Behaviour of Salivary Amylase in Various Reaction Environments with Reference to Km and Vmax. An Overview

Table of contents

Table of figures

Table of tables

List of abbreviations

Behaviour of salivary amylase in various reaction environments with reference to Km and Vmax: an overview

Abstract

1. Introduction
1.1 Industrial use of alpha amylase
1.2 Clinical chemistry and importance
1.3 Objectives

2. Review of literature
2.1 Salivary alpha amylase
2.2 Alpha amylase assay

3. Hypothesis

4. Materials and Methods
4.1 Study area
4.2 Sample collection
4.3 Reagents
4.4 Assay procedure
4.5 Statistical analysis

5. Results and discussion

6. Conclusions

Acknowledgements

References

Table of figures

Figure 1. Sugar backbone linkage. Photo courtesy: Wikipedia.

Figure 2. Description of the salivary glands in human mouth. Photo courtesy: Simplyknowledge.com.

Figure 3. Description of the standard graph of maltose.

Table of tables

Table 1. Protocol for the construction of standard graph of maltose

Table 2. Protocol for the determination of Km and Vmax of salivary amylase.

Table 3. Activity of salivary α- Amylase stimulated by different temperature.

Table 4. Activity of salivary α- Amylase stimulated by different taste.

Table 5. Sample mean and Standard deviations of the Km for different samples of salivary- amylase.

Table 6. Sample mean and Standard deviations of the Vmax for the different samples of salivary- amylase.

List of abbreviations

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ACKNOWLEDGEMENTS

Firstly we thank God Almighty whose blessing were always with us and helped us to complete this project work successfully.

We wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. Joseph V.J, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study. We express our sincere thanks to Mr. Binoy A Mulanthra (lab in charge, Department of Biotechnology) for the support. This research work will not be possible with the co-operation of many farmers.

We are gratefully indebted to our teachers, parents, siblings and friends who were there always for helping us in this project.

Prem Jose Vazhacharickal*, Sajeshkumar N.K, Jiby John Mathew and Twinkle Jose

Cover page photo courtesy: Wikipedia. Human salivary alpha amylase Behaviour of salivary amylase in various reaction environments with reference to Km and Vmax: an overview Prem Jose Vazhacharickal1*, Sajeshkumar N.K1, Jiby John Mathew1 and Twinkle Jose1 1Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India

Abstract

Amylase is an enzyme which catalyzes the hydrolysis of α (1, 4)-glycosidic linkages in amylose (a linear form of starch), amylopectin (a branched form of starch) and glycogen into simpler carbohydrate molecules such as oligosaccharides or disaccharides. Alpha-amylase is the major form of amylase found in human, most prominently in pancreatic juice and saliva. The salivary amylase is an amylolytic enzyme, which can acts on cooked or boiled starch and converts it in to maltose. So it became interesting to study the behaviour of salivary amylase, when it is secreted as result of different stimuli. And thus began to study the effect of five different stimulatory temperatures, and also the effect of four tastes on the behaviour of salivary amylase. For the study of stimulatory effect of temperature on salivary amylase, five different temperatures are selected (4, 27, 37, 55 and 75°C). And likewise four tastes also selected (sweet, sour, salt and bitter). The DNS method was done in the both tests to obtain the absorbance at 520 nm. The samples were collected from three people, of same age. The saliva was collected at same time, after one and a half hour of their breakfast in order to maintain a controlled condition for this study. In each cases the incubation temperature also kept as variable (4, 27, 37, 55 and 75°C). This study was also aimed to determine the behaviour of salivary amylase with reference to the kinetic parameters like Km and Vmax of salivary alpha amylase by incubating the enzyme (stimulated by different stimulatory conditions of temperature and taste) with varying concentration of substrate. The study revealed the consistency in kinetic parameters like Km and Vmax of salivary alpha amylase secreted in response to various stimuli.

