Author: Dharmendra Kumar
Enzymes are globular proteins and like other proteins consist of long chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. Enzymes are responsible for many essential biochemical reactions in microorganisms, plants, animals, and human beings. They differ in function in that they have the unique ability to facilitate biochemical reactions without undergoing change themselves. This catalytic capability is what makes enzymes unique. Enzymes, biological catalysts with high selectivities, have been used in the food industry for hundreds of years, and play an important role in many other industries(washing agents, textile manufacturing, pharmaceuticals, pulp and paper).They without being consumed in the process, can speed up chemical processes that would otherwise run very slowly, or in some cases, not at all [Cavaco-Paulo& Gübitz 2003].After the reaction is complete, the enzyme is released again, ready to start another reaction. Usually most enzymes are used only once and discarded after their catalytic action. All known enzymes are proteins. They therefore consist of one ore more polypeptide chains and display properties that are typical of proteins. Some enzymes require small non-protein molecules, known as cofactors, in order to function as catalysts [Jenkins 2003].
In the green leaves of plants carbon dioxide and water are transformed into glucose and oxygen under the influence of sunlight and with the help of chlorofyl. This process is known as photosynthesis. During the day this starch is deposited as grains in the leaf, the so-called leaf-transition starch. During the night this starch is partially broken down again into sugars which are transported to other areas of the plant. From these sugars the starch arises which is won in the familiar grain shape. The forming of starch is a process which has by far not been clarified yet and during which a number of enzymes play a role.
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. The major industrial sources are maize, tapioca, potato, and wheat, but limitations such as low shear resistance, thermal resistance, thermal decomposition and high tendency towards retrogradation limit its use in some industrial food applications[Goyal et al.2005;M.J.E.C.van der Maarel et al. 2002].With the help of a microscope the grain shape reveals from which plant species the starch derives.Native starch, the starch as it occurs in the plant, can not be dissolved in cold water. When we scatter starch, while stirring, into water we get a milky white suspension which can be stirred without much difficulty. When the stirring is stopped the starch sinks to the bottom (sedimentation), during which process a transparent upper layer is formed. When the suspension is heated the white colour disappears at a temperature characteristic for starch. The starch dissolves into an almost transparent solution. This is what we call gelatinized starch. In comparison with the ungelatinized suspension, stirring takes considerably more difficulty.The temperature at which the resistance during stirring noticeably increases, is called the gelatinization temperature.Gelatinizing starch into viscous substances (swellings) is one of the most, if not the most, important characteristic(s) of starch. This phenomenon lies at the basis of the successful application of starch in a large number of sectors. Among carbohydrate polymers, starch is currentlyenjoying increased attention due to its usefulness in different food products. Starch contributes greatly to the textural properties of many foods and is widely used in food and industrial applications as a thickener, colloidal stabilizer, gelling agent, bulking agent and water retention agent [JaspreetSinghaet al. 2007]. Starch is a polymer of glucose linked to another one through the glycosidic bond. Two types of glucose polymersare present in starch: amylose and amylopectin
(EC 22.214.171.124) The α-amylases are calcium metalloenzymes, completely unable to function in the absence of calcium., α-amylase breaks down long-chain carbohydrates by acting at random locations along the starch chain, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. α-amylase tends to be faster-acting than β-amylase because it can act anywhere on the substrate. In human physiology, both the salivary and pancreatic amylases are α-Amylases.
(EC 126.96.36.199 ) β-amylase is another form of amylase synthesized by bacteria, fungi, and plants. β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond, working from the non-reducing end, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit. Both α-amylase and β-amylase are present in seeds; β-amylase is present in an inactive form prior to germination, whereas α-amylase and proteases appear once germination has begun. Animal tissues do not contain β-amylase.
(EC 188.8.131.52 ) 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, yielding glucose. Unlike the other forms of amylase, γ- amylase is most efficient in acidic environments and has an optimum pH of 3.
Sources of Amylases
Amylases are ubiquitous enzymes produced by plants, animals and microbes, where they play a dominant role in carbohydrate metabolism. Amylases from plant and microbial sources have been employed for
centuries as food additives. Barley amylases have been used in the brewing industry. Fungal amylases have been widely used for the preparation of oriental foods.
