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Reaction Rates of Alpha Amylase Enzyme

Alpha-amylase is a type of enzyme (biological catalyst) which reduces the activation energy required in the hydrolysis of starch which thus speeds up the reaction rate. This reaction is increased roughly by a value of x 10¹° due to the amylase enzymes otherwise this reaction would not happen fast enough. Wang, (2009) says the starch molecules (glucose polymers linked together by the alpha-1,4 and alpha-1,6 glucosidic bonds) need to broken down into sugars and changed in to separate glucose units so the young plant can exploit the carbon and energy stored in the starch.
The aim of the following experiments is to find out the response of alpha-amylase enzymes to a range of pH and temperatures and determine what is the favourable pH and temperature for the highest activity of the enzymes. Enzymes are specialised and only work on a particular substrate(s) (the substance on which the enzyme acts) so if they are changed/damaged in any way the speed of the reaction is significantly reduced. Temperature and pH if wrong can be 2 factors which damage or even denature/destroy the enzymes.
Discussion and conclusion
From the results we can see that the most favourable temperature and pH is 40°C and pH 5.5. Temperature
We also know that the speed of the hydrolysis of starch is significantly reduced at temperature extremes of 0°C and 80°C. According to Bloomfield and Stephens, (1996) the majority of body enzymes including Amylase work best at temperatures of 35°C to 45°C above or below this range the enzymes start to denature and the reaction rate decreases. Bloomfield and Stephens, (1996) also say that above 80°C enzymes are made to be permanently denatured and below 35°C the reaction rate gradually slows till it basically stops and the enzymes become stationary however enzymes are not denatured by low temperatures and will be reactivated by an increase in temperature. According to G and S, Toole, (1999) a rise in temperature has two effects on an enzyme controlled reaction. Firstly the higher temperature increases the kinetic energy of the substrate and enzyme molecules making them move faster and there for collide more frequently with one another which in turn increases the rate of reaction. Secondly as the temperature rises the atoms that make up the enzyme molecules vibrate which breaks the hydrogen bonds and other forces holding the molecules in their particular shape, the enzyme molecules three-dimensional shape is altered so much that their active sites no longer fit the substrate. The enzyme loses its catalytic properties and is referred as being denatured.
Similar to temperature the pH extremes i.e. pH’s 4 and 7 have a detrimental effect on enzyme activity and the rate of reaction speed. Bloomfield and Stephens, (1996) imply that alterations in the pH of the surrounding medium can change both the secondary and tertiary structure of an enzyme possibly causing a change in the geometry of the active site or the surrounding charge distribution. Bloomfield and Stephens, (1996) also state that great alterations in pH denature all enzymes and all enzymes have a particular pH at which they work best but some can abide large shifts in pH while others won’t tolerate the slightest change which significantly decreases there activity. G and S, Toole add that the defined three-dimensional molecular shape that is essential to the working of enzymes is partly to a degree an outcome of hydrogen bonding. These bonds can be broken by the concentrations of hydrogen ions (H?) present. Hydrogen concentration is measured by pH on a scale from 1-14 with 7 being the neutral point. Any pH lower than 7 is called acidic and a pH above 7 is said to be alkaline. Via violating the hydrogen bonds that give enzyme molecules their shape, any alteration in pH will effectively denature enzymes.
What are Buffers
According to Bloomfield and Stepens, (1996) Buffers are substances that when present in solution defy changes in pH. They especially protect against big alterations in pH when acids or bases are added to the solution. Living cells in this case plant cells are exceptionally sensitive to even very minor changes in pH. The reason for this sensitivity is that the enzymes that catalyze metabolic reactions function in only a minute range of pH. Altering the pH slows down or stops the action of the enzyme.
Kent, (2000) points out a few other components that affect the enzyme controlled reaction these being substrate and enzyme concentration and competitive and non competitive inhibitors. These components could be potential limiting factors in the experiment or may explain any anomalies that may have happened.
