Experiment 1final

Table of Contents

No Title Page1. Abstract/ Summary 22. Introduction 2-43. Aims 44. Theory 4-55. Apparatus 5-76. Methodology/Procedure 7-87. Results 8-98. Calculation 9-149. Discussion 14-16

10. Conclusion 1611. Recommendation 1612. Reference 1613. Appendix 17-18

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Experiment 1: Enzyme assays based on UV-VIS spectroscopy: characterization of sweet potato beta amylase.

Abstract

This experiment is conducted to determine the activity of beta amylase in sweet potato dependence on pH and to find out the characterization of beta amylase found in it. In this experiment, starch sample from rice also being used in addition of pure starch. The activity of enzyme is measured dependence on difference in pH (pH 3, 5, 7, 9, 11 and 13). The absorbance value from the spectrophotometer is used to calculate the enzyme activity in a particular condition in this experiment. As a result, only pH 3, 9 and 11 gave the absorbance value while the others were not while the starch from rice is also gave no value. Through the difference in maltose concentration, a standard curve is plot. The equation obtained from the curve is then being used to calculate the activity of beta amylase on starch.

Introduction

Sweet potato

Sweet Potato, common name applied to a perennial, trailing herb of the bindweed family. The plant, which is native to tropical America, is cultivated on sandy or loamy soils throughout many warm regions of the world, and exists as an important food staple in a number of countries. It is planted primarily for its thick, edible roots, called sweet potatoes. The name is due to a sugar content. Two main types are commonly cultivated: a dry, mealy

type, and a soft, light-to-deep-yellow, moist-fleshed type. The species often called wild sweet-potato vine, manroot, or man-of-the-earth is not edible, but is cultivated as an ornamental vine.

Carbohydrates generally make up between 80 to 90 % of the dry weight of sweet potato roots. The sweet potato yields an important starch, which is used as a food starch in noodles etc. and technically for sizing textiles and papers, for the manufacture of adhesives and in laundries. The process of extracting and

refining the starch is similar to the potato starch process. The pink and yellow varieties are rich in carotene, the precursor of vitamin A.

Sweet potato starch granules vary from 4 to 40 microns with 19 micron in average. The amylose content is 19% - 25%. Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store. It is the most important carbohydrate in the human diet and is contained in such staple foods as potatoes, wheat, maize (corn), rice, and cassava.

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Figure 2.1: sweet potato

Starch

Starch is the major carbohydrate reserve in plant tubers and seed endosperm where it is found as granules each typically containing several million amylopectin molecules accompanied by a much larger number of smaller amylose molecules. By far the largest source of starch is corn (maize) with other commonly used sources being wheat, potato, tapioca and rice. Amylopectin (without amylose) can be isolated from 'waxy' maize starch whereas amylose (without amylopectin) is best isolated after specifically hydrolyzing the amylopectin with pullulanase. Genetic modification of starch crops has recently led to the development of starches with improved and targeted functionality.

Figure 2.2:

Starch molecules arrange themselves in the plant in semi-crystalline granules. Each plant species has a unique starch granular size: rice starch is relatively small (about 2μm) while

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potato starches have larger granules (up to 100μm). Although in absolute mass only about one quarter of the starch granules in plants consist of amylose, there are about 150 times more amylose molecules than amylopectin molecules. Amylose is a much smaller molecule than amylopectin. (http://www1.lsbu.ac.uk/water/hysta.html)

What are amylases?

The enzymes that break down or hydrolyze starch or glycogen into the constituent sugars are known as amylases. Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extra-cellular digestion. When they have broken down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed into their cells.

