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Alpha amylase is an enzyme involved in digestion of glycogen and starch. It does this by hydrolyzing the alpha bonds which link these large polysaccharides. These bonds are known as glycosidic bonds, and they link one or two carbohydrate portion with a non carbohydrate moiety. Hence, it breaks down large food molecules into relatively smaller food particles that can be taken up by the small intestines, and subsequently metabolized to give energy in the body. This breakdown of polysaccharides gives rise to maltose and glucose. These form the principal metabolic substrates in the body, thus proving crucial in respiration and energy production.
Human beings consume a given amount of calories in form of carbohydrates. Starch is the most common form of these calories. It is insoluble since it's a long chain polysaccharide. It's often found in foodstuffs like rice, potatoes, and corn. Thus, alpha amylases speed up reaction rates, leading to acceleration of breakdown of starch to produce smaller and soluble particles. Alpha amylase continues with the cleavage to yield maltose, which is made up of two glucose molecules. It is this maltose that is broken down by enzymes in the small intestines to yield glucose.
Alpha amylase is the major form of amylases that are found in other mammals and human beings. It is also found in seeds which contain food reserves in form of starch. Some fungi also secrete alpha amylase. Alpha amylases belong to a family of enzymes known as Glycoside Hydrolase Family 13. They cleave the bonds at position 1 and 4 of the polysaccharide chains hence degrading both the unbranched and branched forms of starch. These alpha 1, 4 bonds serve to connect various glucose monomers hence, leading to formation of polysaccharides. Excess calcium ions may inhibit the catalytic activity of this enzyme by binding to the amino acids required for this catalytic activity.
In human beings, alpha amylase is found a lot in pancreatic juice and also in saliva. These two have their own isoforms of human alpha amylase. On isoelectric focusing, these two alpha amylases behave differently, and thus, they can be separated while testing by use of specific monoclonal antibodies. All amylase isoforms in human beings link to chromosome 1p21.
It is also known as ptyalin. It is found in saliva and it participates in breakdown of starch to dextrin and maltose. This helps in breaking down the large insoluble polysaccharides into small molecules that are insoluble. These include erythrodextrin, achrodextrin, and amylodextrin. Further breakdown of these three leads to production of maltose. Salivary alpha amylase acts on linear glycosidic linkages which occur in starch. This leads to breakdown of the alpha 1, 4 glycosidic linkages occurring in these long chained polysaccharides. Hydrolysis of branched glycosidic bonds requires an enzyme capable of acting and hydrolyzing branched products. When salivary amylase gets to the stomach, it becomes inactivated by the acidic pH of that stomach environment. Total inactivation occurs at a pH of around 3.3 at body temperature. Thus, the higher the acidic nature of the contents of the stomach, the higher the rate of inactivation.
Optimum conditions for salivary alpha amylase
The optimum pH under which this enzyme attains optimum activity is between 5.6 and 6.9. The optimum body temperature should be around 370C. The presence of some activators as well as anions serves to increase the activity of this particular enzyme. These anions and activators include bromide and chloride. These two are the most effective in enhancing enzymatic activity compared to iodide. Phosphate and sulfate also show minimum activity.
Genetic variation occurring in human salivary alpha amylase
During evolution and over the years, salivary alpha amylase gene has undergone significant duplication. Hybridization studies done on the DNA of human beings has indicated that many people have multiple tandem repeat sequences of the gene coding for the enzyme. The study shows that the number of the gene copies identified correlate with the usual levels of alpha amylase. This has been shown by measurements obtained from protein blot assays that used antibodies to human alpha amylase. The number of gene copies is usually associated with the existing evolutionary exposure especially to diets containing high starch content.
This enzyme is also known as ptyalin. This enzyme randomly cleaves glycosidic linkages or bonds existing in amylose to give rise to maltotriose, dextrin, and maltose. For it to retain its normal anomeric configuration, it adopts a mechanism of double displacement. The optimum requirements for its action include an optimum pH under which this enzyme attains optimum activity is between 5.6 and 6.9. The optimum body temperature should be around 370C. The presence of some activators as well as anions serves to increase the activity of this particular enzyme. These anions and activators include bromide and chloride. These two are the most effective in enhancing enzymatic activity compared to iodide. Phosphate and sulfate also show minimum activity. Pancreatic amylase has found application especially in the medical field and also in some industrial applications.
Applications using the enzyme amylase
Diagnosis in medicine
Tests for amylase have been used a lot to monitor and detect certain disease conditions. This is because measurement of pancreatic amylase is easier to perform compared to that of pancreatic lipase. Medical laboratories take either measurements of total amylase or pancreatic amylase. An increase in total serum levels of amylase can be noted in some disease conditions such as salivary gland trauma and also in mumps. These enzymes occur in trace amounts in blood. Therefore, when taking the samples for measurement, proper timing should be done so as to take blood samples capable of giving appropriate results. For example in pancreatitis, blood should be taken immediately after the patient has experienced a bout of pancreatic pain, or else it will be excreted rapidly through the kidneys. Also, salivary amylase has been used a lot as a biomarker in cases of clinical stress states, whereby there is no need to obtain blood samples.
Therefore, increased plasma levels of the enzyme has been indicative of the following disease conditions: salivary trauma and this also includes anaesthetic intubation, mumps as a result of the inflamed salivary glands producing more of the enzyme, renal failure due to decreased excretion of the enzyme, and pancreatitis due to existing damage to the cells that produce the enzyme. Higher readings above 10 times the normal upper limit usually indicate that the patient is suffering from pancreatitis. Five to ten times higher than the normal limit are indicative of gastrointestinal disease especially those affecting the duodenum or ileum. It may also be indicative of renal failure. Slightly lower elevations above the normal limit indicate presence of a disease affecting the salivary glands. Other applications in the medical field especially in diagnosis includes disease conditions such as tumors, trauma, biliary tract disease, acute appendicitis, liver disease, biliary tract obstruction, mesenteric infarction, irradiation leading to damage of the salivary glands, tumors of the testes, prostate, ovary, thymus, esophagus, lung, thyroid gland, renal insufficiency, and ruptured ectopic pregnancy. Other conditions associated with higher amylase levels include HIV/AIDS, macroamylasaemia, diabetic ketoacidosis, and use of some drugs like diuretics, opioids, and steroids.
