Production of Microbial Amylases

Amylase are enzymes that breakdown starch or glycogen. Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans. 

Microbial amylases are  produced by microorganisms to hydrolyze starch. Bacteria and fungi secrete amylases to the outside of their cells to carryout extra cellular digestion. They were first produced in 1894 by the Japanese scientist Jokichi Takamine at Peoria, Illinois (USA) from a fungal source and was used as a pharmaceutical aid- Takadiastase (from A. oryzae) for the treatment of digestive disorders- a pharmaceutical digestive aid.

Starch is a polymer made of linear amylose D-glucose units joined by α -1,4-glycosidic bonds and branched amylopectin with α 1,6 -linkages at branching points. Amylases are important enzymes employed in processing industries for the hydrolysis of starch into simple sugars. 

 There are three types of amylases, namely: 

α amylase (endo-1,4-α-D glucohydrolase), β amylase (β-1,4-glucan maltohydrolase), and glucoamylase (amyloglucosidase). Each of the three types of amylases has a unique way of acting on starch substrate. 

Starch <α-amylase>  dextrins + maltose (liquefying amylase) 

Starch <β-amylase > maltose (saccharifying amylase) 

Dextrins <dextrinase> maltose 

 α amylase (being an endoamylase) cleaves or breaks the α-1,4-glucosidic linkages in starch internally to give glucose, maltose, or dextrins.

β amylase, an exoamylase, cleaves the glycolytic bonds removing two glucose units at a time thus producing maltose. 

Glucoamylase, on the other hand, cleaves both the α-1,4 and α-1,6-glucosidic linkages to yield glucose, maltose, and limit dextrins. 

β-Amylase, is a saccharifying enzyme, forming maltose directly from starch by cleaving disaccharide units from the open ends of chains. The a-amylases have good liquefying ability. 

Amyloglucosidase is a saccharifying enzyme unique in that it attacks starch and 1,4-linked glucose oligosaccharides with direct formation of glucose. 

A range of amylases, suitable for almost any kind or extent of starch conversion, is now available from microbial sources. 

Microbial amylases can be produced by bacteria and fungi (which include molds and yeast). Fungi among many microbes are good sources of amylolytic enzymes. Amongst the bacteria genera, the genus Bacillus (especially Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens) are greatly used for amylase production. However, amongst the fungi, Aspergillus spp and Rhizopus spp are the preferred strains for amylase production. 

 Although amylases can be produced by plants, animals, and microorganisms, microbial amylases are important because of their greater thermal stability, different sugar profiles, and a long history of safe use. 

Application of Microbial Amylases

Microbial amylases (especially thermostable alpha amylase) have various industrial applications because of their greater thermal stability, different sugar profiles, and a long history of safe use. 

 They can be exploited for high fructose corn preparation, for the production of alcohol and brewing, for paper coating, for the preparation of detergents. Industries requiring microbial amylases include: clinical, medicinal, analytical chemistry, textile, food, and distilling industries. 

• Microbial amylases are used for modifying starch in vegetable purees, and in treating vegetables for canning. 
• Other applications of microbial amylases where both fungal and bacterial enzymes are utilized are in processing cereal products for food dextrin and sugar mixtures and for breakfast foods, for preparation of chocolate and licorice syrups and for recovering sugars from scrap candy of high starch content. 
• Fungal amylases are also used for starch removal for flavoring extracts and for fruit extracts and juices, and in preparing clear, starch-free pectin. 
• By proper control of the type and proportion of enzymes used (α -amylase, amyloglucosidase, maltase), syrups of any desired proportions of glucose, maltose, and dextrins may be produced.

Microbial fermentation has been used for amylase production since long time. Manipulation of culture conditions such as temperature and pH, allow greater control of production process. 

The substrate used for the production of microbial amylase is starch.

There are two main methods of amylase production that utilizes the substrate (starch), namely: Solid State Fermentation (SSF) and Submerged Fermentation (SmF).