Amylases are classified based on how they break down starch molecules

i. α-amylase (alpha-amylase)

Reduces the viscosity of starch by breaking down the bonds at random, therefore producing varied sized chains of glucose. . Alpha-amylases are found in plants and in animals. Human saliva is rich in amylase, and the pancreas also secretes the enzyme. Individuals from populations with a high-starch diet tend to have more amylase genes than those with low-starch diets; chimpanzees have very few amylase genes. It is possible that turning to a high-starch diet was a significant event in human evolution.

ii. ß-amylase (Beta-amylase)

Breaks the glucose-glucose bonds down by removing two glucose units at a time, thereby producing maltose. Beta-amylase cuts starch into maltose units. This process is important in the digestion of starch and is also used in brewing, where the amylase from the skin of the seed grains is responsible for converting starch to maltose (Malting, Mashing).

iii. Amyloglucosidase (AMG) - Breaks successive bonds from the non-reducing end of the straight chain, producing glucose

Many microbial amylases usually contain a mixture of these amylases.

Aims/objective

1. To find out the characterization of beta amylase in sweet potato.2. To calculate the activity of beta amylase dependence on pH based on two sample of

starch. 3. To prepare a standard curve of maltose based on difference concentration.

Theory

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http://www1.lsbu.ac.uk/water/hysta.html

One of the form of amylase, β-amylase (alternate names: 1,4-α-D-glucan maltohydrolase; glycogenase; saccharogen amylase) is synthesized by bacteria, fungi, and plants. β-Amylase is found primarily in the seeds of higher plants and sweet potatoes. It yields a single product: maltose. This enzyme is useful in structural studies of starch and glycogen. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond, releases successive maltose units and cleaving off two glucose units (maltose) at a time. The reducing Glc of maltose is in the β-form, hence, the name β-amylase (Kossmann and Lloyd, 2000). The primary function of β-amylase is involvement in starch breakdown in plants (Kossmann and Lloyd, 2000). Based on in vitro studies, the physiological role of β-amylase in starch breakdown has long been controversial, primarily because it was thought to be catalytically inactive on native starch grains without ample prior digestion by other amylolytic enzymes like α-amylase (Beck and Ziegler, 1989). However, a recent study has demonstrated that β-amylase plays a major role in transitory starch breakdown (Scheidig et al., 2002).

During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit. The shortest normal saccharide attacked is maltotetraose (Myrback and Neumuller 1950). Since it is unable to bypass branch linkages in branched polysaccharides such as glycogen or amylopectin, the hydrolysis is incomplete and a macromolecular limit dextrin remains.

β-Amylase is specific for amylose chains of six glucose units (Kainuma and French 1970). It separates maltose disaccharides, or malt sugar, from starch. Some vegetables and many fruits taste sweet in part because of the role that beta-amylase plays to break starch down into sugar. This is one reason why sweet potatoes have their signature sugary taste. The beta-amylase PacMan is very picky and can only "chew" on the ends of a starch molecule, and only on one end and not both. It can only chew on the "reducing" end of starch, and that is the end far off the righthand side of the figure above. When beta-amylase does its job, it bites of maltose units - in other words, two glucose units at a time.

Beta-amylase is also used for industrial purposes, such as in the brewing of beer. The enzyme, secreted from yeast, breaks up the maltose compounds in barley, turning it into malted barley, and thereby aiding the fermentation process. Its presence in yeast is also the reason why amylase often plays an important role in bread making, where it breaks down starch, adds flavor and leavens dough.

Materials and apparatus

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http://www.ncbi.nlm.nih.gov/pubmed/12047632

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC519081/#bib2



Incubator shaker Sigma 3-18K rotor Boiler Spectrophotometer

pH meter

Sweet potatoes DNSA reagent Acetate/acetic acid buffer (pH 3, 5, 7, 9, 11 and 13)Sodium hydroxide (NaOH)Ammonium sulfate Maltose Boiling water bath Distilled waterTest tubes Measuring cylinder

BeakerStarch Rice Measuring cylinder Pipette Digital pipette with pipette tipMagnetic stirrerAnalytical balanceUniversal bottleGlass rodDropper

Procedures

The preparation of the enzyme from sweet potato.

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1. Two sweet potatoes is grinded in a meat grinder and then mixed in a blender for two minutes with a quality of water equal to half the mass of pulp.