Keywords: Amylase; Vmax; Km; 3, 5-dinitrosalicylic acid method; Environment.

1. Introduction

Amylase - one of a group of amylolytic enzyme that catalyzes the hydrolysis of starch into simpler carbohydrate molecules. There are three forms of amylase enzyme. The α-amylase EC 3.2.1.1 occur in saliva, pancreatic juice, malt, and certain bacteria. It is also present in seeds containing starch as a food reserve, and is secreted by many fungi. It catalyzes the hydrolysis of alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. The β-amylases EC 3.2.1.1 occur in grains, vegetables, malt, and bacteria, is involved in the hydrolysis of starch to maltose. However all amylases are glycoside hydrolases and act on α-1, 4 -glycosidic bonds.

Alpha-amylase is the major form of amylase found in humans and other mammals. Although found in many tissues, amylase is most prominent in pancreatic juice and saliva. Each of which has its own isoform of human α-amylase. They behave differently on isoelectric focusing, and can also be separated in testing by using specific monoclonal antibodies. In humans, all amylase isoforms link to chromosome1p21 (Mandel et al., 2010) Alpha-amylase is one of the major protein components of saliva. Among other proteins, alpha-amylase is synthesized and secreted by acinar cells, after neurotransmitter stimulation (Baum, 1993). Acinar cells are innervated by sympathetic and parasympathetic branches of the autonomic nervous system (Emmelin et al., 1981).

The main function of salivary alpha-amylase is the enzymatic digestion of starch into maltose and dextrin. This form of amylase is also called "ptyalin". Ptyalin acts on linear α (1, 4) glycosidic linkages and, it will break large, insoluble starch molecules into soluble starches (amylodextrin, erythrodextrin, and achrodextrin) producing successively smaller starches and ultimately maltose. Salivary amylase is inactivated in the stomach by gastric acid. In gastric juice adjusted to pH 3.3, ptyalin was totally inactivated in 20 minutes at 37°C. In contrast, 50% of amylase activity remained after 150 minutes of exposure to gastric juice at pH 4.3 (Fried et al., 1987). It is also important for mucosal immunity in the oral cavity, as it inhibits the adherence and growth of bacteria (Bosch et al., 2002).

The salivary amylase gene has undergone duplication during evolution, and DNA hybridization studies indicate many individuals have multiple tandem repeats of the gene. The number of gene copies correlates with the levels of salivary amylase, as measured by protein blot assays using antibodies to human amylase. Gene copy number is associated with apparent evolutionary exposure to high-starch diets. For example, a Japanese individual had 14 copies of the amylase gene (one allele with 10 copies, and a second allele with four copies). The Japanese diet has traditionally contained large amounts of rice starch. In contrast, a Biaka individual carried six copies (three copies on each allele). The Biaka are rainforest hunter-gatherers who have traditionally consumed a low-starch diet. So the variation in starch intake correlates with the number of copies of the salivary amylase gene and amylase saliva in human populations. Increased copy number of the salivary amylase gene may have enhanced survival coincident to a shift to a starchy diet during human evolution (Perry et al., 2007).

The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and tri saccharides which are converted by other enzymes to glucose to supply the body with energy. The pancreatic α-amylase randomly cleaves α(1-4) glycosidic linkage of amylose to yield dextrin, maltose, or maltotriose. It adopts a double displacement mechanism with retention of configuration. Genes that are responsible for the production of salivary alpha amylase are AMY1A, AMY1B and AMY1C. The genes responsible for the production of pancreatic amylase are AMY2A and AMY2B (Perry et al., 2007).

Another form of amylase, β-amylase (EC 3.2.1.2) (alternative names: 1, 4-α-D-glucan maltohydrolase; glycogenase; saccharogen amylase) is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second α-1, 4 glycosidic bond, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavour 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. Cereal grain amylase is key to the production of malt. Many microbes also produce amylase to degrade extracellular starches. Animal tissues do not contain β-amylase, although it may be present in microorganisms contained within the digestive tract. The optimum pH for β-amylase is 4.0-5.0.

