HOW TO EXTRACT ENZYMES FROM AN ORANGE SKIN
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HOW TO EXTRACT ENZYMES FROM ORANGE
This experiment investigates the extraction of protein from an orange skin. Oranges constitute a lot of citrus on the planet. The primary piece of orange is utilized as a part of commercial enterprises for extraction of squeezed orange which leaves extensive sum as buildup including the ring which is rich in many enzymes. These proteins have a significant application in the biotechnological and synthetic commercial ventures. The orange skin contains esterase and protease compounds which are hydrolase catalysts which can part as corrosive and liquor by hydrolysis. The point of completing this trial is to concentrate compounds from the orange utilizing hydrolysis strategy (McMahon, 1993).
Introduction
Protease are type of enzymes that breaks down or changes the composition of proteins or peptides, to release the amino acids needed by the body. They cleavage proteins by a hydrolysis reaction-the addition of a molecule of water to a peptide bond in the following manner:
Figure 1: Hydrolysis of proteins by protease
From the structure above, it can be observed that, through hydrolysis, protease break down the bonds between proteins hence converting them into smaller chains called peptides or even much smaller units known as amino acids.
Commonly found in plants such as pawpaw and pineapple, protease also plays a very vital role in numerous physiological processes. For instance, proteins are known to have a complex folded structure that demands these types of enzymes to break down in very specific manners. For many years, protease found in the leaves of papaya has been used to tenderize meats.
They are used commercially in food, detergents, diagnostics, and fine chemical industries. Stabilizing proteins and enzymes against inactivation that is irreversible is of major concern; hence minimizing the protein inactivation is key in the storage and purification of the protein. Irreversible inactivation may be due to extensive heat, unsuitable PH and detergents and the nature of the protein hence the biological source of the enzyme ought to be processed quickly with procedures that will ensure minimal degradation. For instance, fresh extraction tissue should be chilled in ice, minced, and cleaned quickly.
Orange is a fruit of species Citrus sinensis whose bulk composition is made up of citrus acid. These species originated from East Asia. Currently, these fruits are consumed all over the world as an excellent source of vitamin C, which is a strong natural antioxidant that helps to build the immune body system. Also, essential phytochemicals like limonoids, thiamine, magnesium, and potassium are also present. The biologically active compounds aid in the prevention of arteriosclerosis, kidney stones, stomach ulcers, cancer and a reduction in cholesterol level thereby promoting human health (Obsbeck, 2006).
Aims of the experiment
The aim of the examination is to confirm protease compound creation or level in the orange products of the orange peels. It additionally goes for showing the easy technique for protease and protein estimation utilizing the Lowry method that is utilized to focus the aggregate synthesis of proteins in an answer. The aggregate piece can be set up and indicated by a color change of a specimen arrangement in rate to the centralization of the protein and afterward it can be measured by colorimetric methods (Oliver H. 1940).
Material
Orange, knife, funnel, pestle and mortar, centrifuge, muslin cloth, hand blender and weighing balance.
Method
Preparation of the buffer
While conducting this experiment, the theoretical PH (6.5) of orange was adopted in the fruit extraction, purification steps, and the assays.
Preparation of an orange extract
The weight of the orange was determined and recorded. The skin of the oranges was then removed and the weighed determined. The measurements were tabulated as they are shown in table 1below.
Specimen Weight Volume of the specimen extract
Orange Orange: 14.66g
Skin: 16.3g Orange: 14ml
Skin:12ml
Table 1: Orange Extract measurements
A piece of orange was then put in a blender that contained ice-cold 100mm phosphate buffer solution. They were then homogenized for a few minutes. The homogenate was then emptied to plastic centrifuge tubes and centrifuged for 3 minutes at a speed of 3000 rpm. This solution was then poured off through glass wool (filters) in a funnel and into the volumetric flask. The same procedure was repeated in the case of the orange fruit skin.
Protease assay method
About 730- μl 100 mm buffer solutions, and 250 μl 2% azo casein were added in a 5ml Eppendorf and pre-incubated for about 5 minutes at a temperature of about 37 0C. Here, the point was to hydrolyze casein so that it could release a dye into the solution hence causing detection by the absorbance at 440 mm. The reaction was initiated by adding 20 μl dilute Neutrase and incubated for about 10 minutes at a steady temperature of 370C. The reaction was then halted by adding 200 μl 10% TCA. This was then frozen for 10 minutes on ice and then span in a microfuge at a maximum speed for another 10 minutes. A 250 μl duplicate of the supernatant was then taken and added to 750 μl Sodium Hydroxide (NaOH). Lastly, the reading of the absorbance was taken at 440 nm against a zero enzym
The Protein estimation using the Lowry method
The protein focus was additionally dictated by utilizing Lowry method by corrosive hydrolysis took after by amino examination. This area condenses in an even way, the aftereffects of different analyzes directed utilizing Lowry’s technique. As prior said the point of the Lowry technique is to focus the level of protein in an answer. It helps know the extent of protein for catalyst planning as it is the situation in this investigation Furthermore in cell parts, chromatography fractions, and so forth. In the beneath tables, we are going to make a determination of the protein levels in an orange arrangement.