When the substrate concentration increase so does the rate of an enzyme -controlled reaction up to the point when the enzyme starts to work to its maximum capability. This is when the enzyme molecules attain there turnover number and providing that all the other conditions like temperature are idyllic, the only to amplify the speed of the reaction even more is to add more enzymes.
Enzyme concentration
All reactions catalysed by an enzyme the amount of enzyme molecules at hand are much smaller than the amount of substrate molecules. A molecule of an enzyme can change millions of substrate molecules into products every minute. If there is a plentiful supply of substrate the rate of reaction is restricted by the number of enzyme molecules. In this case adding more enzymes will increase the rate of reaction.
Inhibitors reduce the speed or stop enzyme action. Normally enzyme inhibition is a natural process that can turn enzymes on and off if needed. Normally inhibition is reversible and enzyme activity resumes full activity when the inhibitor is removed. Numerous ‘external’ chemicals like drugs or poisons can inhibit certain enzymes. These inhibitions are frequently non-reversible and if so bind permanently to the enzyme making it ineffective so it is no surprise that organisms rarely make this type of inhibitor for their own enzymes. Reversible inhibitors are either competitive or non competitive.
Competitive inhibitors
Competitive inhibitors fight with ordinary substrate molecules to inhabit the active site. The inhibitor molecules must be a compatible shape and size to the substrate to fit the active site but cannot be changed into the correct product. Basically competitive inhibitors ‘get in the way’ and decrease the amount of interactions that can occur between enzyme and substrate.
Non-competitive inhibitors
Non-competitive inhibitors attach to the enzyme away from the active site but alter the general shape of the molecule, changing the active site so that it may no longer turn substrate molecules into product. Non-competitive inhibition posses this name due to their non-competitiveness for the active site. The existence of a non-competitive inhibitor has the same effect as reducing enzyme concentration: all inhibited molecules are taken out of action completely.
How inhibitors help to control metabolism
Numerous metabolic pathways are self-controlling: when a substance is needed, a certain pathway is turned on to produce it. When a sufficient amount has been produced the pathway is turn of. This is due to some enzymes in a metabolic pathway being inhibited by the end product. When too much product starts to build up a enzyme in the pathway is inhibited. When the product is again in short supply the inhibition is lifted and the pathway becomes active again. This self-regulation is an example of negative feedback.
Enzyme structure and function
According to G and S Tool (1999) Enzymes are sophisticated three-dimensional spherical proteins and some may have additional related molecules. Although the enzyme molecule is usually bigger than the substrate molecule it acts upon -only a minute fraction of the enzyme molecule in fact comes into contact with the substrate. This area is known as the active site. Only a small number of the amino acids of the enzyme molecule make up the active site. These supposed catalytic amino acids are frequently some distance apart in the protein chain but are gathered close together by the folding of that chain.
Enzymes and activation energy
G and S Tool, (1999) also say Prior to a reaction can start it must overcome an energy barrier by exceeding its activation energy. Enzymes function by reducing this activation energy and thus allow the reaction to happen more readily. As heat is usually the basis of activation energy, enzymes often remove the need for this heat and thus allow reactions to happen at lower temperatures. Numerous reactions which wouldn’t normally happen at the temperature of an organism happen easily with the company of enzymes.
Mechanism of enzyme action
The properties of enzymes can be explained in relation to the lock and key mechanism of enzyme action and the theory of induced fit and has been explained by many such as Bloomfield and Stephens, (1996); Boyle and Senior, (2002); Kent, (2000); G and S Tool (1999).
Enzymes are believed to function like a lock and key mechanism. Like a key fits a lock very accurately, the substrate fits precisely into the active site of the enzyme molecule. The two molecules make a momentary structure know as the enzyme-substrate complex. The products have a dissimilar shape from the substrate and so once made they break away from the active site leaving it available to attach to another substrate molecule.