2. Insoluble debris is removed by centrifugation for 15 minutes in a Sigma 3-18K rotor at 12000 rpm

3. The enzyme is separated from the supernatant by precipitation using ammonium sulfate.

4. Ammonium sulfate is added in the amount of 0.47g per mL of supernatant and the precipitate is harvest by centrifugation at 12000 rpm for 10 minutes.

5. Pellets were suspended with 1mL of water for each 8mL of original pulp and recentrifuged (12000 rpm, 10 minutes).

6. Supernatant were saved cold and should contain active enzyme.

Determination of the amount of amylase activity dependence on pH.

1. 0.1mL of the preparation is mixed with 0.1mL of 1% starch and 0.1mL of different pH values buffer (around 0.1 M acetate/acetic acid) which are at pH 3, 5, 7, 11 and 13.

2. The mixture is incubated at 37 degrees for 15 minutes.3. 0.3mL of DNSA reagent is added and the mixture is heated in a boiling water bath for

10 minutes.4. The mixture then being diluted with 4mL of water and the absorbance is determined

at 540 nm.5. Blank solution is prepared by replacing the preparation in step 1 by distilled water

with the same volume.

Preparation of standard curve based on maltose concentration.

1. Maltoses with different concentrations ranging from 0.01g/mL to 0.1g/mL are prepared.

2. 0.3mL of DNSA reagent is added and the mixture is heated in a boiling water bath for 10 minutes.

3. The mixture then being diluted with 4mL of water and the absorbance is determined at 540 nm.

4. Graph of absorbance versus concentration is plot.

Preparation of DNSA reagent(being used during the experiment)

1. 2.5g of DNSA is weighted and dissolved in 50mL of 0.4M of NaOH.2. 125mL of distilled water is added into the mixture.3. Next, 75g of sodium potassium sulphate is added.4. Solution is diluted with distilled water until 250mL of solution is reached. 5. The mixture is stirred using magnetic stirrer.

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a) 1g DNSA powder = 100 mL DNSA reagent

2.5g DNSA powder = 250mL DNSA reagent

b) 20mL 0.4M NaOH = 100 mL DNSA reagent

50mL 0.4M NaOH = 250mL DNSA reagent

c) 50mL distilled water = 100mL DNSA reagent

125mL distilled water = 250mL DNSA reagent

d) 30g sodium potassium sulphate = 100mL DNSA reagent

75g sodium potassium sulphate = 250mL DNSA reagent

Results

Determination of the amount of amylase activity dependence on pH

Volume of supernatant obtained from the sweet potato = 10mL

We are divided into 5 groups. Each group is assigned to determine the activity of enzyme based on respective pH.

pH

group

Enzyme source

Blank 3 5 7 9 11 13

1 Starch 0.000 0.003 - - - 0.042 -2 Starch 0.000 0.008 - - 0.042 - -3 Rice 0.000 0.042 - 0.000 - - -4 Rice 0.000 0.045 - - - - 0.0005 Rice 0.000 0.044 0.000 - - 0.042 -

The difference in maltose concentration to create a standard curve

Concentration of maltose (g/mL) Absorbance value

0.01 0.000

0.02 0.000

0.03 0.000

0.04 0.058

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0.05 0.000

0.06 0.040

0.07 0.065

0.08 0.002

0.09 0.003

0.10 0.000

Blank solution 0.000

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.450.000

0.050

0.100

0.150

0.200

0.250

0.300

f(x) = 0.648725266362253 x + 0.0128453196347032R² = 0.983993585556275

AbsorbanceLinear (Absorbance)

Concentration of maltose (g/mL)

Abso

rban

ce

Calculation

Enzyme activity = µmoleof maltose released

Reaction time×volumeof enzyme∈reactionmixture

Reaction time = 15 minutes

Volume of enzyme in reaction mixture = 0.1mL

Group 1

Starch source: pure starch

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I. Absorbance value at pH 3 = 0.003

y = 0.6487x + 0.0128

0.003= 0.6487x + 0.0128

X = -0.0098

µmoles of maltose released = massof maltoseMW of maltose

=−0.0098 g360.31g /mol

= -2.72× 10-5 moles × 1 µmoles

1.0×10−6

= -27.199µmoles

Thus,

Enzyme activity = −27.199µmoles15min×0.1ml

= -18.133 U/mL.

