Amylase enzyme.

1.    AMYLASES

Amylase is a group of enzymes whose catalytic function is to hydrolyze (breakdown) starch to give progressively smaller polymers composed of glucose units. They are found in animals and plants as well as produced by many microorganisms.

In order to make use of the plant-based starches present in food, large starch molecules first must be split into smaller units. This breakdown of starches is performed by amylases present in saliva and pancreatic juice.

All amylases are glycoside hydrolyses and act on α-1,4-glycosidic bonds.

Classification:-

1.    α-Amylase

 It is also called as glycogenase. It is the major form of amylase found in humans and other mammals. It is also present in seeds containing starch as a food reserve, and is secreted by many fungi and bacteria.

The α-amylases are endogenous calcium metalloenzymes, completely unable to function in the absence of calcium.

It cleaves α-1,4 bond in amylose and amylopectin.

2.    β-Amylase

β-amylase is synthesized by bacteria, fungi, and plants.

Working from the non-reducing end, It catalyzes the hydrolysis of the second α-1,4 glycosidic bond to give limit dextrin and β-maltose.

3.    γ-Amylase

It is also called as amyloglucosidase and glucoamylase.

It cleaves the α-1,4 glycosidic linkage as well as α-1,6 glycosidic linkage of amylose and amylopectin, yielding β-glucose.

Production of Amylases:-

Although many microorganisms produce this enzyme, the most commonly used for their industrial application are Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger.

Amylase from the Bacillus amyloliquifaciens species is active up to 90 degree Celsius.

 The fermentation of the bacterial amylase enzyme is accomplished in submerged culture at neutral pH and at temperature between 30-40 degree Celsius.

Cereal meal and starch rich medium are used along with an organic source of nitrogen. After 10 to 20 hours, formation of amylase starts and continues for another 100 hours.

pH of the medium must be below 6 during the fermentation to prevent the denaturation of the enzyme.

After incubation the fermentation medium was harvested by centrifugation at 5000 rpm for 20 minutes at 4°C. The supernatant was collected and subjected to estimate the amylase activity.

Application of Amylases:-

1.    Bacillary amylase is also used in clothing and dishwasher detergents to dissolve starches from fabrics and dishes.

2.    The most important applications of amylases are in the starch industry and in the production of sugars from starch.

3.    Amylases transform maize or potato starch in several steps to syrups containing sugar like glucose syrup and fructose syrup. These are subjected to a wide variety of uses as in sweets, ice-creams, tomato ketchup etc. Furthermore, glucose syrup is a basic ingredient of many other food ingredients and additives.

4.    Many spirits are produced from starches (e.g. in cereals or potatoes). These starches first must be split by amylases into sugar units, which then may be fermented into alcohol.

5.    Large quantities of amylases are used in cake manufacturing. In baked goods produced using yeast, this leads to an enhanced yeast performance and increases the volume of the product.

Enzyme immobilization.

            ENZYME IMMOBILIZATION

As enzymes are biological catalysts that promote the rate of reactions but are not themselves consumed in the reactions; they may be used repeatedly for as long as they remain active. However, in most of the processes, enzymes are mixed in a solution with substrates and cannot be economically recovered after the reaction and are generally wasted. Thus, there is an incentive to use enzymes in an immobilized or insolubilized form so that they may be retained in a biochemical reactor for further catalysis. This is done by Enzyme immobilization which may be defined as-

                   “The process whereby the movement of enzymes, cells, organelles, etc. in space is completely or severely restricted usually resulting in a water-insoluble form of the enzyme.”

Immobilized enzymes are also sometimes referred to as sound, insolubilized, supported or matrix-linked enzymes.

SALIENT FEATURES OF ENZYME IMMOBILIZATION:-

1. The enzyme phase is called as carrier phase which is water insoluble but hydrophilic porous polymeric matrix, e.g. agarose, cellulose, etc.
2. The enzyme phase may be in the form of fine particulate, membranous, or microcapsule.
3. The enzyme in turn may be bound to another enzyme via cross linking.
4. A special module is produced employing immobilization techniques through which fluid can pass easily, transforming substrate into product and at the same time facilitating the easy removal of catalyst from the product as it leaves the reactor.
5. The support or carrier utilized in immobilization technique is not stable at particular pH, ionic strength, or solvent conditions. Hence, may be disrupted or dissolved releasing the enzyme component after the reaction.

Advantages of enzyme immobilization:-

• Multiple or repetitive use of a single batch of enzymes.
• Immobilized enzymes are usually more stable.
• Ability to stop the reaction rapidly by removing the enzyme from the reaction solution.
• Product is not contaminated with the enzyme.
• Easy separation of the enzyme from the product.
• Allows development of a multienzyme reaction system.
• Reduces effluent disposal problems.

Disadvantages of enzyme immobilization:-

• It gives rise to an additional bearing on cost.
• It invariably affects the stability and activity of enzymes.
• The technique may not prove to be of any advantage when one of the substrate is found to be insoluble.
• Certain immobilization protocols offer serious problems with respect to the diffusion of the substrate to have an access to the enzyme.