Recent analysis of the lock and key mechanism implies that in the presence of the substrate the active site may change to become compatible with the substrate’s shape. The enzyme is flexible and moulds to fit the substrate molecule like clothing is flexible and can mould itself to fit the shape of the wearer. The enzyme at first has a binding configuration which draws the substrate. On binding to the enzyme the substrate disturbs the shape of the enzyme and makes it take up a new configuration. This new configuration is there for catalytically active and affects the shape of the substrate and lowers its activation energy. This is known as the induced fit of the substrate to the enzyme.
According to G and S Tool, (1999) and Kent, (2000) starch is a polysaccharide which is located in most parts of the plant in the structure of small granules. It is a storage food made from any surplus glucose made during photosynthesis. It is common in the seed of plants e.g. Maize or in this case Barley where it makes the food supply for germination. Obliquely these starch stores make an important food supply for animals.
Starch is a mixture of two substances: amylose and amylopectin. Starches differ a little from one plant species another but generally consist of 20% amylase 79% amylopectin and 1% of other substances such as phosphates and fatty acids.
Amylose-unbranched chain of alpha glucose with 1-4 glycoside links Amylopectin- branched chain of alpha glucose units with 1-4 and 1-6 glycoside links. a-Amylose is an endoglacoside which randomly hydrolyses a-(1 4) linkages of the side chains of a glycogen and amylopectine. It can cut either side of a branch point excluding very highly branched regions Amylo-a (1-6)-glucosidease the debranching enzyme catalyses the hydrolysis of the a (1 6)glycosidic bonds of the limit dextrins thereby allowing additional breakdown by a-and ß – amylase. For information on Monosaccharides and Polysaccharides see appendix.
Why is barley seed a rich source of a-amylase?
Like many other seeds Barley seed is a rich a source of alpha-amylase because large molecules of stored starch need to be broken down into sugars before they can be absorbed and digested by the embryo to sustain the growing seedling until it is able to photosynthesis. (Ingram; Vince-Prue; Greegory, 2008)
Why is the reaction studied in this experiment important in the process of germination of barley seeds?
According to Janick, Schery, and Ruttan (1996) the production of malt is a fairly straightforward process. Grain is steeped and then germinated under controlled humidity, temperature and atmosphere. Initial sprouts build up a high content of amylase and other enzymes prized for later digestion of starch adjuncts in brewing. Germination is stopped when the enzyme content is at its maximum by heating and drying the sprouting seed. In brewing, malt is mixed with additional grains to provide the mash, the enzymes of the malt reduce starch to simple sugars in the resulting wort, which is afterwards fermented by yeast to make the beer.
From this we know that brewers need to know which varieties of barley have the most amylase and are there for better for brewing. And when in germination is the enzyme content at its maximum so you know when to stop germination and thus maximise the hydrolysis of starch. Brewers also need to know what are the most favourable conditions i.e. temperature and pH for a-amylase so they can again get maximum hydrolysis of starch.
To what extent do the results explain the importance of temperature on barley seed germination?
We know that the a-amylase enzymes will start to work just above 0°C below this and germination won’t happen however the maximum rate of hydrolysis of starch won’t happen till the temperature reaches 40°C this will be further explained on the next page.
Why the optimum temperature for a-amylase, obtained in this experiment, may not correlate with the optimum temperature for germination of barley seeds in a field situation?
(Koning, 1994; uni-hamburg, 2009) states that with the imbibition of water, the hormone signal in barley, gibberellic acid (GA), is transported from the embryo to the aleurone layer of the endosperm. The GA activates the DNA for the gene encoding alpha-amylase in the aleurone cells. Transcription and translation of that gene results in the manufacture of alpha-amylase inside the aleurone cells. This enzyme is transported by ER into the Golgi, sorted and packaged into vesicles, and exported through the cell membrane by exocytosis. The amylase is then dumped into the endosperm region. There the amylase breaks down starch into the sugar maltose which is taken to the embryo. The sugar fuels respiration in the embryo so it can grow. The radicle comes out from the seed coat, and germination is accomplished. Once plant physiologists found this out, we could easily inform the brewing industry how to produce more beer per bushel of barley i.e. by treating the barley seeds with a solution of gibberellic acid (instead of plain water) the increased level of hormone signal causes extra synthesis of amylase. This causes a large acceleration of maltose release.
