Enzymes Are Highly Specific Macromolecules

Published Date: 02 Nov 2017

Introduction

Enzymes are highly specific macromolecules that catalyze and accelerate chemical reaction rates without being depleted in the process. The enzyme reaction rate is influenced by a number of factors, e.g., enzyme concentration, substrate concentration, temperature, pH, and the existence of inhibitors (competitive and non-competitive). For instance, the increase of the substrate concentration increases the rate of the reaction reaching a maximal reaction velocity at a high substrate concentration (Rogers and Gibson, 2009). Enzymes also act on the conversion of substrates into products by reducing the free energy of the reaction activation. For example, enzymes can help convert charge repulsions and give the reacting molecules the opportunity to form new chemical bounds. In contrast, the enzyme can apply stress on a substrate molecule, reproducing a specific bound easy to be broken if the reaction requires that. Normally, enzymes are regulated by a complex set of positive and negative feedback systems that control the reaction rates (Gonze and Kaufman, 2011).

The principle of enzyme kinetics is based on the measurement and mathematical description of their reaction rates and their associated constants (Rogers and Gibson, 2009). The model that is used for understanding the kinetic features of most enzymes is known as the Michaelis-Menten model. This model suggests the following mechanism for enzyme catalysis: the enzyme (E) and substrate (S) merge to form an enzyme-substrate complex (ES). Therefore, the reaction occurs, and the substrate is converted to the product of the reaction. In this case, the enzyme-substrate complex is broken separately, yielding enzyme (E) plus product (P), as shown in the following equation (Washington University, 2004).

E+S ES

ES E+P

E+S E+P

Catalytic reactions in enzymes are vital to various biological pathways, and their arrangements are necessary in discovering drugs because the numerous drug targets are enzymes. In addition, catalytic reactions may be used in comparing enzymes from unalike organisms or under conditions that are not the same or in order to know how these shown differences are related to the organisms physiologic/function (Functional Application Areas, 2008). Moreover, the enzymes that exist in the serum are vital for making a diagnosis of several diseases. This is because their existence shows the cellular damage or tissue damage has taken place and that intracellular mechanisms are in the blood stream. For example, an increase in the amino transferase alanine transaminase levels (ALT) indicates damages of the liver cells (The Medical Biochemistry Page, 2012).

β-Galactosidase (E.C 3.2.1.23) is one of the enzymes used in catalyzing the lactose hydrolysis to galactose and glucose. This lactose is plentiful disaccharide found in milk. It has potential significance because of the many applications in the dairy and food industries that involve themselves with ingredients having reduced lactose (Gekas and Lopez, 1985). Major β-galactosidase applications include making the sensory and technological food characteristics to be good by accumulating their solubility.

This is by assimilation of foods having lactose for lactose intolerant inhabitants, creation of galacto-oligosaccharides and the change of ways into value-added products that are not the same (Braga et al, 2012). Galacto-oligosaccharides (GOS) are oligosaccharides that are not digestible. They comprise of 2–20 galactose molecules and one glucose molecule (Mahoney, 1998), that are known as prebiotics. This is because they can kindle the lactic acid bacteria proliferation and bifidobacteria found in the human intestine (Sako et al, 1999). Due to this, when producing GOS, much attention is given. Specifically by using enzymatic transgalactosylation, because the GOS chemical synthesis is too monotonous (Sear and Wong, 2001). Different sources have β-Galactosidases. These sources may be microbial, animal and vegetable. Additionally, the characteristics of the enzymes differ with their origin. The β-galactosidases that are more technologically fascinating are created by Kluyveromyces yeasts. These β-galactosidases are intracellular. The synthesis of β-galactosidases is repressed by glucose and encouraged by galactose.

They are mainly achieved by immersing cultivation (Ladero et al, 2000. Szczodark, 2000). Knowledge of the enzyme’s stability is a vital aspect to consider when allowing for its use in biotechnological processes. This is because it can give information about the enzyme’s structure and enable an inexpensive production design. The mechanisms used in deactivation may be difficult, because many enzymes have vastly defined structures. These enzymes also have the least deviation from their inherent form which can have an impact on their exact activity.