To meet the demand of industries, low-cost medium is required for the production of a-amylase. Both SSF and submerged fermentation (SmF) could be used for the production of amylases, although traditionally these
have been obtained from submerged cultures because of ease of handling and greater control of environmental factors such as temperature and pH. Mostly synthetic media have been used for the production of bacterial amylase through SmF. The contents of synthetic media such as nutrient broth, soluble starch, as well as other components are very expensive and these could be replaced with cheaper agricultural by-products for the reduction of the cost of the medium. SSF resembles natural micro biological processes such as composting and ensiling, which can be utilized in a controlled way to produce a desired product. SSF has been used for long to convert moist agricultural polymeric substrates such as wheat, rice, soy, cassava, etc. into fermented food products including industrial enzymes.
Optimization of various parameters and manipulation of media are one of the most important techniques used for the overproduction of enzymes in large quantities to meet industrial demands. Production of a-amylase in fungi is known to depend on both morphological and metabolic state of the culture. Growth of mycelium is crucial for extracellular enzymes like a-amylase . Various physical and chemical factors have been known to affect the production of a-amylase such as temperature, pH, period of incubation, carbon sources acting as inducers, surfactants, nitrogen sources, phosphate, different metal ions, moisture and agitation with regards to SSF and SmF, respectively. Interactions of these parameters are reported to have a significant influence on the production of the enzyme.
PURIFICATION OF AMYLASE
Industrial enzymes produced in bulk generally require little downstream processing and hence are relatively crude preparations. The commercial use of _-amylase generally does not require purification of the enzyme, but enzyme applications in pharmaceutical and clinical sectors require high purity amylases. The enzyme in the purified form is also a prerequisite in studies of structure-function relationships and biochemical properties. Different strategies for purification of enzymes have been investigated, exploiting specific characteristics of the target biomolecule. Laboratory
scale purification for _-amylase includes various combinations of ion exchange, gel filtration, hydrophobicity interactions and reverse phase chromatography. Alternatively, _-amylase extraction protocols using organic
solvents such as ethanol, acetone and ammonium sulfate precipitation and ultrafiltration have been proposed. These conventional multi-step methods requires expensive equipments at each step, making them laborious, time consuming, barely reproducible and may result in increasing loss of the desired product.
Amylases are among the most important enzymes used in industrial processes. Although, the use of amylases, a-amylases in particular, in starch liquefaction and other starch based industries has been prevalent for many decades and a number of microbial sources exist for the efficient production of this enzyme, the commercial production of this enzyme has been limited to only a few selected strains of fungi and bacteria. Moreover, the demand for these enzymes is further limited with specific applications as in the food industry, wherein fungal a-amylases are preferred over other microbial sources due to their more accepted GRAS status. Structural conformation plays an important role on amylase activity .
- Aehle, W., Misset, O., (1999). Enzymes for industrial applications. In:Rehm HJ, Reed G, editors. Biotechnology, 2nd ed. Germany:Wiley-VCH, 189-216.
- Ahlawat, S., Dhiman, S.S., Battan, B., Mandhan, R.P., Sharma, J., (2009).Pectinase production by Bacillus subtilis and its potential application inbiopreparation of cotton and micropoly fabric. Process Biochemistry,44,521–526.
- Amritkar, N., Kamat, M., Lali, A., (2004). Expanded bed affinity purificationof bacterial α-amylase and cellulase on compositesubstrate analogue–cellulose matrices, Process Biochem.,39, 565–570.
- Arauza, L.J., Jozalaa, A.F., Mazzolab, P.G., Penna, T.C.V., (2009). Nisinbiotechnological production and application: a review. Trends Food SciTechnol.,20, 146-154.
- Babu, K.R., Satyanarayana, T., (1995). α-Amylase production by thermophilicBacillus coagulansin solid-state fermentation, Process Biochem., 30, 305–309.
About Author / Additional Info:
Dharmendra kumar ICAR-IARI, Division of Microbiology, New Delhi-12