This means that temperature is not the activator for the germination of barley but water we also know that barley’s optimum germination temperature is 20°C the min is 4°C and the max is 30°C (Forbes and Watson, 1992). So once water has entered the seed germination will start. Water is a liquid above 0 so above this germination should start and the a-amylase start to function so the plants can start to grow in early spring or autumn and have chance to grow and set seed. From the results 40°C was the optimum temperature for a-amylase but in this in country in outside field condition the temperature doesn’t get this high and if it did most of the growing season would have been and gone so the plants wouldn’t have chance to set seed. Also the seed doesn’t want to use all its energy reserves in one go i.e. it wants a steady supply so it has enough time to make leaves and start to photosynthesize and make more energy but in the brewing process we want to maximise production i.e. speed it up so we need the optimum temperature for a-amylase not germination. Another thing to note is the variety used in crop production and brewing i.e. the varieties are more than likely different for their required purpose and will have different optimum temperatures.
Why is a-amylase important to other living organisms
Like plants animals will have starch stored away but in the form of fat which will need to be broken down by a-amylase and other enzymes. a-amylase are also needed in digestion e.g. seeds are a food source for animals and as we all ready know contain starch which is broken down by a-amylase. (Enzyme essentials, 2008; G and S Tool, 1999) and Kent, 2000).
What where the main sources of potential error in this experiment?
It would appear that there are no obvious errors or anomalies however there were many occasions where things could have gone wrong due to human error for starters it was very easy to accidently add the wrong buffer to the wrong sample a amylase solution. It was also easy to lose track of time and there for allow the a-amylase to work longer on the starch and there for give inaccurate results. The concentrations and amounts of a-amylase and starch buffer solutions were also subject to human error.
G and S Tool, (1999) and Kent, (2000) say that monosaccharides are a collection of sweet soluble crystalline molecules of comparatively low molecular mass. They are named with the suffix-ose. Monosaccharides contain either an aldehyde sugar or they contain a ketone group (C=O), and they are termed ketoses or keto-sugars. The general formula for a monosaccharide is (CH2O)n where n=3, the sugar is called a triose sugar, n=5 a pentose sugar, and n=6 a hexose sugar.
Structure of monsaccharides
According to G and S Tool, (1999) and Kent, (2000) Possibly the most well know monososaccharide glucose , has the formular C6H12O6. All but one of the six carbon atoms has a hydroxyl group (-OH). The other carbon atom forms part of the aldehyde group. Glucose may be represented by a straight chain of six carbon atoms. These are numbered starting at the carbon of the aldehyde group. Glucose like other hexoses and pentoses readily forms stable ring structures. At any one time most molecules rather than a chain exist as rings. In the case of glucose, carbon atom number 1 may join with the oxygen atom on carbon 5. This makes a six-sided structure called a pyranose ring. In the case of fructose, it is carbon atom number 2 that links with the oxygen on carbon atom 5. This makes a five-sided structure know as a furanose ring. Both glucose and fructose can exist in both pyrunose and furanose forms.
Glucose like most carbohydrates can exist as a number of its omers (they possess the same molecular formula but differ in the arrangement of their atoms).
Different isomers occur when a carbon atom has four different groups attached to it. This is known as an asymmetric carbon atom. An asymmetric carbon atom occurs when glucose forms a ring structure. This gives rise to two isomers, a form and the ß form. Both types occur naturally.
Numerous monosaccharides may join together by condensation reactions to give a polysaccharide. The amount of monosaccharides that join together varies and the chain created can be branched or unbranched. The chains may be folded thus making them compact and therefore perfect for storage. The size of the molecule makes them insoluble-another characteristic which suits them for storage as they apply no osmatic influence and do not readily diffuse out of the cell. In hydrolysis, polysaccharides can be changed to their constituent monosaccharides ready for use as respiratory substrates.