Knowing the stability of the enzyme better in the operating conditions could assist in optimizing the enzymatic processes profitability (Miller and Whistler, 2000). The enzyme activity and thermal stabilities are influenced by different environmental factors (pH, temperature, shaking and reaction medium) that may affect the precise spatial conformation or three-dimensional structure of the protein strongly (Jurado et al, 2004). It is also essential in analyzing the expected thermodynamic parameters, since this assists in knowing the likely denaturation mechanism. This denaturation mechanism is significant in enzymatic processes (Ustok et al, 2010).

Materials and methods

Materials

Methods

RESULTS

pH 3.5

Figure 1. The Optical density (OD) over time (5 min) at various ONPG concentrations with β-galactosidase enzyme at pH 3.5. The absorbance of samples containing the indicated concentrations of ß-galactosidase enzyme were determined in 30 second intervals for a total of 5 minutes. Absorbance data were then plotted versus time.

This graph was created to collect data in order to look at how the product is produced over the time and get this information to be able to create the substrate versus the velocity. By measuring the rate at which the color intensity increases we can calculate the activity of the enzyme. Optical density (OD) is a measure of the amount of light absorbed by a solution. O-nitrophenol, the yellow product of the breakdown of ONPG, absorbs light maximally at 420nm. This OD 420 in this experiment was a measured of the amount of O-nitrophenol present in the sample and could be used to calculate ß-galactosidase activity. The more yellow in the solution, the more ONPG has been degraded, and higher the absorbance.

Figure 2. Effect of substrate concentration on enzyme velocity at pH 3.5.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 3. Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the maximum rate (VMax) and Km values by using the equation (Y=mx + b) =[] + ,where the y-intercept ‘b’ defines the 1/Vmax, while the slope is equal to Km divided by Vmax and x defines [].

pH 4.5

Figure 4. Kinetics of ONPG hydrolysis at pH 4.5. The absorbance of samples containing the indicated concentrations of ONP were determined in 30 second intervals for a total of 5 minutes. Absorbance data were then plotted versus time .

This graph was created to collect data in order to look at how the product (ONP) is produced over the time and get this information to be able to create the substrate versus the velocity. By measuring the rate at which the color intensity increases the activity of the enzyme can be calculated.

Figure 5. Depicts the activity of ß-galactosidase enzyme as a function of the substrate concentration at pH 4.5.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 6: Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the maximum rate (VMax) and Km values .

pH 6.5

Figure 7: The Optical density (OD) over time (5 min) at various ONPG concentrations with β-galactosidase enzyme at pH 6.5. The absorbance of samples containing the indicated concentrations of ß-galactosidase enzyme were determined in 30 second intervals for a total of 5 minutes. Absorbance data were then plotted versus time.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 8: Effect of substrate concentration on enzyme velocity at pH 6.5.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 9: Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the maximum rate (VMax) and Km values.

pH 7.5

Figure 10: Kinetics of ONPG hydrolysis at pH 7.5. The absorbance of samples containing the indicated concentrations of ONP were determined in 30 second intervals for a total of 5 minutes. Absorbance data were then plotted versus time .

This graph was created to collect data in order to look at how the product is produced over the time and get this information to be able to create the substrate versus the velocity. By measuring the rate at which the color intensity increases the activity of the enzyme can be calculated.

Figure 11: Depicts the activity of ß-galactosidase enzyme as a function of the substrate concentration at pH 7.5.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 12: : Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the maximum rate (VMax) and Km .

Class pH 7.5

Figure 13: shows our result of the activity of ß-galactosidase enzyme as a function of the substrate concentration at pH 7.5 compared to result of class pH 7.5.

Figure 14: shows our result of Lineweaver-Burk plots of 1/enzyme activity and 1/[S] compared to the class pH 7.5 result.

As figure 14 shown there was different between our result and the class result at pH 7.5 regarding the coefficient of determination (R2) value for both results 0.8728 and 0.9574 respectively , it seems that the class result was the best fitted between data and the drawn line. However , in fig 13 the differences between our result and the class result not significantly

pH 8.5

Figure 15: The Optical density (OD) over time (5 min) at various ONPG concentrations with β-galactosidase enzyme at pH 8.5. The absorbance of samples containing the indicated concentrations of ß-galactosidase enzyme were determined in 30 second intervals for a total of 5 minutes. Absorbance data were then plotted versus time.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 16: Depicts the activity of ß-galactosidase enzyme as a function of the substrate concentration at pH 8.5.

This graph was created to measure velocity of ß-galactosidase enzyme -catalyzed reaction at different concentrations of substrate in order to get information to be able to create 1/enzyme activity versus 1/[S] , therefore determine the Km (the Michaelis constant) and Vmax .