Gamma-Amylase (EC 3.2.1.3 ) (alternative names: Glucan 1, 4-α-glucosidase; amyloglucosidase; Exo-1, 4-α-glucosidase; glucoamylase; 1, 4-α-D-glucanglucohydrolase) will cleave α(1-6) glycosidic linkages, as well as the last α (1-4) glycosidic linkagesat the nonreducing end of amylose and amylopectin, yielding glucose. The γ-amylase has most acidic pH optimum because it is most active around pH 3.

1.1 Industrial use of alpha amylase

Alpha and beta amylases are important in brewing beer and liquor made from sugars derived from starch. In fermentation, yeast ingests sugars and excretes alcohol. In beer and some liquor, the sugars present at the beginning of fermentation have been produced by "mashing" grains or other starch sources (potatoes). In traditional beer brewing, malted barley is mixed with hot water to create a "mash," which is held at a given temperature to allow the amylases in the malted grain to convert the barley's starch into sugars. Different temperatures optimize the activity of alpha or beta amylase, resulting in different mixtures of fermentable and unfermentable sugars. In selecting mash temperature and grain-to-water ratio, a brewer can change the alcohol content, mouth feel, aroma, and flavour of the finished beer.

Alpha-Amylase is used in ethanol production to break starches in grains into fermentable sugars. The first step in the production of high-fructose corn syrup is the treatment of cornstarch with α-amylase, producing shorter chains of sugars called oligosaccharides. An α-amylase called "Termamyl", sourced from Bacillus licheniformis, is also used in some detergents, especially dishwashing and starch-removing detergents.

1.2 Clinical chemistry and importance

Measurement of serum α-amylase activity is an important diagnostic test for pancreatitis and acute attacks of chronic pancreatitis. Medical laboratories will usually measure either pancreatic amylase or total amylase. The test for amylase is easier to perform than that for lipase, making it the primary test used to detect and monitor pancreatitis. Amylase is measured in Pts with suspected pancreatitis; serum and urine levels peak 4-8 hrs after onset of acute pancreatitis, and normalize within 48-72 hrs; parotitis due to mumps or radiation therapy also increase serum amylase; in cases of increased serum amylase without pancreatitis or parotitis, requires quantification of amylase isoenzymes Ref range Varies by laboratory; 25-90 U/L, serum; 4-30 U/2 hrs, urine; amylase is increase in acute pancreatitis, obstruction of common bile duct, pancreatic duct or ampulla of Vater, pancreatic injury from perforated peptic ulcer and acute salivary gland disease. Amylase is decreased in chronic pancreatitis, pancreatic CA, cirrhosis, hepatitis and eclampsia.

In molecular biology, the presence of amylase can serve as an additional method of selecting for successful integration of a reporter construct in addition to antibiotic resistance. As reporter genes are flanked by homologous regions of the structural gene for amylase, successful integration will disrupt the amylase gene and prevent starch degradation, which is easily detectable through iodine staining.

Salivary α-amylase has been used as a biomarker for stress that does not require a blood draw (Noto et al., 2005). Previous studies revealed that the marked increases in salivary alpha-amylase following psychosocial stress or stress-dependent activation of salivary alpha-amylase (Kirschbaum et al., 1994). The latest research suggests that alpha-amylase is linked to our emotions and our health. In the growing field of amylase research, recent studies have underscored the usefulness of salivary alpha-amylase in this regard. However, some methodological issues have to be resolved in order to integrate salivary alpha-amylase measurements as a standard tool into psycho-physiological research (Rohleder et al., 2006).

1.3 Objectives

The objective of this study was The purpose of this study is to determine whether or not the influence of different stimulatory temperature and tastes correlates with the enzymatic activity of amylase and was studied by the hydrolysis of starch by salivary amylase under various stimulatory conditions (temperature and taste).The activity of salivary amylase is determined by the DNS method (Bernfeld, 1955). The absorbance is read at 520 nm activities in various conditions were calculated from the absorbance using the maltose standard graph.