 TECHNIQUE OF ENZYME IMMOBILIZATION:-

1.     Carrier binding.

·  Physical adsorption.

·  Covalent bonding.

·  Ionic bonding.

2.     Cross linking.

3.     Entrapment.

·  Occlusion within a cross linked gel.

·  Microencapsulation.

PHYSICAL ADSORPTION:-

This method is based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers. Examples of suitable adsorbents are ion-exchange matrices, porous carbon, clay, hydrous metal oxides, glasses and polymeric aromatic resins.

The bond between the enzyme and carrier molecule may be ionic, covalent, hydrogen, coordinated covalent or even combination of any of these.

Immobilization can be brought about by coupling an enzyme either to external or internal surface of the carrier.

The external surface binding method is advantageous as it does not involve conditions like pore diffusion. The disadvantages, however, include exposure of enzymes to microbial attack, physical abrasion of enzyme due to turbulence associated with the bulk solution.

The major disadvantage of the internal immobilization method is the pore diffusion.

Advantages of adsorption:-

·       Little or no confirmation change of the enzyme.

·       Simple and cheap.

·       No reagents are required.

·       Wide applicability and capable of high enzyme loading.

Disadvantages of adsorption:-

·       Desorption of the enzyme protein resulting from changes in temperature, pH, and ionic strength.

·       Slow method.

Methods of immobilization by adsorption:-

The absorptive immobilization of enzymes can be done by following methods:

1.     Static Process:- This is most efficient technique but requires maximum time. In this technique, enzyme is immobilized by allowing it to be in contact with the carrier without agitation.

2.     Dynamic Process:- This process typically involves the admixing of enzyme with the carrier under constant agitation using mechanical shaker.

3.     Reactor loading:- This process is employed for the commercial production of immobilized enzymes. The carrier is placed into the reactor and enzyme solution is transferred to the reactor with agitation of the whole content in the reactor.

4.     Electro-Deposition:- In this technique, carrier is placed in the vicinity of one of the electrode in an enzyme bath and electric current is applied leading to migration of enzyme towards the carrier. This results in deposition of enzyme on the surface of the carrier.

Covalent bonding:-

Covalent binding is the most widely used method for immobilizing enzymes. The covalent bond between enzyme and a support matrix forms a stable complex. The functional group present on enzyme, through which a covalent bond with support could be established, should be non essential for enzymatic activity.

The most common technique is to activate a cellulose-based support with cyanogen bromide, which is then mixed with the enzyme.

The protein functional groups which could be utilized in covalent coupling include:

• Amino group
• Carboxylic group
• Phenol ring
• Indole group
• Imidazole group

On the other hand examples of the polymeric supports include:

    ·       Amino and related groups of polysaccharides and silica gel etc.

·       Carboxylic acid and related groups of polyglutamic acid, carboxy methyl cellulose.

·       Aldehyde and acetal groups of polymers.

·       Amide gr. Of polypeptide.

The polymers may be engaged in direct coupling as well as could be modified by other coupling groups or activating groups. The most commonly used polymers are polysaccharides, polyvinyl alcohol, silica and porous glasses.

Advantages of covalent coupling:-

• The strength of binding is very strong, so, leakage of enzyme from the support is absent or very little.
• This is a simple, mild and often successful method of wide applicability

Disadvantages of covalent coupling:-

·       Enzymes are chemically modified and so many are denatured during immobilization.

·       Only small amounts of enzymes may be immobilized (about 0.02 grams per gram of matrix).

Cross linking:-

This method is based on the formation of covalent bonds between the enzyme molecules, by means of multifunctional reagents, leading to three dimensional cross linked aggregates.

The most common reagent used for cross-linking is glutaraldehyde.

Advantages of cross linking:-

·       Very little desorption(enzyme strongly bound)

·       Best used in conjunction with other methods.

Disadvantages of cross linking:-

·       Cross linking may cause significant changes in the active site.

 Entrapment:-

In entrapment, the enzymes or cells are not directly attached to the support surface, but simply trapped inside the polymer matrix. Entrapment is carried out by mixing the biocatalyst into a monomer solution, followed by polymerization initiated by a change in temperature or by a chemical reaction.

Polymers like polyacrylamide, collagen, cellulose acetate, calcium alginate or carrageenan etc are used as the matrices.

Advantages of entrapment:-

• Loss of enzyme activity upon immobilization is minimized.

Disadvantages of entrapment:-

• The enzyme can leak into the surrounding medium.
• Another problem is the mass transfer resistance to substrates and products.
• Substrate cannot diffuse deep into the gel matrix.

1. Occlusion within a cross linked gel:-

In this entrapment method, a highly cross-linked gel is formed as a result of the polymerization which has a fine "wire mesh" structure and can more effectively hold smaller enzymes in its cages.

Amounts in excess of 1 g of enzyme per gram of gel or fibre may be entrapped.

Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and natural polymer (starch) have been used to immobilize enzymes using this technique.

2. Microencapsulation:-

This entrapment involves the formation of spherical particle called as “microcapsule” in which a liquid or suspension of biocatalyst is enclosed within a semi permeable polymeric membrane.