Alpha amylase

α -Amylase is an enzyme which has ability to catalyze the hydrolysis of internal α-1, 4-glycosidic linkages in starch to yield products like glucose and maltose.

1. Amylase production by solid-state fermentation of agro-industrial wastes using Bacillus sp . Saxena , R. and Singh, R., 2011. BGE 503 Enzyme Technology Effat Jahan Tamanna Department of Biotechnology and Genetic Engineering, Jahanginagar University . α -Amylase

2. Introduction Enzymes are biological catalysts which are indispensable components of reactions and work at milder conditions than chemical catalysts. Amylases are important hydrolase enzymes which have been widely used since many decades. R andomly cleave internal glycosidic linkages in starch molecules to hydrolyze them into dextrins and oligosaccharides . T hree types of amylase enzymes: α -amylase, β -amylase, γ -amylase. Among these α -amylase is mostly used.

3. α -Amylase is an enzyme which has ability to catalyze the hydrolysis of internal α-1, 4-glycosidic linkages in starch to yield products like glucose and maltose. It was first discovered and isolated by Anselme Payen in 1833. Mostly extracellular . EC (Enzyme Commission) number : 3.2.1.1 Molecular weight: 51 - 55.4KDa. α -amylase is produced by plants, animals and microorganisms . It is a major digestive enzyme secreted in pancreas and salivary glands . Other names: glycogenase , endoamylase , 1,4- α -D- glucan glucanohydrolase . α -Amylase

4. α -Amylase Active sites contains trio of acidic groups – white and red. A short chain of five sugars – yellow and orange. The site of cleavage – pink. A calcium ion – grey. A chloride ion – green.

5. Sources of α -amylase There are mainly two methods which are used for production of α-amylase. These are: 1) Submerged fermentation and 2 ) Solid state fermentation. Bacteria Fungi Bacillus subtilis Aspergillus oryzae Bacillus licheniformis Penicillium fellutanum Bacillus cereus Thermomyces lanuginosus Bacillus amyloliquefaciens Aspergillus niger Bacillus coagulans Penicillium roquefortii Chromohalobacter sp. Engyodontium album Halobacillus sp. Penicillium chrysogenumm Halomonas meridiana Penicillium janthinellum Rhodothermus marinus Pycnoporus sanguineus Genetic Engineering of Microbes α-Amylase e nzyme can be produced from genetically engineered microorganisms . Methods Used: Conventional mutagenesis (UV or chemical exposure) or Recombinant DNA technology.

6. Materials and Methods Isolation of b acteria: 50 strains were isolated and characterized. Screening of bacterial isolates: 3 strains showed the biggest zone of clearance in starch hydrolysis. Selected strain : Bacillus subtilis RSA-27, RSB-75 and RSE-162. Inoculum preparation: The selected bacterial strains were inoculated in nutrient broth followed by incubation at 37°C for 24 h. Substrate: Four agro-industrial wastes - w heat bran, gram husk, rice bran and mustard o ilseed cake . Fermentation media : Media was prepared and s terilized.

7. Optimization: Substrate: Four substrate material were made particle size of 1.0 to 2.0 mm. Moisture: 1:2 , 1:3, 1:4 and 1:5 . Temperature and pH: 37°C and 7.0. Inoculum size: 1 %, 5%, 10% and 20% of bacterial culture. SSF technique: The flasks inoculated with inoculum, thoroughly mixed and followed by incubation at 37°C for 5 days . Enzyme assay: C arried by DNSA ( 3, 5-dinitro salicylic acid) method . Partial purification of enzyme: precipitation. Centrifugation at 12000g for 20 minutes at 4°C . Characterization of p urified enzyme  

8. Table – 01 Strain Activity (U/g) (on the third day) RSA-27 900 RSB-75 200 RSE-162 626 Results and Discussions Strain RSA-27 , a gram positive rod shaped bacterium, was selected for further testing and optimization. Table – 02 Substrate Activity (U/g) Mustard oilseed cake 5166 Wheat bran 1233 Gram husk 900 Rice bran 933 Mustard o ilseed c ake was selected as substrate for further optimization.

9. Figure: α -Amylase production by three strains RSB- 75 (▥), RSA- 27 (▦) and RSE 162 (▤ ).

10. Table – 03 Moisture Content Activity (U/g) 1:2 1233 1:3 5366 1:4 2166 1:5 2966 Table – 04 I noculum size Activity (U/g) 1% 226 5% 1733 10% 3333 20% 5133 Best Result Strain RSA-27 produced about 5400 U/g of α - amylase at 1:3 moisture content, 20% inoculum, after 72 h of incubation with m ustard o ilseed cake as the substrate.

11. Figure: Effects of variation in substrate , moisture c ontent and inoculum s ize in enzyme production.

12. Various parameters that affects enzyme activity were optimized: Optimum temperature : 50°C Optimum pH: 6 Figure: (A) Temperature optimization for enzyme production [ 30°C to 70°C ]. (B) pH optimization for enzyme production [5 to 9 ].

13. Thermostability and pH stability of the enzyme: The enzyme was found to be stable mostly at 30°C for 2h. The enzyme was very stable at neutral pH (6- 7 ). Figure: (A) Stability of enzyme at temperatures 30°c to 70°C (◆) 30°C , (●) 40°C , ( ▲) 50°C , (■) 60°C , (□) 70°C . (B) Stability of enzyme at pH 5 – 9 (◆) 5, (■) 6, (▲) 7, (●) 8, ( Ж) 9

14. Effects of m etal i ons: The presence of salts as , , and at 5mM and 10mM concentration enhanced the activity of the enzyme.   Effect of ions on the thermal stability of the enzyme at 70°C (▦) Crude , ( ▧) 5Mm , ( ▥) 10mM  

15. Conclusion Among all enzymes, α-amylase covers 25% of total enzyme market . α-amylase is used in Starch conversion Bakery Industry Detergent Industry Textile Industry Fuel alcohol Production Pharmaceutical industry Textiles and apparel industries capture the leading position in Bangladesh and α-amylase enzyme can minimize these costing. Living in an era of depleting fossil fuels with a desperate need to produce alternative forms of energy, this enzyme is a ray of hope. As enzyme helps to keep the environment clean, more researchers are focusing on the microbial production of the enzyme.