This study was also aimed to determine the Km and Vmax of salivary alpha amylase by incubating the enzyme (stimulated by different stimulatory conditions of temperature and taste) with varying concentration of substrate. And three incubation temperatures (27°C, 37°C, and 55°C) is setup for each set of reaction in which secretion of salivary alpha amylase is stimulated in response to different stimulatory conditions. By analyzing the obtained Vmax and Km values it is possible to understand the velocity of substrate conversion by the salivary amylase on different reaction conditions and also the affinity of the enzyme with its substrate under various reaction conditions.

And thus analyse the behaviour of salivary alpha amylase secreted in response to various type of stimuli possess any behavioural changes when it undergo in different reaction environments (temperatures).

The scope of the present study is to provide, a basic information on the kinetic parameters like Vmax and Km of salivary alpha amylase secreted in response to various stimuli under different reaction environments, for development of enzyme systems in vitro for various applications of research and commercial importance in future.

2. Review of literature

2.1 Salivary alpha amylase

Amylase is a calcium dependent enzyme which hydrolyzes complex carbohydrates at α (1, 4)-glycosidic linkages to form maltose and glucose. Amylase is a major component of human saliva, plays a role in the initial digestion of starch. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. It may also be involved in the colonization of bacteria involved in early dental plaque formation. Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins.

The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. More surprisingly, α-amylase is also found in blood, sweat and tears, possibly for anti-bacterial activity Plants and some bacteria also produce amylase. Specific amylase proteins (isoforms of enzyme) are designated by different Greek letters (α, β, γ –amylase).All amylases are glycoside hydrolases and act on α-1, 4-glycosidic bonds.

The α-amylases (EC 3.2.1.1) (alternative names: 1, 4-α-D-glucan glucanohydrolase; glycogenase) are calcium metalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" (oligo saccharide) from amylopectin. Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase. In animals, it is a major digestive enzyme, and its optimum pH is 6.7-7.0.

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In vitro, α -Amylase is also able to hydrolyze the α (1,4) linkages in glycogen, but has no activity on the α (1,6) linkages responsible for the more highly branched structure of glycogen. These branched structures also reduce the activity of α-Amylase toward glycogen by limiting the accessibility of the target α (1, 4)-glucan bonds.

The amount of amylase present in saliva varies with the composition of the diet and is present in largest amounts when the diet contains a large amount of the carbohydrates that are hydrolyzed by this enzyme. The salivary amylase levels found in the human lineage are six to eight times higher in humans than in chimpanzees, which are mostly fruit eaters and ingest little starch relative to humans.

The first enzyme that the food encounters in our mouth is called salivary amylase. It is released by salivary glands and is the most abundant enzyme in saliva (Ramasubbu et al., 1996). There are three main pairs of salivary glands - the parotid glands, the submandibular glands (also called the submaxillary glands) and the sublingual glands. In addition, there are between six hundred and a thousand minor salivary glands located in the mouth, throat and lips.

The parotid glands are the largest salivary glands. One parotid gland is located in each cheek, in front of the ear. The parotid glands produce a watery liquid containing protein. The two submandibular glands (or submaxillary glands) are located under the floor of the mouth. These glands produce a liquid that is a mixture of water and mucus. The two sublingual glands are located under the tongue, in front of the submandibular glands, produce a liquid that contains more mucus than the secretions of the other salivary glands. Saliva leaves the glands in tubes called salivary ducts. More saliva is made when the mouth contains spicy, sour or acidic foods. When taste buds are stimulated by these chemicals they trigger the release of saliva.