The cost of enzyme production in submerged production is high, which necessitates reduction in production cost by alternative methods. The contents of synthetic media are very expensive and these contents might be replaced with more economically available agricultural by products for the reduction of cost of the medium.

SSF is a technique for growing microorganisms such as fungi and bacteria on a moist solid substrate. The use of agricultural wastes makes solid state fermentation an attractive alternative method Researchers have reported amylase production in solid state fermentation with wheat bran and rice husk as substrates.

Industrially important enzymes have traditionally been obtained from submerged fermentation because of the ease of handling and greater control of environment factors such as temperature and pH. 

Enzymatic hydrolysis of starch has now replaced acid hydrolysis in over 75% of starch hydrolyzing processes due to many advantages.

Bacterial Amylases

Bacillus species – B. subtilis, B. cereus, B.coagulans, B. stearothermophilus, Pseudomonas, Serratia, Clostridium etc

Bacteria produce more of α-amylase. It can be done either through solid state fermentation (SSF) and submerged fermentation (SmF).

Media can be natural raw materials such as wheat bran, CSL, corn starch, which contain trace elements, vitamins, growth factors etc. Organic and inorganic nitrogen may be added if required. Stillage from alcohol production from grain or soluble/hydrolyzed starch supplemented with ammonium salts and buffer can be used. Hydrolyzed soy bean cake, pea nut cake, casein act as sources of nitrogen and mineral salts. Wheat bran supplemented with salts may be used.

Incubation is done at 25-37^oC for 2-6 days, in tray method of solid state fermentation and 24-48 ^oC for 24-48 hours for submerged method. pH can increase during fermentation, so maintained using buffers or media components like urea, peptone etc.

Bacterial α-amylases require minimum purification. After fermentation, extracellular enzyme is harvested and recovered using precipitation, concentrated by dialysis and purified using different chromatographical/adsorption techniques. Other contaminating enzymes are removed by selective adsorption or inactivation.

α-amylases from B. subtilis and especially B. stearothermophilus, are thermostable with industrail applications.

Fungal Amylases

Fungi like Rhizopus, Mucor rouxii, Penicillium spp, Candida spp, etc can be used for amylase production. Aspergillus spp are widely used in both solid state fermentation (SSF) and submerged fermentation (SmF) processes for amylase production.

Starchy grain mash is inoculated with the molds. Amylases are produced by saccharification of mashed substrate and this process is termed Amylo process.

Aspergillus oryzae is used in solid state fermentation and Aspergillus niger is generally used in submerged fermentation. Moistened, steamed rice/wheat bran is used for amylase production from  Aspergillus oryzae by tray method. In which the mold is grown on thin layers of the medium in trays. Alternatively, drum method can also be used, where the bran is filled loosely in a rotating drum. Maximum yield is obtained in about 48 hours at 30^oC in tray method with proper humidity and adequate ventilation.

Moisture content is critical in SSF and it depends on the substrate thickness and incubation temperature, mainly. Incubation can be done from overnight to 4 days. This depends on the organism employed. Fungal α-amylase accumulates largely in the stationary phase and even during autolysis.

After fermentation, amylases are extracted from the fermented mycelium and purified using precipitation, chromatography and finally concentrated. Contaminating enzymes in the fermented broth may be removed in the purification process.

SSF can also be carried out in deep tanks with flour/starch as raw material supplemented with inorganic salts. Addition of stillage, corn steep liquor and yeast extract is found to increase the yields.

 SSF has the upper hand due to its advantages, namely:

i. High volumetric productivity is obtained.

ii. Relatively higher concentration of the enzyme is produced.

iii. Less effluent is generated.

iv. Low cost of production is required.

v. It is easy to use.

vi. Raw starch is efficiently digested

    
Applications 

    Amylases have played a key role in both biotechnological and pharmaceutical industries. Thermophilic bacteria and fungi are efficient producers of thermostable amylases. For an efficient large scale production of amylases, the structural and functional relationships of these enzymes have to be known in detail. An understanding these relationships will lead to improving the stability of the existing enzymes as well as the discovering of many new ones.