Theory: The enzyme, Invertase is a yeast derivative.

It breaks up sucrose into glucose and fructose also is used to develop shelf time of confections (A.E.T. Ltd. 2007). Its enzyme classification is E.C. 3.2.1.26 and its reaction is separation of a water molecule of terminal non-reducing -Dfructofuranoside residues in -D-fructofuranosides (A.E.T. Ltd. 2007).

FIG.1- Structure of Invertase. (IDSwater 2012) The use of baking yeast to extract and partially purify its enzyme Invertase where different procedures used are the extraction, heat treatment and alcohol fractionation. Further purification in the lab is done by ion exchange chromatography using DEAE- cellulose column. In the alcohol fractionation, Invertase is precipitated by an alcohol to make the enzyme solidified reducing its activity to be further purified (Hudson and Paine 2007). To achieved this is to use large proportions of a strong alcohol for example 95% ethanol to precipitate the enzyme under low temperatures. Precipitation done by a very strong alcohol can destroyed the enzyme (Hudson and Paine 2007). A Clinistix test is a qualitative test for glucose done by a strip containing a reagent spot that tests particularly for glucose resulting in a change of colour when glucose is current in the solution (Southern biological, n.d.). Under certain conditions like at room temperature the test is performed and the colour of the reagent spot at about ten seconds following wetting of the strip. Next the result is compared to the colour diagram on the bottle sticker (Southern biological, n.d.). The qualitative outcomes are interpreted from the colour diagram as a negative or changeable degrees of positive which point out the qualified amounts of glucose at hand also protein and the pH of the tested sample (Southern biological, n.d.). Colour blocks are designated as Negative, Light, Medium or Dark. This test is based on a binary of chronological

enzyme reactions. The enzyme, glucose oxidase catalyzes the structure of gluconic acid and hydrogen peroxide from the oxidation of glucose (Southern biological, n.d.). A second enzyme known as peroxidase, catalyzes the reaction of hydrogen peroxide with chromogen orthotolidine in the direction of generating colours ranging from purple all the way through to dark blue (Southern biological, n.d.).

FIG.2- Diagram of different charged column groups in Ion exchange chromatography. (Berg et al 2002). Ion exchange chromatography is a method used to take apart proteins based on their net charge (Berg et al 2002). Proteins with a net positive charge at pH 7 join to a column of beads consisting of carboxylate groups. These cationic proteins can be divided on negatively charged carboxymethyl-cellulose columns known as CM-cellulose columns (Berg et al 2002). As for negatively charged proteins which cant bind to carboxylate groups, these anionic proteins are separated through the same purification method on positively charged diethylaminoethylcellulose columns known as DEAE-cellulose columns (Berg et al 2002). The positively charged protein bound to such a column can be able to then be eluted by escalating the concentration of sodium chloride or a different salt in the eluting buffer (Berg et al 2002). The sodium ions in the buffer challenge the positively charged groups on the protein for the binding to the column (Berg et al 2002). Proteins that have a little density of net positive charge will be likely to come out foremost, followed by those having a higher charge density (Berg et al 2002).The same done for negatively charged proteins. The yield and purification level of this method of purification is 77% and 9 respectively (Berg et al 2002).

FIG.3- Diagram of the mechanism of protein flow in Ion exchange chromatography. (Berg et al 2002). It is important not to let the DEAE cellulose resin to dry because splitting and breaking of the resin occurred. This causes the formation of spaces between the resin to become not uniform resulting disfunctionality in the resin. The sample must be added slowly to prevent any disturbance of the resin in column. Next the addition of increasing salt concentrations to the column is to reduce the interaction and can be used to elute the proteins with a higher net charge by competing with the protein groups for binding to the charged groups on the matrix (U.A. 2003). The fractions that will contain the enzyme of interest are the ones from the eight elute buffer consisting of 150mM NaCl, 0.05M Tris buffer and 200mM NaCl with Tris buffer. This is because the higher concentration of sodium ions in the resin will compete with the protein for the matrix eluting the protein/enzyme of choice first due to its high net charge (U.A. 2003). Fractions from elute buffer 5 and 6 consisting100mM and 150mM NaCl respectively. The reason is the buffers concentration of sodium ions is the start point of retaining the enzyme of choice which on the URISCAN strip the colours produce will be optically descriptive and vivid. The fractions from elute buffers 6, 7 and 8 will show a positive glucose test on the URISCAN strip due the level of purification by the high concentrations of NaCl added in the buffer. Discussion: In the experiment the enzyme Invertase was extracted and was purified by the method of Ion exchange chromatography. The first part of the procedure was the extraction and alcohol fractionation of the enzyme. Where two out of the four fractions for analysis were collected, they were the crude and the heat extract. The crude fraction was the of the homogenized and extracted sample from the baking yeast. By the use of deionized water and centrifugation the yeast and sand mixture was separated leaving a pellet and a layer of toluene behind. The aqueous layer transferred as the crude was the sample where the enzyme was contained. The next fraction, heat extract was the crude liquid sample being treated with acetic acid and was

incubated at an appropriate temperature. This was followed by a precipitation by the use of ethanol which solidifies the liquid sample to a pellet for the next step in purifiying the enzyme (Hudson and Paine 2007). The Clinistix test shown in table 2 shows there was high protein and glucose activity at 100 for the second fraction. The next part of the experiment was the purification by Ion exchange chromatography. Where the pellet from the first session was resuspended in Tris buffer and centrifuged. The supernatant was the protein containing the enzyme to be used as the sample to be purified in the chromatography. This was slowly added to the DEAE column first with and the collection of the first eluates were the proteins of low net charge due to lack of sodium ions present. The following additions of elute buffers of increasing salt concentration resulted in collections of eluates which were increasing in net charge competing with the sodium ions in the resin (U.A. 2003). For each eluate the absorbance was measured and they were diagnosed by the Clinistix test as shown in table 3. Then the two fractions chosen for their high activity were fractions 13 and 14. They both have the same glucose and protein activity. Also shown in the first graph above their absorbances taken were closed enough and represents high activity. The results of the fractions shown gave the evidence that the enzyme, Invertase was contained due to the activity. Due to the high concentration of sodium ions in the buffer at 150mM which helped elute the protein of high net charge at time the fractions 13 and 14 have to be collected. The next graph represents the glucose activity of each eluted fraction where the activities of 13 and 14 is the same at 1000. The fractions were combined to be stored as the last fraction, column fraction for further use. The fraction will able to be used in the next session due to its high activity and level of purity.