BIOCHEMISTRY PRACTICALS PRACTICAL 6 ISOLATION AND ENZYME ACTIVITY OF YEAST INVERTASE

ABSTRACT

The experiment involved the isolation and analysis of enzyme activity produced from baker’s yeast. The activity is based on the measurement of glucose or reducing sugar produced due to them hydrolysis of sucrose (table salt). It involves the examination of enzyme activity and determining protein concentration using standard curves which are also useful for determination of Enzyme activity and specific enzyme activity. After, the organic solvent precipitate was analyzed and total protein was determined by Branford assay and after plotting the graph of absorbance against concentration, protein concentration in crude extract and in redissolved precipitate were determined and enzyme activity were determined by incubation enzyme with sucrose and the amount of reducing sugar formed was determined spectrophotometrically from the absorbance at 540nm and standard glucose was prepare followed by several dilutions and addition of DNS and after placing the sample into several condition absorbance was measured and lastly the graph was obtained and after calculated enzyme activity redissolved precipitate appear to has high enzyme activity.

INTRODUCTION

Enzymes are large biological molecules responsible for the thousand processes that sustain life. They are highly selective catalyst, greatly accelerating both the rate and specificity of metabolic reactions from the digestion of food to the synthesis of DNA. Most enzymes are protein although there are also some RNA molecules which act as catalysts. Enzymes may include organic or inorganic cofactors to assist in catalysis.

In this practical, the activity involved was the analysis of yeast extract for enzyme invertase. This analysis was about the invertase specific activity and then the purification of extract by organic solvent. Invertase is a yeast derived enzyme that can generally cleave peptide bonds and specifically hydrolyze sucrose to glucose and fructose. The official name for invertase is beta-fructofuranosidase (EC3.2.1.26), which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal non-reducing beta-fructofuranoside residues in beta-fructofuranosides. Sucrose, commonly known as table sugar, is a disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,4-glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equimolar mixture of glucose and fructose is generated. This mixture of monosaccharides is called invert sugar, which is derived from the fact that sucrose rotates plane polarized light to the right whereas the hydrolysis products rotates plane polarized light to the left.

The specific activity of yeast invertase was also determined and compared between the specific activity of invertase in Yeast crude extract and in the redissolved precipitates. Enzyme specific activity defined as the amount of substrate the enzyme converts (reactions catalyzed), per mg protein in the enzyme preparation, per unit of time. It is a measure of enzyme purity. The value becomes larger as an enzyme preparation becomes more pure, since the amount of protein (mg) is typically less, but the rate of reaction stays the same (or may increase due to reduced interference or removal of inhibitors).Because both the reactants and product of the invertase reaction are colorless, the experiment involves spectrophotometer by using the monosaccharides produced from the reaction to subsequently reduce brightly yellow- colored solution of 3,5-dinitrosalicylate to dark orange-colored solutions of 3-amino-5-nitrosalicylate which will be detected with spectrophotometer at wavelength of 540nm.

MATERIAL AND METHODS

Solutions:

· 0.1 M Sodium bicarbonate

· 0.1 M acetate buffer, pH 4.8 (2.3 mL glacial acetic acid and 4.92 grams of sodium acetate in 1 L)

· DSN reagent (Made by dissolving , with warming, 5g of 3,5-dinitrosalicyclic acid in 100 mL of 2 M NaOH (8g NaOH/100 mL). Add 150 g sodium potassium tartrate to 250 mL of water and warm to dissolve. The two solution were mixed and diluted to 500 mL with water.

· Bradford reagent (5x) (85% Ortho-phosphoric acid, 500 mL, 95% ethanol, 250 mL and Brilliant Blue G-250 dye, 500 mg, these are mixed and diluted to1L). Stock solution is stored at 4 ^oC. To prepare 1x reagent, 1 volume concentrate is mixed with 4 volume distilled water, mixed well.

· 95% ethanol solution ( ice cold)

· 1000 mg/L standard glucose solution

· 100 µg/ml standard BSA

EXPERIMENTAL PROCEDURES

(A) Preparation of Yeast extract

1. Dried baker’s yeast is dissolved in a 0.1 M NaHCO₃solution at the rate of about 4 mL solutions per gram of dry yeast.

2. The mixture was incubated for a day at 35- 40 ^oC

3. Then it was centrifuged at 1000xg for 15 minutes and save the supernatant as crude invertase enzyme

(B) Analysis of organic Solvent Precipitate

1. 9.0 mL of yeast extract was obtained from the centrifuge tube.