Also, some disease conditions are associated with lower than normal amylase values. Conditions such as exocrine pancreatic insufficiency are associated with low serum levels of amylases.
Genes coding for amylases
The genes coding for salivary amylase include AMY1B, AMY1A, and AMY1C. The ones coding for pancreatic amylase include AMY2B and AMY2A.
Alpha amylase has some industrial applications. Some of these include production of ethanol by breaking down starch in grains used so as to produce fermentable sugars. In this process, there is production of high fructose syrup from the corn after treatment with alpha amylase leading to production of oligosaccharides. The source of this enzyme is Bacillus licheniformis.
Research on amylases
Below, I have taken a look at an article on alpha amylases from microbial sources and looked at an overview of recent developments. It also covers progress that has been made in research in regard to microbial alpha amylase. The paper gives a little bit more detailed aspect of the alpha amylases. The content of the paper is not contrary to the existing information on alpha amylases, but rather more detailed information on the same. This enzyme has a wide range of applications in pharmaceuticals, food, and detergents. That article took a look at the characteristics of the enzyme, its sources, downstream processing, production aspects, biochemical properties, enzyme engineering, industrial applications, and recent research developments.
Alpha amylases form part of a group of starch degrading enzymes responsible for the internal hydrolysis of glycosidic bonds in polysaccharides to give smaller fragments. Most of these alpha amylases require metallic cations to act as catalysts hence, accelerating their activities. Calcium cations are the most common. Their industrial application majorly revolves around conversion of starch into sugar syrups and also production of cyclodextrins especially in the pharmaceutical industry. This enzyme accounts for approximately 30% of the enzyme production in the world. This group of enzymes known as amylases is majorly classified into two main groups: the hydrolyzing enzymes and the transglycosylating enzymes. Most industrial applications prefer enzymatic hydrolysis to acidic hydrolysis due to the many advantages that come with it.
Alpha amylases fall under a class of enzymes comprising of debranching enzymes, amylases, transferases, exomylases, and endomylases. Sources of amylases range from animals, plants, and microbes. Those from microbial and plant sources have been used over the centuries as food additives. Some like barley amylases have been employed widely in beer brewing.
Characteristics of the catalytic domain
The alpha amylase family was identified using the four sequence regions that cover the strands beta 3, 4, 5, and 7 of the beta/alpha domain. The two dimensional structure contains three domains called domain A, domain B, and domain C. Domain A consists of the N terminal while B consists of a long loop which protrudes in between alpha strand 3 and beta strand 3 helix. The whole barrel consists of 8 alternate beta strands and alpha helices.
To meet the required demands in industrial production, low cost medium is used to produce alpha amylase. The most commonly used medium include SSF and SmF (submerged fermentation). Recent advancements in biotechnology have led to use of synthetic media comprising of soluble starch, nutrient broth and other products. For fermentation to occur well, optimum conditions must be in place so as to ensure maximum activity of the enzyme. These conditions include conditions such as temperature and pH. Others include moisture requirements, nitrogen and carbon sources among others.
The influence temperature has is related to growth of microorganisms. Hence, the best or optimum temperature depends on whether the cultures used are thermophilic or mesophilic. Most fungi used are mesophilic and the optimum temperature ranges from 250C to 300C. For thermophilic fungi, the optimum temperature recorded lies between 50 to 550C. Thus, different microbes have different requirements in regard to temperatures under which optimum activity is observed.
PH determines the growth rate and morphology of the microorganisms, since most of them are sensitive to concentration of hydrogen ions which may be present in the media. Earlier studies carried out revealed that fungi might be slightly acidic while bacteria require a neutral environment for proper growth. Other requirements include moisture, supplementation of metal ions, nitrogen sources, carbon sources, surfactants and presence of oxidative stress.
Advancements that have been made
Cloning of alpha amylase genes
In the recent years, extensive research has been done and with use of the latest technology, scientists have tried to come up with enzymes that have novel properties. One of the ways that has been explored extensively is cloning of genes under protein engineering and hyperproduction. Cloning has been done in a bid to study hyperproduction, sequences, characteristics and expression of this family of enzymes. This has been made possible through enzymatic engineering. This was necessitated by requirement of amylases with different biochemical and physiological properties capable of being applied in various industries. Hence, this led to the production of novel enzymes. This form of engineering includes introduction and integration of desired properties into the appropriate gene. These properties include wide pH profile, thermo stability, and calcium independency, high starch degrading capability, high activity at higher starch concentrations, protease resistance, hyperproduction, and ability to be insensible to catabolite repression.
These novel enzymes have found use in food and beverage industries which utilize approximately 90% of these enzymes worldwide.
Alpha amylases form part of the group of enzymes that are widely used especially in preparation and production of fermented foods. Their demand is on a rise, and apart from the food and beverage industrial application, they also find use in other industries like textile, paper and pulp industries. With increase in their application and activity spectrum, there is a greater demand for enzymes with specificity. Research has now been focused on the development of pH tolerant and thermo tolerant enzymes from microorganisms. This has been made possible by genetic modification or by applying mutagenesis at the sites making them acquire desired properties. Most of the commercial productions carried out occur in submerged fermentation but scientists are currently looking at a possibility of solid state fermentation.