Experiment: Isolation of enzyme Invertase from Baker’s Yeast: A two- step process experiment in enzyme Purification and characterization.

Aim of the Experiment

Then aim of this experiment is to:

i)                     Extract enzyme Invertase from the baker’s yeast, purify, and characterize the enzyme using a number of methods as outlined in this paper.

ii)                  Perform systematic procedures for the characterization and separation of enzyme Invertase found on the preplasmic membrane of the Baker’s yeast cell wall.

iii)                Determine the native mass and the sub-unit mass of the protein enzyme

Introduction

An enzyme is defined as conjugated proteins or numerous proteins that are synthesized by the living organism and it serves to speed up (catalyses) various biochemical reactions within a living biological cell. An enzyme functions to break down and convert the molecules of a substrate into different simpler constituent basic components. Invertase (β-fructofuranosidase E.C. 3.2.1.26) is the common name of the enzyme that cleaves sucrose (a non-reducing disaccharide) into glucose and fructose (reducing monosaccharides) as presented in the diagram below

invertase

Sucrose + H2O                     Fructose + glucose (“hydrolyzed sucrose” or “inverted sugar”)

Enzymes play a vital role in various speeding up and initiating biochemical reactions and metabolic pathways within the living biological cells so that they (reactions) take place at an adequate rate necessary to sustain life.

Step 1:  Hydrolysis of Sucrose

Step 2:  Isomerization

Just like the natural organics catalysts, enzymes increase the rate of biochemical reaction within the cell by reducing the activation energy that is required to initiate and sustain the activities of the reacting molecules. Consequently, the rate at which the products are formed and reaction equilibrium reached within a shorter time than it would have taken without the involvement of the enzyme. Enzyme Invertase specifically cleaves the carboxyl (O-C) bonds in fructose.

Invertase is extracted from baker’s yeast (Saccharomyces cereviase) and it could also be synthesized by honey making bees which relies on it to produce honey from the nectar. The enzyme is known to work best at an optimum temperature of 600C and a pH medium of 4.5. It is also called sucrose glycosidase given that it hydrolyzes acetal or glycosidic linkages of the preferred sucrose substrate.

Invertase is compartmentalized between periplasmic and cytoplasmic membranes of the yeast cell where it actively cleavage the non-transportable disaccharide sucrose into its constituent monosaccharides that could be easily transported across the cell cytoplasm.

Once enzyme Invertase has been extracted from the yeast cells under extremely gentle condition since disruption of periplasmic membrane occur under very mild condition (overnight incubation in a 0.10M solution of NaHCO3), the proteins (enzymes) will be conjugated with polysaccharides (glycosylated) before they get transported out of the eukaryotic cells: Saccharomyces cerevicae. The glycosylation increases the mass of Invertase by nearly 50% of its original mass hence the need to purify the enzyme after its production from the yeast cells.

 

Under the normal circumstances, the extensive degree of Invertase glycosylation helps in the protection of the protection of the enzyme against proteolytic degradation and thermal denaturation during the incubation period. This explains the loss enzyme after the incubation of yeast to extract the enzyme at 370C for several hours. The structural stability of the Invertase makes it ideal for extraction within 4-5 weeks under normal library conditions. However, high sugar content lowers enzyme’s specific response in the SDSPAGE and Bradford dye-binding assay and results into a smeared band for Invertase but not other proteins.

 

Yeast (Sacchromyces cerevisae)

Sacchromyces cerevisae is the kind of a eukaryotic cell that is capable of synthesizing enzyme Invertase within the structures of its biological cells. The cells of the micro-organism are round to ovoid in shape and their sizes range from 5-10 µm in diameter. The eukaryotic cells are capable of synthesizing many proteins that are crucial to the understanding of human biology. Such proteins include protein processing, signaling proteins and cell cycle proteins. This type is capable of synthesizing large volumes of the enzyme Invertase for commercial purposes.