2. The 9.0 mL of yeast was placed on ice and while on it, 10.0 mL of cold 95% ethanol solution was added drop wise taking around ten minutes

3. Then again the mixture was centrifuged at 1000xg for 10 minutes.

4. The supernatant was decanted and the precipitates were redissolved in 2 mL of the acetate buffer.

1. This method is suitable for measurement of protein concentrations in range of 0- 100µg protein/mL. So, to prepare a calibration curve, we prepared standard protein solution at five different concentrations in a range of 0 -100µg/ml by diluting BSA (bovine serum albumin) stock solution with distilled water in test tubes.

2. Six test tubes for standard curve were prepared. i.e. 0, 5, 10, 15, 20 and 25

3. 10 mL of Bradford reagent was prepared by adding 2 mL of stock solution to 8 ml of distilled water.

4. 500µL of working Bradford reagent prepared above was added to each labeled standard tubes and the tubes for unknown.

5. An approximate volume of standard BSA was transferred to each of the standard curve tubes (i.e. 2.5 µL of standard to tube labeled “5” 5µL to tube labeled “10”, 7.5µL to tube labeled “15”, 10µL to tube labeled “20” and 25µL to tube labeled “25”.

6. The crude extract and redissolved precipitate (both 1:5 diluted), are protein solution samples.

7. 2.5µL of solution sample was added to 500µL of diluted Bradford reagent and the mixture were mixed vigorously using vortex mixer.

8. We waited for 10 minutes

9. Then absorbance was measured against the blank at 595 nm by using spectrophotometer.

10. The standard curve was drawn and protein concentration in crude extract and in redissolved precipitate was estimated.

(D) Determination of Specific Activity

1. Three tests tubes were needed for each assay (a control and 5 assays were performed). The table below shows the contents to each tube. After getting ready to start the assay, sucrose solution from the second test tube was poured into the first test, mixed well and beginning timing using timer capable of measuring seconds.

2. After exactly 3 minutes, the third tube (DNS solution) was poured into reaction test tube(mixed well). Put the tube in boiling water bath for 10 minutes, then cool. After cooling, the mixture was diluted by adding 5.0 mL of distilled water and 5.0 mL of buffer followed with reading the absorbance at 540nm.

Solution to Assay	First	Test Tube		Second Test tube	Third test tube
	Acetate buffer	Distilled water	Undiluted Enzyme solution	Sucrose solution	DNS reagent
Control (blank)	3.0 mL	2.0 mL	0.00 mL	1.0 mL	3.0 mL
Crude extract	3.0 mL	2.0 mL	1.00 mL	1.0 mL	3.0 mL
Redissolved ppt	3.0 mL	2.0 mL	1.00 mL	1.0 mL	3.0 mL

3. Glucose standard was prepared by suitable dilution of stock solution. Concentration in the range of 100-1000mg/ml was suggested as starting point.

4. A dilution of 2:10 was prepared as follows:

a. 2.0 mL of stock solution was pipette into a clean 10 mL test tube, and distilled water was added to a calibration mark of 10 mL.

b. The test tube was covered and well shaken. In similar fashion 4:10, 6:10, 8:10 dilutions were prepared.

5. 2 mL of DSN reagent was into test tube and the tube was placed in the boiling water bath for 10 minutes, then the mixture was cooled and after cooled it was diluted with 5.0 mL of distilled water and 5.0 mL of buffer and finally the absorbance was measured at 540nm.

6. The standard curve was drawn and the enzyme activity of the sample determined.

RESULTS

By dilution formula concentration after dilution was calculated as follows;

c₁v₁ =c₂v_2, from this

V₁=500µl

C₁=100mg/ml=0.1µg/µl

In test tube labeled 5: v₂=2.5µl hence c₂= c₁v₂/v_t

0.1×2.5/502.5=0.000498E

In test tube labeled10:v₂=5µl hence c₂₌0.1mx5/505=0.00099

In test tube labeled 15: v₂=7.5µl hence c₂= 0.1x7.5/507.5=0.0015

In test tube labeled 20:v₂=10µl hence c₂=0.1x10/510=0.0019

In test tube labelled25:v₂=12.5µl hence c₂=0.1x12.5/512.5=0.0024

Table of results for part C

Test tube	Concentration (µg/µL)	Absorbance
0	0	0.048
5	0.000498	0.178
10	0.0009	0.222
15	0.0015	0.320
20	0.0019	0.356
25	0.0024	0.411
Crude extract	0.00048	0.159
Redissolved ppt	0.000598	0.179