1. The accompanying weakening solution was set in triplicate of standard ox-like serum egg whites (BSA) arrangement (600 µg/ml).
BSA soln (ml) 0 0.1 0.2 0.3 0.4 0.5
Water (ml) 1.0 0.9 0.8 0.7 0.6 0.5
Protein conc.
(µg /ml) 0 60 120 180 240 300
2. To 1 ml of protein solution, 5 ml of the alkaline copper reagent was added.
It was then left for 10 minutes at room temperature.
3. 0.5 ml Folin was added and mixed immediately andLeft for 30 minutes.
4. The results at 750 nm against 1 ml water were treated with reagents as above.
Using Excel, the graph of the data was plotted, and the gradient determined in the above table represent the standards (i.e.; 0 and 120 µg /ml BSA) using a number of dilutions of each sample in distilled water. Both the standard samples and the dilute samples were processed simultaneously.
Results
This section summarizes in a tabular manner, the results of various experiments conducted using Lowry’s method. As earlier mentioned the aim of the Lowry method is to determine the level of protein in a solution. It helps know the proportion of protein for enzyme preparation as it is the case in this experiment and also in cell fractions, chromatography fractions, etc. In the below tables, we are going to make a determination of the protein levels in an orange solution
Table 1: Dilution of the fruit extract in ug/ml
Protein concentration (ug/ml) 1st Dilution 2nd Dilution 3rd Dilution
0 0.034 0.039 0.04
120 0.439 0.480 0.510
The results in this table symbolize the dilution of the orange extracts in ug/ml. The results depict a certain trend in that the concentration of proteins in the solution increases with each dilution. According to the table, there is an increase in the protein
concentration with each progressive dilution. On the first row concentration was 0.034 after 1st dilution the concentration of protein increases to 0.04 on the 3rd dilution. On the 2nd-row concentration increases from 0.439 to 0.510 throughout the dilution.
Table 2: The results of freezing the absorbance at 750nm
Protein concentration (ug/ml) 1st Dilution 2nd Dilution 3rd Dilution
0 0.104 0.210 0, 330
120 0.438 0.490 0.550
Results shown in this table are in ug/ml is what was established after freezing the absorbance at 750 nm. According to the table, there is a rise in the protein concentration with each successive dilution.
Table 3: Dilution of the fruit extract
Protein concentration (ug/ml) 1st Dilution 2nd Dilution 3rd Dilution
0 0.034 0.039 0.04
120 0.439 0.480 0.510
This table depicts the concentration of protein in the orange extract after the progressive dilutions of the solution
The outcomes in Table 3 symbolize the weakening of the orange concentrates in ug/ml. The outcomes delineate a certain pattern in that the centralization of proteins in the arrangement increments with every weakening. As per the table, there is an increment in the protein fixation with every dynamic weakening. On the first line fixation was 0.034 after 1stdilution the convergence of protein increments to 0.04 on the 3rd weakening. On the 2nd-column, focus increments from 0.439 to 0.510 all through the dilution
Table 4: The results of freezing the absorbance at 750nm
Protein concentration (ug/ml) 1st Dilution 2nd Dilution 3rd Dilution
0 0.104 0.210 0, 330
120 0.438 0.490 0.550
This table shows that there is a considerable increase in concentration of each consecutive concentration
Results indicated in this table 4 are in ug/ml is what was built up in the wake of solidifying the absorbance at 750 nm. As indicated by the table, there is an ascent in the protein focus with each progressive weakening.
Table 5: Determining the protein concentration in the orange extract
Dilution 750nm Concentration A Concentration B
½ 0.532 0.618
1/5 0.520 0.609
1/10 0.490 0.590
1/50 0.450 0.518
1/100 0.370 0.480
Table 5 depicts the concentration of protein in the orange extract after the progressive dilutions of the solution
Table 6: Determining the protein concentration in an orange after freezing
750nm Concentration A Concentration B
½ 1.150 1.120
1/5 1.009 1.001
1/10 1.009 0.958
1/50 0.932 0.890
1/100 0.800 0.850
According to Table 6 below, there is a considerable increase in concentration on each consecutive concentration
All the results in the tables depicted a considerable change upon being exposed to extremely low temperatures. The rationale is that at these points the process solely depended on the aqueous extraction and not the method (Pandey 74). The reaction due to enzymes reduced and even halted due to inactivation of the enzymes by extremely low temperature. The yields at this point were low or happened after a longer duration of incubation.
