Scientific and Technical ST0032 Educational Enzyme Kit Amylase User Guide

Scientific and Technical ST0032 Educational Enzyme Kit Amylase

Product Information

Specifications:

• Part Number: ST0032
• Manufacturer: Scientific and Technical Limited
• Address: John Eccles House, OX4 4GP, England
• Registered in England and Wales: 15611682
• Email: Sales@carboncapture-instruments.com
• Website: www.carboncapture-instruments.com

Product Usage Instructions

Background:

Enzymes are nature’s machines that play a crucial role in various life processes. They are composed of proteins, made up of amino acids, and convert starting materials into products. Enzymes are widely present in the body and are essential for digestion, energy production, and other biological functions. They have been used for centuries in various industries and are now being explored for their biomedical properties.

Industrial Enzymes:

Enzymes like amylase, cellulase, lipase, protease, pectinase, and xylanase are commonly used in different industries for processes like brewing, baking, biofuels production, paper manufacturing, textiles, food processing, and more. These enzymes help in breaking down starch, cellulose, fats, proteins, and other substances into useful products.

Educational Enzyme Kit:

We have developed an educational enzyme kit to introduce students to the world of enzymes. This kit will help students understand the importance and applications of enzymes in various fields.

Carbohydrates, Oligosaccharides, and Simple Sugars:

Enzymes play a crucial role in breaking down carbohydrates into oligosaccharides and simple sugars, which are essential for energy production and various metabolic processes.

Frequently Asked Questions

• Q: How can I purchase the educational enzyme kit?
  • A: You can purchase the educational enzyme kit directly from our website or contact our sales team via email at Sales@carboncapture-instruments.com for more information.
• Q: What age group is the educational enzyme kit suitable for?
  • A: The educational enzyme kit is designed for students of various age groups, typically suited for middle school to high school levels.

Background

Enzymes are nature’s machines and play a huge role across all life. Enzymes are composed of proteins (which are themselves composed of amino acids) and convert starting materials into products. Their biggest role is to breakdown food in the digestive system to unlock essential nutrients and energy. However they are also found in the mouth, eyes, liver, and pancreas, in addition, to every single cell inside your body.
These machines have been used by humans for thousands of years to curdle milk to produce cheese, or for making beer from malt grains. For the last 80 years, they have played a big role across many industries, including food processing, brewing, baking, distilling, paper manufacturing and cleaning. More recently scientists have started to investigate their biomedical properties to help cure diseases including certain types of cancers. A list of the main industrial enzymes are shown in table 1. Enzymes also have excellent green credentials and reduce the carbon footprint in almost all processes they are used in when compared to conventional methods. This is because enzymes are highly efficient and can operate a low or ambient temperatures, after all they have had millions of years to evolve to perform specific chemical processes. Enzymes are beginning to play a central role in a more sustainable future, this is perhaps most visible in the production of biofuels from food waste. Given their hugely important role across many domains, we have developed an educational enzyme kit to introduce students into the world of enzymes.

Table 1. Overview of major industrial enzymes.

Enzyme| Class| Starting material| Product/s| Main industrial

use

---|---|---|---|---
Amylase| Glycoside hydrolases| Starch| Oligosaccharides, small chain sugars| Brewing,    baking,

biofuels,      starch processing

Cellulase| Glycoside hydrolases| Cellulose| Oligosaccharides, small chain sugars| Biofuels,       paper and pulp, textiles, animal           feed, agricultural, laundry,

detergents,

Lipase| Lipid

hydrolases

| Fats & lipids| Fatty       acid       &

glycerol

| Food processing
Protease| Protein

hydrolases

| Protein| Amino acids| Food processing,

nutraceutical,

Pectinase| Pectin

hydrolases

| Pectin| Pectic acid| Fruit               juice clarification,  jam

production.

Xylanase| Glycoside hydrolases| Xylan| Xylose| Biofuels,     animal feed, laundry,

detergents

---|---|---|---|---

Carbohydrates, Oligosaccharides and Simple Sugars:

Sugar is the energy molecule for life. Sugars are pumped around the body and broken down to release energy enabling life to function. Sugars can exist in many different forms, from simple single units, two units joined together, to long chains called polymers, examples of each are shown in Figure 1. In polymer form, sugars are known as carbohydrates and are found in many important foodstuffs, including pasta, rice, flour and grains. In contrast, simple sugars, taste sweet and can be found many fruit juices, honey, jams and confectionaries. Oligosaccharides on the other hand are composed of three to ten simple sugars joined together.

