Enzymes are proteins that are formed in the body and act as catalysts. They break materials down by increasing the rate of chemical reactions and biological processes. However, those chemical or biological processes do not change enzymes. Enzymes play a key role in lowering the activation energy for a reaction enhancing the rate of reaction. Comparing enzymes and catalysts, catalysts catalyze many biochemical reactions without any selectivity while enzymes are highly selective to a reaction. Enzymes do not affect the equilibrium of the reactions. However, enzymes are chemical-specific to their functions. Invertase is a yeast-derived enzyme that catalyzes the breakdown of sucrose. This results in the production of fructose and glucose mixture referred to as inverted sugar syrup.
Enzymes have two major parts that form them; they include non-protein and protein parts. The non-protein part is crucial part of the enzyme for any reaction to occur, it may consist of metal ions including Mn2+ or Zn2+. These metal ions are referred to as co-factors that are covalently bound to protein sections to assist in the reduction of activation energy. The protein section is where reactant bind to it lowering the activation energy hence increasing reaction (Dietmar and Ida 89).
They are two major categories of Yeast invertase; they include internal and external invertase. There is a clear similarity and marked disparities shown by properties of purified preparations if external and internal invertases of yeast that are designated by their location in relation to the cell membrane. There is resemblance between specific activity and molecular weight of both internal and protein moiety of external invertase.
However, they are differences between external and internal invertase; their molecular weights vary (external invertase MW=270,000 Daltons, internal invertase MW=135,000 Daltons). The compositions of amino acids are dissimilar with external invertase having cystine or cysteine. The internal enzymes do not have the same amino acids composition. However, the general structure and differences of enzymes are the same with differences in amino acids composition.
There are various sources of invertase where it is produced including bacteria, fungi (saccharomyces, cerevisiae, Fusarium solani, Candida utilis, and Penicillium species), and higher plants and animals. The external invertase from Saccharomy cerevisiae is in most instances is used in food industry because it is non-toxicogenic and non-pathogenic.
One of the crucial enzymes in the food industry is external invertase (B-fructofuranosid fructohydrolose) from Saccharomyces cerevisiae. Sucrose sugar is catalyzed into an equimolar mix of fructose and glucose referred to as inverted sugar. The invert sugar gotten from this enzyme reaction is colorless with a higher yield of conversion than the product obtained by acid hydrolysis. The key functional state of the external invertase is a homodymer with a molecular mass of 270 kDa.
A glycoprotein is stable and dissociates only under denaturation conditions. Polymannan is an external invertase mass that forms 50% of the mass with glucosamine forming 3%. They are four mixtures of isoforms in external invertase that exhibit diversified stabilities and chemical reactivities. Furthermore, these four isoforms are found in the external invertase preparations of various S. cerevisiae industrial strains. To improve the efficiency of the food industry, a thorough study and research need to be carried out.
The anomalous migration rates of glycoproteins usually make it difficult to get their accurate molecular weights on electrophoresis in SDS-polyacrylamide gels. Another reason is that partial specific volumes utilized for them in sedimentation equilibrium analysis are of questionable accuracy. This is predominantly right for glycoproteins like external invertase from yeast whereby, half of molecular weight is contributed by associated oligosaccharide chains. Nevertheless, on taking away nearly all of the oligosaccharide chains of this enzyme with the endo-B-N-acetylglucosaminidase H from Streptomyces plicatus, it has been probable to illustrate that carbohydrate-free invertase is made of two 60,000-dalton sub-units.
Using Shaffer and Hartmann reagent, Hoffman and Seegerer’s work was the most popular method used for assaying invertase activity in determining the reducing sugar content. However, they were other methods used in determining the amounts of reducing sugars including the Lehmann-Maquenne titration method and a colorimetric method based on the color appearing in Fehling’s solution.
In the colorimetric method, the non-reducing β-d-fructofuranosid residue of sucrose is hydrolyzed by invertase to produce invert sugar. 3.5 dinitrosalicylic acid (DNS) is reacted with the released invert sugar to reduce it to 3-amino-5nitrosalicylic acid. The change of color is proportional to the inverted sugar amount produced that is similar to the invertase activity within the sample. The absorbance is measured at 540nm. It is then changed into micromoles of reducing sugar created by the use of a calibration curve. One invertase unit is the quantity of enzyme that generates 1 micromole of reducing sugar in terms of invert sugar per minute under the procedure’s particular circumstances (Marangoni 56).
Lineweaver-Burk transformation of Michaelis-Menten equation was used to determine the Michaelis-Menten constant (Km) for sucrose and a sucrose concentration in the range from 2.5 to 300mM. All invertase isoforms’ Km in 50mMacetate buffer (pH 4.5) were determined to be 25.6mM. An identical pH optimum from 3.5 to 5.0 is exhibited by all invertase isoforms and temperature optimum with maximum activity at 600 C. Due to lack of significant difference in pH, Km, and temperature optima as well as the molecular weight between external invertase isoforms, this is an indication that there is no difference in catalytic activity between isoforms.
It is vital to know the kinetics properties and parameters of the invertase enzyme in order to understand the rate of reaction catalyzed by enzyme yeast invertase. The highest rate of speed for an enzyme to interact with the substrate is Vmax. This is towards making the product per time to reach the saturated rate. This kinetic parameter was reported to be 2.3mM.min-1 for 1 µg/ml concentration. Km is the affinity of substrate for enzyme that is described as (k2+k1)/k1.
The last section of this experiment was carried out to examine the mechanism of inhibition of the invertase in certain molecules. An inhibitor is a molecule that tends to block the activity of enzymes. These inhibitors play a major role in the drug industry like killing pathogens and correcting metabolic pathways. Inhibition can be categorized into major parts that include irreversible and reversible inhibitors. The chemical reaction with enzyme, irreversible inhibitors is known to cause permanent inhibition. Reversible inhibitor has three types of reversible inhibitors and it does not have a chemical reaction.
The inhibitor and substrate compete for the enzymes’ active sites during competitive inhibition. After the inhibitor occupies the active site of the enzyme, it would stop the substrate from binding. These inhibitors are known to have the same shape as the substrates. However, this can be treated through the increase of substrate amounts to be more than inhibitors. This lowers the chances of the inhibitor molecule that binds to the enzyme. This type of inhabitation increases the Km while Vmax remains unchanged, as more substrate would be required to overcome the inhibition.
Works Cited
Marangoni, Alejandro. Enzymes Kinetics: a modern approach. London: John Wiley and Sons, 2003.
Dietmar and Schomburg, Ida. Springer handbook of enzymes, Volume 12. New York: Springer, 2003.