Figure 17: Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the maximum rate (VMax) and Km .

Table 1. Results summary obtained from Lineweaver-Burk plots.

pH

Vmax (µM/min)

Km (mM)

3.5

0.0613

0.79

4.5

0.0058

1.53

6.5

1.08

5.72

7.5

0.365

0.56

7.5 class

0.478

0.611

8.5

0.35

0.421

Discussion

The aim of this experiment was to investigate the interactive effect of pH changes in the activity of β-galactosidase enzyme and on its Km and Vmax values.

β -galactosidase catalyzes the breakdown of the substrate lactose, a disaccharide sugar found in milk into two monosaccharide sugars, galactose and glucose. The oxygen bridge connecting the two sides of the lactose molecule is cleaved through the addition of a water molecule. Since it is difficult to assay for the activity of β-galactosidase when lactose is the substrate, ONPG (o-nitrophenyl β-D-galactopyranoside) was used in the experiment,because the similarity in the structure of ONPG and lactose.therefore, the enzyme does not distinguish between lactose and ONPG. In addition to that it is easy to determine the amount of ONPG cleaved by using a spectrometry assay. ONPG is colorless, but the product ONP (o-nitrophenol) is yellow, so that as the β -galactosidase continues to work, more and more ONPG is degraded, and the solution turns more and more yellow.

The enzyme activity was measured at different concentrations of the substrate ONPG. The amount of the substrate used in the reaction medium were 4 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.062mM, 0.031mM and blank. The enzyme β-galactosidase follows a parabolic Michaelis-Menten kinetics. Km and Vmax values of β-galactosidase were calculated from the reciprocal plots of substrate concentration versus reaction velocity.

By looking at β-galactosidase enzyme activity figures at all pH (3.5, 4.5, 6.5 and 8.5) there were a different between their absorbance results in comparison to the absorbance result at pH 7.5 , i.e. pH 7.5 is the most optimal for beta-galactosidase enzyme. At pH 7.5 more products (ONP) were obtained in comparison to other pHs.

Using a polynomial regression (Fig. 3,6,9,12 and16) analysis of these data an equation describing this relationship can be used with a high degree of confidence, as the coefficient of determination (R2) was almost the same. In statistics, a value is often required to determine how closely a certain function fits a particular set of experimental data. R2 values range from 0 to 1, with 1 representing a perfect fit between the data and the line drawn through them, and 0 representing no statistical correlation between the data and a line.

As shown in table 1 there was different value for both Km and Vmax at all different pHs. It indicated that best Km and Vmax values were at pH 6.5. However, it found in the literature that the optimum pH for β-galactosidase enzyme activity was between pH 6.5-7.5 ( ).This could be due to some technical errors such as, Inaccuracy in pipetting, enzyme may kept for a long time at room temperature, and air bubble formation on the cell walls when using spectrophotometry which interfere with the passage of light to the detector and will potentially cause significant false positive errors in the absorbance reading. The enzyme is stable for at least 30 min at 40°C within pH 6-8 (Wallenfels and Weil, 1972), but its stability at 40°C falls sharply below pH 6.0 and slowly above pH 8.0. Heat stability is considerably lowered by various intermediates in the glucose metabolic cycle (Brewer and Moses, 1967). ( Book).

Conclusion

This experiment went successfully in all most and we discovered the affect of pH on the enzyme according to the results were obtained.

Amylases are enzymes which hydrolyze starch molecules in giving different products including dextrin and gradually smaller polymers that have glucose units (Windish and Mhatre, 1965). Alpha amylase acts on starch by breaking starch into sugars. Starch is a source of carbohydrate that consists of two molecules; amylopectine and amylase. Amylase is made from glucose chains linked α1, four. The amylopectine is made from α1, four linked glucose chains in one, six branch points that are linked. These amylases are enzymes working by hydrolyzing the straight chain bonds amid the distinct glucose molecules making up the starch chain.

A distinct chain starch which is straight is referred to as an amylase. A starch chain that is branched is referred to as an amylopectin. The enzymes described are of importance in biotechnology used nowadays with applications that range from fermentation, food and textile to paper industries distillery and detergent industries and starch syrup industries (Pandey et al, 2000). Amylase was the first enzyme to be revealed and insulated . Although it can be derived from numerous sources including animals, microorganisms, and plants.