Extracellular and intracellular Invertase

Most plants cells have both the intracellular and extracellular Invertase. The intracellular (vacuolar) Invertase accumulates in the vacuoles of plant cell as a soluble protein (Sturm et al. 1999). The soluble acid Invertase is very vital in the conversion of sucrose into its constituent monosaccharides within the lumen of acidic cell compartment (vacuole). According to Sturm (1999), intracellular Invertase can also be useful in the cleavage of other disaccharides such as sucrose and starch though to a smaller extent.

Extracellular Invertase is also referred to as the cell wall Invertase because they are mainly synthesized on the cell wall of plant cell. Similarly, their operations are restricted within the cell walls where they are ionically bound to carry out various bio-physiological synthetic functions of the plant cell.

Biochemistry and enzyme function of the Invertase

The yeast Invertase exists in various isoforms within their respective sub-cellular locations. The larger percentages of the plant secreted enzyme are highly constituted of the N-glycosylated polypeptides and their expressions are controlled by the Glc repression (Carlson & Botstein 1982).  Nevertheless, the cytoplasmic yeast Invertase is made up of the nonglycosylated polypeptide chains that are relatively expressed at low levels compared to those of the N-glycosylated enzymes. It is most important to note that the two enzyme isoforms are encoded by the same gene but their respective transcripts are derived from totally different strands of the heteronuclear mRNA. This results into two distinct classes of polypeptides namely basic pl polypeptides of the cell wall Invertase and acidic pl of the vacuolar Invertase (Carlson & Botstein 1982).

The functions of enzyme Invertase are many in plant cell as far as synthesis of metabolites and various plant functions are concerned. The enzyme is very useful in the synthesis of nectarines in the flowering plants. According to Baker & Baker (1983), the synthesis of nectar, complex solution of natural sugars and other groups of carbohydrates, that is secreted by the members of angiosperm to attract cognate pollinators into the flower is a direct function of the enzyme Invertase.

Secondly, the main enzyme situated on the unloading pathways of apoplasmic phloem which is responsible for the speeding up the hydrolytic cleavage of sucrose that is transported and further deposited into the apoplast. The unloading pathways of the apoplasmic phloem acts are the only supply of substrates that generate metabolic signals that regulate various processes of defense response and primary metabolism. It further supports heterotrophic growth within the plants. Additionally, the extracellular Invertase plays an important role in the synthesis of phytohormones and giving of common reactions to all kinds of stress-related stimuli for instance mineral stress and pathogen infection.

 

Discussion

Enzymes increase the rate of biochemical reaction within the cell by reducing the activation energy that is required to initiate and sustain the activities of the reacting molecules. Consequently, the rate at which the products are formed and reaction equilibrium reached within a shorter time than it would have taken without the involvement of the enzyme. Enzyme Invertase specifically cleaves the carboxyl (O-C) bonds in fructose.

Invertase is extracted from baker’s yeast (Saccharomyces cereviase) and it could also be synthesized by honey making bees which relies on it to produce honey from the nectar. The enzyme is known to work best at an optimum temperature of 600C and a pH medium of 4.5. It is also called sucrose glycosidase given that it hydrolyzes acetal or glycosidic linkages of the preferred sucrose substrate.

Sucrose + H2O                                                            Fructose + glucose

Invertase is compartmentalized between periplasmic and cytoplasmic membranes of the yeast cell where it actively cleavage the non-transportable disaccharide sucrose into its constituent monosaccharides that could be easily transported across the cell cytoplasm.

 

Extracellular Invertase is the main enzyme situated on the unloading pathways of apoplasmic phloem which is responsible for the speeding up the hydrolytic cleavage of sucrose that is transported and further deposited into the apoplast. The unloading pathways of the apoplasmic phloem acts are the only supply of substrates that generate metabolic signals that regulate various processes of defense response and primary metabolism. It further supports heterotrophic growth within the plants. Additionally, the extracellular Invertase plays an important role in the synthesis of phytohormones and giving of common reactions to all kinds of stress-related stimuli for instance mineral stress and pathogen infection.

Enzymes play a vital role in various speeding up and initiating biochemical reactions and metabolic pathways within the living biological cells so that they (reactions) take place at an adequate rate necessary to sustain life.