(NO3)2 |
Application of α![]()
-amylase:
- Amylases have potential application in a number of industrial processes such as in food, textiles, paper industries, bread making, glucose and fructose syrup, detergents, fuel, ethanol, from starches, fruit juices, alcoholic beverages, sweatness digestive aid, spot remover in dry cleaning.
- Bacterial
α-amylase are now also used in areas of clinical, medicinal and analytical chemistry.
- In most wildly used thermostable enzymes are the amylases in starch industry.
| Industry (field) | Applications |
| Starch industry | Liquefaction of starch for production of glucose, fructose, maltose |
| Milling | Modification of |
| Alcohol | Liquefaction of starch before the addition of malt for saccharification |
| Bakery | Increase in the production of fermentable carbohydrates |
| Brewing | Preparation of barley, liquefaction of additives, enhanced fermentability of grains, modification of beer characteristics |
| Paper industry | For the liquefaction of starch without sugar production for sizing of paper |
| Textile industry | Continuous de-sizing at high temperature |
| Feed industry | Improvement of utilization of enzymatically barley in poultry and calf raising |
| Sugar industry | Improvement of filter-ability of can sugar juice through breakdown of starch in juice. |
| Laundry and detergent | Increase in cleaning power additive in dish washer detergent |