Generally, microbial enzymes meet the demands of the industry. Alpha amylase is endogenously produced in many organisms that are different e.g. B. stearothermophilus, B. subtilis and B. licheniformis. As well as, A.orzyae and Aspergillus niger. Currently, many microbial amylases exist commercially and they have nearly complete starch replaced chemical hydrolysis in starch processing industry (Bernfeld, 1955). This is due to amylase effecting quick length reductions of the starch polymer. The subsequent fragments are oligosaccharides. These oligosaccharides are freely soluble in water and are too short to preserve the noteworthy adhesive capability. Thus, in coping with the ever growing demand, different projects have concentrated on increased enzyme production and enzyme stability. One of the advantages of using microorganisms in the producing amylase is the fact that economical substance production capacities and microbes are easy to deploy in obtaining enzymes of preferred characteristics (Lonsane et al, 1990).

The amylases history started in 1811 when the first starch degrading enzyme was revealed by Kirchhoff. Ohlsson proposed the starch digestive enzyme classification in malt as beta- and alpha-amylases according to the numeric sugar type created by the enzyme reactions. Due to their biotechnology significance, amylases have gained a lot of attention. They constitute a class of industrial enzymes that has nearly 25% of the enzyme’s market internationally.

A lot of thermostable Actinomycetes like, Bacillus species, Actinomycetes thermoactinomyces and Actinomycetes thermomonospora are useful amylase producers. The genus Bacillus creates a great range of extracellular enzymes whereby proteases and amylases are of Industrial significance .Currently, two categories’ of amylases alpha-glucosidase and glucoamylase are significant for starch hydrolysis. Glucoamylase attacks one, four bonds, discharging D-glucose molecules. This type of enzyme also attacks one, six bonds at diverging points in the amylopectin molecule. This process is slower than -one, four linkages.

α -amylase distribution in microorganisms

Worldwide α -Amylases are distributed all over the plant, microbial and animal kingdoms. During the last few decades, substantial research has been embarked on with the production of extracellular a-amylase by using various microorganisms (Fogarty et al 1979). α -Amylase has been originated from quite a lot of fungi, bacteria actinomycetes and yeasts. On the other hand, enzymes originated from bacterial and fungal sources have conquered applications found in industrial areas.

α -amylase activity determination

α -Amylases become assayed generally by the use of modified starch or a soluble starch like the substrates. The α -1 hydrolysis is catalyzed by α –Amylase which is four starch glycosidic linkages in producing dextrin and glucose. This reaction is examined with increasing the levels of reducing sugar or decreasing the color of iodine in the treated substrates (Priest et al 1977).

Physiology of α -amylase production

Producing α -amylase using solid state fermentation (SSF) and submerged fermentation (SmF) has been investigated thoroughly affected by various physicochemical factors. Most notable factors among these are the structure of pH of the medium, the growth medium, phosphate concentration, temperature, inoculum age, aeration, nitrogen source and carbon source (Fogarty et al 1979). Among the different factors are; nitrogen, carbon and trace supplemented elements such as the glucose, calcium chloride and peptone respectively. This enhanced production of enzymes.

Sources of α–Amylases

One of the ubiquitous enzymes plants, microbes and animals produce is the amylases. The amylases play prevailing roles in the metabolism of carbohydrates. Amylases from microbial sources and plant have been used for many years as food additives. In the brewing industry, Barley amylases have been used for a long time. Fungal amylases have been extensively used in making oriental foods despite of the wide microbial sources, amylases distribution, namely fungal and bacterial amylases. They are mainly used in industrial productions because of the advantages like consistency, cost effectiveness, less space and time required for production and easing optimization and modification process. In bacteria, Bacillus sp. is the widely used in producing thermostable a-amylase for it to meet the needs of the industry. B. stearothermophilus, B. subtilis , B. licheniformis and B. amyloliquefaciens are well known to be good amylase producers. They have been used widely for profit-making production of the enzyme in several applications (Vihinen et al 2000).

Characteristics of α–Amylases

The enzyme is a glycoprotein. It is a single polypeptide chain having nearly four hundred and seventy five residues that have four disulphide bridges and the SH group containing a tightly bound Ca2+. This enzyme is found in two forms (I &II) that have the same enzymatic properties. These properties differ only in electrophoretic flexibility. A binding site for Cl-has been described which have effects on conformational changes enhancing various activities.