Examples of storage polysaccharides are starch and glycogen. However not all polysaccharides are used for storage e.g. cellulose is a structural support to cell walls.
Bloomfield, M. M. and Stephens, L. J. (1996) Study Guide: Chemistry and the living organism (6th edition). New York: John and Sons.
Delvin, R.M. and Witham, F.H. (1983) Plant Physiology (4th edition.). New Delhi: CBS Publishers

Methods to Test Liver Function

The liver is as an organ in the body and its duty is to destroy toxin, store and produce bile and responsible for thousands of biochemical reaction. The liver is located in the upper right part of the abdomen, the colour of the liver is a dark brown tinted with red. Also known as the largest gland and the largest solid organ in the body, whose weight differs from both sex, in human the liver approximately weighs 1.4-1.6 kg (3.1-3.5 lb)
Live function test as known it carries out a thousands of function, the true function liver test is not the blood it is just the standard test used to detect cell damage in the liver and biliary obstruction (the sign and symptoms of biliary obstruction occurs when the bile fails to reach its proper destination). Serum bilirubin is considered as the true or real test of the liver function because it shows the ability to process and secret bilirubin into bile. There is most common test carried out when looking at the liver function test, which is going to be discussed in a brief note they are as follow, bilirubin, Albumin, Globulin, Ck (creatinine kinase), Gamma GT (gamma glutamyl transpeptidase), Total protein, Alanine Transferase, Aspartate Transferase and Alkaline Phosphatase.
Bilirumbin is produced by the liver, it is found in bile and has a yellowish pigment. Bilirubin is the chemical and physical process of conversion of the breakdown of heme (the deep red, nonprotein ferrous component of hemoglobin (C34H32FeN4O4)), bilirubin as a cellular antioxidant serves as its physiological role , which enables haemoglobin in the red blood cell(Erythrocyte) to be broken into haem and back to unconjugated bilirubin It is in a lipid soluble form which is not soluble in water and in that case it has to be made soluble in the reticuloendothelial cell of the liver. The new produced bilibrubin is tightly bounded to albumin, this unconjugated bilibrubin formed is also called free bilirubin.
Bilibrubin that has not been conjugated by the liver is called an unconjugated bilibrubin. It produces an indirect reaction to the van den bergh test. When there is a high level of unconjugated bilibrubin in the blood it shows a lack of bilibrubin clearance by the liver. Unconjugated bilirubin is released from the carrier molecule of albumin in the process of moving some to the liver where it is converted to conjugated (water soluble) by an enzyme called glucurnosyltranseferase. During this process, the testing laboratory may be able to identify conjugate unconjugated when they react to certain dyes. And that’s when you get to understand why so-called direct (conjugated) and indirect (conjugated).
Conjugated bilirubin is bilirubin, which were picked up by liver cells and conjugated to form water soluble (conjugated) can be excreted into the bile through the bile into the small intestine and then share combined with bacterial urobilinogen, some of these fabric is excrected in stool, and some absorbed and excreted in bile or urine. Several of urobilinogen absorbed is returned to the liver to be re-excreted in bile and eliminated some amountof urobilinogen excrete in the urine, lack of human urinary urobilinogen in the system causing obstructive jaundice.
Jaundice is not a disease but a symptom that shows high accumulation of bile pigment (bilirubin) in the body, the yellow color of the skin and whites of the eyes. Jaundice becomes evident in a patient when the serum bilirubin exceeds the normal range. There are three main things that cause jaundice, which is pre-hepatic or hemolytic, hepatic and post hepatic or extrahepatic. Pre-hepatic jaundice is used when too many red blood cells break down and the liver is incapable of removing the excess
Albumin is a protein produced by the liver, serum albumin binding activity, and many include drugs such as bilirubin binding. Serum test the amount of protein in the clear liquid blood test to determine if the body is not absorbing enough protein. It also keeps the fluid leakage of blood, tissue, difficult piece of that injury, and blood capillaries, increasing capillary permeability caused by burns may be the loss of serum proteins. low albumin level may reflect disease, the kidneys can not prevent the leakage of albumin from the blood in the urine and is lost, the loss of albumin in the urine due to renal failure (nephotic syndrome) can cause human albumin. Protein in urine is a sign of kidney disease, and pre-eclampsia. Albumin indicate liver problems, the most common cause of liver failure caused by low albumin chronic liver cirrhosis (cirrhosis of the liver does not function properly as a result of chronic liver disease). Albumin concentration usually depends on the rate of synthesis, the volume and distribution and the rate of catabolism, so that the concentration is normal in chronic disease to cirrhosis and other serious damage occurs.