II. Absorbance value at pH 11 = 0.042

y = 0.6487x + 0.0128

0.042 = 0.6487x + 0.0128

X = 0.045


=0.045 g

360.31g /mol

= 1.249 × 10-4 moles × 1 µmoles

1.0×10−6

= 124.9µmoles

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Thus, Enzyme activity = 124.9 µmoles15min×0.1ml

= 83.267 U/mL.

Group 2:

Starch source: pure starch

I. Absorbance value at pH 3: 0.008

y = 0.6487x + 0.0128

0.008 = 0.6487x + 0.0128

X = -0.00739


=−0.00739 g360.31g /mol

= - 2.054 × 10-5 moles × 1 µmoles

1.0×10−6

= -20.54µmoles

Thus,

Enzyme activity = −20.54 µmoles15min×0.1ml

= -13.69 U/mL.

II. Absorbance value at pH 9 = 0.042

y = 0.6487x + 0.0128

0.042 = 0.6487x + 0.0128

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X = 0.045


=0.045 g

360.31g /mol

= 1.249 × 10-4 moles × 1 µmoles

1.0×10−6

= 124.9µmoles

Thus, Enzyme activity = 124.9 µmoles15min×0.1ml

= 83.267 U/mL.

Group 3

Source of starch: rice

I. Absorbance value at pH 3: 0.042

y = 0.6487x + 0.0128

0.042 = 0.6487x + 0.0128

X = 0.045


=0.045 g

360.31g /mol

= 1.249 × 10-4 moles × 1 µmoles

1.0×10−6

= 124.9µmoles

Thus,

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Enzyme activity = 124.9 µmoles15min×0.1ml

= 83.267 U/mL.

Group 4

Starch source: rice


y = 0.6487x + 0.0128

0.045 = 0.6487x + 0.0128

X = 0.0496


=0.0496 g

360.31g /mol

= 1.378 × 10-4 moles × 1 µmoles

1.0×10−6

= 137.8µmoles

Thus,

Enzyme activity = 137.8 µmoles15min×0.1ml

= 91.843 U/mL.

Group 5

Starch source: rice


y = 0.6487x + 0.0128

0.044= 0.6487x + 0.0128

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X = 0.048


=0.048 g

360.31g /mol

= 1.335× 10-4 moles × 1 µmoles

1.0×10−6

= 133.48µmoles

Thus, Enzyme activity = 133.48µmoles15min×0.1ml

= 88.99 U/mL.

After being calculated.pH

group

Enzyme source

Blank 3 5 7 9 11 13

1 Starch 0.000 -18.133 U/mL.

- - - 83.267 U/mL.

-

2 Starch 0.000 -13.69 U/mL.

- - 83.267 U/mL.

- -

3 Rice 0.000 83.267 U/mL.

- - - - -

4 Rice 0.000 91.843 U/mL.

- - - - -

5 Rice 0.000 88.99 U/mL.

- - - - -

Discussions

In this experiment, we were investigating the β-amylase activity on starch where at the end of this experiment, the enzyme activity is calculated dependence on pH. The β-amylase activity is tested through two types of samples which are rice and starch. In observing the reaction dependence on pH, pH ranging from 3 to 13 is used and absorbance value from the spectrophotometer is taken. Based on the absorbance value obtained only the starch in rice gave the negative value or we assumed it as zero. Based on this situation we can conclude that the reaction of β-amylase on the starch in rice cannot take place as rice is not

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completely break down into starch yet and the process might take a longer time as the rice is not crushed completely.

At the pH of 9 and 11 the absorbance value obtained showed that the activity of enzyme is occurred. From the calculation the enzyme activity only occur in pH 9 and 11. β-Amylase is specific for amylose chains of six glucose units (Kainuma and French 1970). It separates maltose disaccharides, or malt sugar, from starch. Some vegetables and many fruits taste sweet in part because of the role that beta-amylase plays to break starch down into sugar. This is one reason why sweet potatoes have their signature sugary taste. The beta-amylase is very picky and can only "chew" on the ends of a starch molecule, and only on one end and not both. It can only chew on the "reducing" end of starch, and that is the end far off the right hand side of the figure above. When beta-amylase does its job, it bites of maltose units, in other words, two glucose units at a time.