Protein Concentration in Crude Extract and in Redissolved Precipitate

Concentration of Crude extract;

From the standard curve above the equation of the graph is;

y=145.8x+ 0.080

But the graph is plotted for Concentration versus Absorbance and hence,

y= absorbance and x= concentration

Therefore; x=

The concentration for Crude extract was 0.00054

Concentration for Redissolved precipitate;

= 0.00067

The concentration for Redisssolved precipitate was 0.00067

Results for part C: Determination of specific Activity

Solutions to assay	Absorbance
Control (Blank)	0.013
Crude Extract	0.576
Redissolved Precipitate	0.092

Table of Result for Glucose Standard Curve

Dilutions	Concentrations	Absorbance
2:10	0.2	0.031
4:10	0.4	0.035
6:10	0.6	0.071
8:10	0.8	0.088

Calculation of Enzyme Activity;

The slope and y- intercept from above curve are:

Slope = 0.103

y-intercept= 0.004

The formula for enzyme activity is as follows;

i. µmole glucose liberated=

ii. Units/mLenzyme =

(DF)

Ø Enzyme activity for Crude Extract;

Change A540=Crude extract absorbance – Blank absorbance

Change A540= 0.576- 0.013=0.563

µmole glucose liberated=

= 5.427 µmole glucose liberated

Units/mL enzyme=

× 1DF = 1.809 µmole/ min

The enzyme activity for Crude Extract is 1.809 µmole/min

Ø Enzyme activity for Redissolved precipitate;

Change A540=Redissolved ppt absorbance-Blank absorbance

Change A540= 0.092-0.013=0.079

µmole glucose liberated=

= 0.728 µmole glucose liberated

Units/mL enzyme=

×1Df = 0.243 µmole/min

The enzyme activity for redissolved ppt is 0.243µmole/min

Calculation for Specific activity of the Solution

Recall: Protein Concentration for Invertase in Crude extract = 0.00054 µg/µL =5.4×10^-4 mg/ml

Specific Activity =

= 1549.63µmol min^-1mg^-1

Specific activity of invertase in Crude Extract is 1549.63µmol min^-1mg^-1

Recall: Protein Concentration for invertase in Redissolved ppt = 0.00067 µg/µL= 6.7×10^-4mg/ml

Specific Activity =

= 362.68 µmol min^-1mg^-1

DISCUSSION

The results above shows the standard curves of concentration against absorbance helped in the determination of concentration of crude extract and the concentration of redissolved precipitate. Consequently the curves helped in the calculations for enzyme activity and enzyme specific activity. From the above results it shows that the enzyme specific activity from enzyme invertase in crude extract is larger as compared to the one in the Redissolved ppt. This means that there is more invertase enzyme in the crude extract of the centrifuged beakers yeast as compared to the one present in the redissolved precipitates. This is so because enzyme specific activity measures the purity of enzymes.

The isolation of enzyme invertase is not an easy task and is accompanied with some complications throughout the isolation process. Some of the common concerns in the isolation of invertase enzymes are that the protein may be denatured due shearing forces or chemicals used during the initial stages, Proteins can become insoluble or inactivated due to differences between the composition of the cellular fluid and extraction medium (including pH, ionic strength, and the concentration of reducing agents or other specific solutes)

To reduce these complications the initial raw materials should be the simplest biological system which contains large concentration of the targeted protein so as to maximize final yield and reduce the amount of other proteins during the homogenization process.

CONCLUSION

This practical was very useful since it involved the isolation of specific protein the invertase enzyme from initial biological source, the baker’s yeast. The initial starting material the Bakers yeast is relatively inexpensive and readily available in most local grocery stores, so it was enough for the whole class to perform the experiment and participate in the activities involved. Invertase is an important enzymes and is widely used in food industry preferably candy industry. This industry exploits it for breaking glucose to fructose which is much sweeter and doesn’t crystallize easily. When it becomes expensive to get invertase it is preferably to get fructose from glucose using glucose isomerase.

REFERENCE

C. Webb, C. R. Soccol, A. Pandey, C. Larroche. (2008). Enzyme technology. Springer

http://biotech.about.com/od/glossary/g/SpecActivity.htm

www.wikipedia.com

http://askville.amazon.com/calculate-specific-activity-enyzme/AnswerViewer.do?requestId=6371390