Aspartate transferase AST is also known as SGOT (serum glutamic oxolacetic), an enzyme that is often present in liver cells are damaged, this enzyme is mainly caused by the cell in the liver. High levels of this enzyme implicates the liver is damage, AST is released into the blood the amount of protein is proportional to the amount of damaged cells. The height of AST relies on how long the blood is tested after the accident, the results of correction of circulatory disorders often shows a very rapid decrease in levels of AST.
Serum alanine aminotransferase is also known as ALT, which measures the amount of the enzyme ALT in the blood. ALT is found in liver and muscle, ALT does not necessarily indicate the severity of liver damage. ALT tends to be slightly higher than the level of AST and this may be important in the detection of necroinflammatory activity in patients with chronic diseases hepatisis.
Alkaline phosphatase (ALP) is an enzyme located throughout the body, like all enzymes, it is necessary and required in a small amount to trigger specific chemical reactions. ALP is measured by combining the individual serum with a specific substance in which alkaline phosphatase is known to react. ALP produce different from liver, bone and placenta are called isoenzymes, liver isoenzyme is found in the surface region of the bile canalicular membrane hepatkovyte. ALP accumulate and escape into the bloodstream, in the presence of the disease of bile from the liver obstructive nature (cholestatic) differnciate ALP is used to identify what type of disease is present in the liver , cholestatic or hepato Cell. However, the leak of a pregnant woman during pregnancy is normal and ALP is produced in the placenta of a pregnant woman.
Antitrypsin is a glycoprotein synthesized in the liver and prevents the serine proteases, especially elastase. The normal concentration of 1 to 1.6 g / L. It is an acute phase protein that increases in serum inflammatory diseases, pregnancy and after oral contraceptives (OCP). The liver disease is generally seen as a deficiency of antitrypsin, an inherited disease. Depletion should confirmed by quantitative measurement.
Glutamyl transpeptidase (GGT) is a membrane glycoprotein that catalyzes the transfer of γ-glutamyl group to other peptides, amino acids and water. Large amounts can be found in kidney cancer, pancreas, liver, intestine and prostate. Glutamyl transpeptidase gene is on chromosome 22 glutamyl transpeptidase levels are high in infants and children up to 1 year and can increase after 60 years of life. Men have higher values. Children over 4 years are the values ​​of the serum in normal adults. The normal range is 0-30IU/L1. Glutamyl transpeptidase levels of acute viral hepatitis may reach its peak in the second or third week of illness, and in some patients remain elevated for 6 weeks. From a diagnostic test the primary purpose of glutamyl transpeptidase is narrowed to just the exclusion of bone diseases such as γ-glutamyl transpeptidase is not in the bone.
A test of liver function alone is of little value in detecting liver disease that many serious liver disease may be associated with normal and abnormal levels are found in asymptomatic healthy patients. Using the battery of tests of liver function, however, is a very sensitive procedure. The number of false negatives should be reduced by this technique. Using the battery of tests of the liver is also associated with high specificity, especially when more than one test is abnormal. The pattern of enzyme abnormality, interpreted in the context of patient characteristics, can help direct the diagnostic work up. Knowledge of the prevalence of certain liver disease in specific populations and possible liver damage in systemic diseases or drug treatments can help the doctor identify the cause of the deterioration effectively