Figure 9.1: The arrows show the bond in which the β-Amylase will cut.

Figure 9.2: The process of conversion of starch into glucose

Theoretically, the characterization of beta amylase in sweet potatoes has the following criteria:

1. Molecular weight

206,000 ± 1,000 (Colman and Matthews 1971).

2. Composition:

The enzyme is tetrameric with molecular symmetry (Colman and Matthews 1971). Thoma et al. (1965) report on the amino acid composition. See also Uehara et al. (1970). It has been

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indicated that tryptophan residues are involved at the active center (Uehara et al. 1971). The four binding sites are independent of each other.

3. Optimum pH:

pH of 4-5.

4. Extinction coefficient:

= 17.7 (Takeda and Hizukuri 1969).

5. Specificity:

β-Amylase is specific for amylose chains of six glucose units (Kainuma and French 1970). See also Lee (1971). Thoma et al. (1971) has reviewed substrate structural requirements of both α- and β- amylases. See Kainuma and French (1970) for comparison of specificity with hog pancreas α-amylase, and also the review by French (1960).

6. Inhibitors:

The enzyme is sulfydryl sensitive, and inhibited by heavy metal ions, p-mercuribenzoate, iodoacetamide and urea. Ascorbate inhibition has been attributed to cupric ion reduction and subsequent formation of an inactive cuprous enzyme complex (Rowe and Weill 1962). Cyclohexaamylose is a competitive inhibitor.

During this experiment there might be some errors occurred that affect our results. That might be from the DNSA reagent prepared at the beginning of the experiment in which it has been contaminated by other substance came from the pipette used to take an amount of DNSA reagent. Not only that the surface of cuvette used in spectrophotometer which is not being wiped properly also affect our results.

In industry, the activity of beta amylase mostly being used in brewing and malting. β-Amylase plays a central role in the complete degradation of starch to metabolisable or fermentable sugars during the germination or malting of cereal grains. It also finds considerable application, together with starch debranching enzymes, in the production of high maltose syrups.

Conclusions

Based on this experiment we are actually investigating the activity of beta amylase on starch. Through this experiment we can somehow get the idea on how the beta amylase work in breaking down the starch molecules into maltose and the optimum condition it will work in. Based on the result obtained only pure starch at pH 9 and 11 gave the absorbance values while the others were not. Although we make some mistakes during this experiment

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but we somehow get a lot of knowledge from it and learnt to be more careful in conducting more experiment after this. In conducting biological laboratory we need to be extra careful with the apparatus, material and condition we worked in because enzyme, living things and etc are very sensitive to certain conditions. In conclusion, the objectives of this experiment are achieved at the end of this experiment and through this experiment we get the familiarity with some procedures in biological laboratory and on how to handle certain apparatus.

Recommendations

1. The rice used in this experiment should be crushed completely until it turns into a paste because in this form the rice is readily being broken down into starch and can be easily reacting with the beta amylase.

2. Instead of using rice we can also use corn as a replacement as corn also containing starch.

3.

References

1. Mc Murry, John. Organic Chemistry, 7th edition.2. Trudy, McKee. The molecular basis of life, 3rd edition.3. Dr Shakinaz, Desa. College Matriculation Biology.4.

Appendix

Plate 13.1: Sweet potato after the insoluble debris is removed by the centrifugation for 15 minutes.

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Plate 13.2: Supernatant after being separated from the liquid and being added by ammonium sulphate.

Plate 13.3: The condition of supernatant after being cold and the solution obtained is actually the active enzyme.

Plate 13.4: The mixture of enzyme with starch after being incubated.

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Plate 13.5: The mixture of enzyme with starch after DNSA reagent is added.

Plate 13.6: Preparation of DNSA reagent

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Experiment 1final

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