Saliva is thick, colourless and glistening liquid consisting of about 98% to 99% water. It kills bacteria, helps to prevent tooth decay, begins the digestion of food, helps to speak and helps to swallow food as well. Saliva contains many chemicals in addition to water, including mucus, salts, antibacterial substances (lysozyme, lactoferrin, peroxidase and immunoglobulin A), enzymes, other proteins and buffering agents (Sodium bicarbonate) to keep the pH at the correct level in the mouth. It also contains bacterial cells, since bacteria live in our mouths, and human cells shed by the lining of the mouth, the tongue and the gums. The salivary glands generally make between one and two litres of liquid a day (between two and four pints). During an average lifetime they produce about 10,000 gallons of saliva. Saliva is released continuously from the salivary glands, although the amount varies during the day. The quantity increases when taste, smell or even think of food, as well as when we eat certain kinds of food, but it decreases during the time of sleep.

Saliva contains an enzyme called salivary amylase or ptyalin, which digests starch into a sugar called maltose. (Maltose is later broken up into glucose molecules in the small intestine). When chewing, teeth break down the food into physically smaller pieces that can be acted on by digestive enzymes. The first enzyme that the food encounters in our mouth is called salivary amylase. It is released by our salivary glands and is the most abundant enzyme in our saliva (Ramasubbu et. al, 1996).

2.2 Alpha amylase assay

Several methods are available for determination of α-amylase activity, and different industries tend to rely on different methods. The starch iodine test, a development of the iodine test, is based on colour change, as α-amylase degrades starch and is commonly used in many applications. A similar but industrially produced test is the Phadebas amylase test, which is used as a qualitative and quantitative test within many industries, such as detergents, various flour, grain, and malt foods, and forensic biology. The Nelson-Somogyi (NS) and 3, 5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides.

Maltose can be used as a standard for estimating reducing sugar in unknown samples. Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing sugars present in an unknown sample and for determining the activity of amylase enzyme in forthcoming experiments. The standard curve for maltose is usually constructed using DNS as the reagent. Maltose reduces the pale yellow coloured alkaline 3, 5-Dinitro salicylic acid (DNS) to the orange- red coloured, 3 amino, 5 nitro salicylic acid.

Saccharolytic activity of alpha -amylase was measured with the DNS method (Bernfeld, 1955). The 3, 5-Dinitrosalicylic acid (DNS or DNSA, IUPAC name 2-hydroxy-3, 5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. The DNS procedure uses 1 % soluble starch as substrate.100 µl of the enzyme were incubated for 30 min at 37°C with 2.5ml of phosphate buffer (0.02 M, pH 7.1) and 2.5ml of soluble starch. A blank without substrate but with α-amylase extract and a control containing no α-amylase extract but with substrate were run simultaneously with the reaction mixture. The reaction was stopped by addition of 0.5 ml of DNS and heated in boiling water for 5 min prior to read absorbance at 540 nm. One unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg of maltose in 30 min at 37 °C.

A standard curve of absorbance against amount of maltose released was constructed to enable calculation of the amount of maltose released during α-amylase assays. Amylase activity can be defined in international (IUB) units of micromoles of product/min per litre of saliva. It was first introduced as a method to detect reducing substances in urine and has since been widely used, for example, for quantifying carbohydrates levels in blood. It is mainly used in assay of alpha-amylase. However, enzymatic methods are usually preferred due to DNS lack of specificity (Miller, 1959).

The study about the activity of salivary amylase, it role and concentration in saliva are now a day’s widely subjected to the various fields of study and research works. The salivary amylase is now used as the biomarker for stress. Its concentration variation in the saliva is considered as disease conditions. A large number of studies are reported about the salivary amylase day by day. And most of them are about the relations between stress and increase of salivary amylase concentration. ‘The psychosocial stress-induced increase in salivary alpha-amylase is independent of saliva flow rate is the one kind of study’ done by Rohleder et al. (2006) and Kirschbaum et al. (1994). They reported that flow rate is not confounder of stress-induced salivary amylase activation.

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