Graph 1: The graph of a standard curve of absorbance shown below depicts the function of initial protein concentration and will be used to determine the unknown protein concentrations.
A graph showing the determination of protein concentration against the absorbance at 750nm
Table 7: Determining the protein concentration from the skin with each progressive dilution
Protein Concentration (µg/ml) Duplicate A Duplicate B
1/2 0.591 0.599
1/5 0.520 0.550
1/10 0.409 0.480
1/50 0.400 0.450
1/100 0.385 0.389
On the table 7 above, the point is to focus the level of protein from skin removes with every movement weakening.
Table 8: Determining the protein concentration from skin extracts (Freezing Method)
Protein Concentration (µg/ml) Duplicate A Duplicate B
1/2 1.162 1.140
1/5 1.054 1.002
1/10 1.010 0.947
1/50 0. 937 0.889
1/100 0.821 0.849
In table 8, the point is to build up the level of protein from skin extracts. The freezing method was used that to save new tissue for quite a while at temperatures inside of the scope of -80OC preceding presenting them to temperatures once more.
Table 9: protease assay at 440nm orange absorbance
Protein concentration Dilution 1A Dilution 1B Dilution 1C Dilution 1D
0.051 0.098 0.112 0.113
0.052 0.105 0. 19 0.126
In Table 9 where the squeezed orange was utilized, the level of protein fixation increments, when the squeezed orange is weakened while with respect to the skin of the orange protein focus, is diminishing when weakening happens
Table 10: Protease assay at 440nm skin absorbance
Protein concentration Dilution 1A Dilution 1B Dilution 1C Dilution 1D
0.023 0.025 0.026 0.027
0.021 0.023 0.022 0.023
Table Ten above indicates that fluorescein were the principle case in and was taken as the primary substrate of the protease. In this table where the squeezed orange was utilized, the level of protein focus increments, when the squeezed orange is weakened while with respect to the skin of the orange protein fixation, is diminishing when weakening happens.
Discussion
The outcome attained from this experiment confirmed the hypothesis that an Orange, especially its peels, contain a large amount of enzymes. The enzymes that can be obtained from an orange are very important in biochemistry because of their medical and industrial significance. The aqueous extraction that relied on the enzymatic activity required some time for incubation for the process to be concluded effectively. There was need for the total recovery of the system during the extraction process, maximum time, preferably twice the incubation time, was essential. As per the the results, the introduction of enzymes improves the experiment because the enzymatic actions increase the efficiency with which the process occurs, (Aneja 25). The entire process is effectively enabled due to the improvement by the enzymes action. The advantages to this method are that it could be scaled up or down, doesn’t require any special equipment and it is relatively cheaper (Yada 2004). Due to the necessity of obtaining results that were closely related to the theoretical expectations, there was need to seek more channels of improving the results. The data represented in throughout this experiment offers more meaning to the results and making it possible to analyse the effect of errors on the results.
An orange contains protease enzyme which is alkaline hence a serine protease with the best activity with azo-casein and highly inhibited by PMSF. It was therefore important that an alkaline PH maintained for optimal activity and at optimum temperature (Yada 2004). Temperature played a significant role in enabling the process of extracting enzymes from the Orange. The rate at which the extraction occurred accelerated with the increasing temperature, a trend that characterized the aqueous extraction, (Uhlig 137). Notably, the effectiveness of the aqueous extraction significantly fell below the expectations when the temperature went high 80oC. The direction started to indicate a downward trend and even began to fall at temperatures higher than this level. The reason behind the above observation is the fact that several differences characterized the treatment adopted in preparing the system substances for extraction. As for the case of extraction supported by enzymatic action, the rate increased as the temperature increased with the 80oC being the temperature at which the process was fastest. From the observations, its evident that when the temperature in increased at a continuous trend, it leads to contnious activation of enzymes. Enzymatic action stopped because of the inactivation which was caused by freezing temperatures. Significantly, the choice of the composition of the extraction buffer is very important. For proteins that are soluble, a 20-50mM phosphate composition would be the most recommended at a pH range of 6.5 with stabilization additives.
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