Figure 1. Chemical structures of the main types of sugars and their macrostructure. A) Monosaccharides; glucose, fructose and galactose, B) disaccharide sucrose, or C-I) polymer (long chain) forms cellulose, starch and glycogen showing their subunit connectivity. C-II) The polymers can interact with themselves to generate a higher structure, cellulose forms crystalline sheets with interact via hydrogen bonding (dashed lines), whilst starch forms overlapping long chains, and glycogen forms bundles

Amylase Enzyme

The digestion of carbohydrates into sugar is one of the most important processes in nature. Amylase enzymes play a central role in fragmenting carbohydrates into oligosaccharides and sugars to help unlock energy. Amylases also play a big role across industry, widespread applications include brewing, baking, starch and food processing, detergents and biofuels. Amylase is typically added in brewing, baking and food processing products to increase sweetness by unlocking sugars. In detergents this enzyme helps breakdown carbohydrate-based stains. For biofuels, amylase can help maximise final ethanol product yield by increasing the concentration of fermentable sugars.Scientists have isolated amylases from nature and been able to crystallise them in order to determine the chemical shape and size. The X-ray crystal structure of Amylase derived from a fungal strain containing four glucose molecules is shown in Figure 2.

Figure 2. X-ray crystal structure of the enzyme amylase, left) space filling representation, right) volume representation, scale bar denotes 1 nanometre. Four glucose molecules can be seen inside the representations (light green outlines). The different types of protein folding structure are also annotated, alpha-helix and beta-pleated sheets. Scale bar 1 nanometre (10-9 meter).

Enzyme Activity and Reaction Kinetics

Enzymes are catalysts that convert starting materials (also called substrates) into products. The chemical reaction can be defined according to the process diagram shown in Figure 3A. When an enzyme is added to a starting material, the enzyme binds to generate a complex – this is because the enzyme has a binding pocket specific for this starting material. Next, the enzyme is able to perform its specific function on the complex to yield the product. The reactions progress can be plotted on a graph as shown in Figure 3B. Typically time is plotted against the percentage of product formed. Initially the reaction proceeds quickly in a linearly fashion, then starts to slow down as the starting material concentration decreases. Reaction rates, also known as reaction kinetics, can be modelled and predicted using a famous equation called the Michaelis–Menten equation (see further reading). Many factors influence the enzyme reaction rate, including the concentration of enzyme, the concentration of substrate, temperature, salt concentration, pH and the presence of any inhibitors. In this kit we explore the effect of two different substrates, potato starch and crushed malt.

Figure 3. Overview of enzyme reaction. A) Starting materials (blue circles) are mixed with an enzyme (orange circle with binding pocket) to form an enzyme-starting material complex, the enzyme converts the starting material into a product (green circles). B) Reaction progress can be plotted on a graph of time (in minutes) versus product % formed. The graph shows four different enzyme concentrations, typically reactions follow a linear (straight line) profile, then slow down as the concentration of available starting material decreases (only visible in the fastest profile).

For more information about enzymes and reaction kinetics see:

• Bell, E.L., Finnigan, W., France, S.P. et al. Biocatalysis. Nat Rev Methods Primers 1, 46 (2021).
• Cooper, G.M. The Cell: A Molecular Approach. 2nd edition, 2000.

Alternatively, check out these YouTube videos below:

• YouTube video here, here and here.

Kit Overview

Figure 3. Photograph of Educational Enzyme Kit – Amylase contents.

Table 2. Itemised contests of educational kit with defined usage.

enzyme,       100      mL bottle

| 1| Active    enzyme     for    digesting

starches     to    oligosaccharides and simple sugars

200    g    potato

starch

| Dried potato starch| 1| Powdered     starch      containing

high percentage carbohydrate.

200 g crushed malt| Crushed malt|  | Crushed         malt       containing moderately high percentage of

carbohydrates

Lugol’s     Iodine Solution| 1% Lugol’s Iodine 100 mL bottle| 1| Iodine solution used to stain starch         via iodine starch

complex

Portable

Refractometer

| BRIX refractometer| 1| Used         to        monitor         the concentration       of     sugar     in

solution

Pasture Pipette| Pasture Pipette| 3| Used to transfer liquids, note can be reused after washing

with water

500 mL beaker| 500 mL beaker| 1| Large     volume     container     to

perform experiments

6-well

sampling trays

| 6-well sampling trays| 3| 3 mL volume sampling trays to

work up reactions

Experimental Plan

Figure 4. Suggested 6-well sampling plate layout. #1 is the control well for reference (starch sample without enzyme), followed by the numbers 2 to 8 which are sequential timepoints at the stated time intervals.

Safety information:

Always wear protective equipment, including laboratory glasses, safety gloves and a laboratory coat and clean up spills promptly.

Table 1. Safety information.

cause respiratory irritation. May cause allergy or asthma symptoms or breathing difficulties if inhaled. In case of inadequate ventilation, wear respiratory protection. Avoid breathing dust or mist. Wear suitable protective clothing, gloves and eye / face protection. Rinse eyes immediately and copiously with clean water for 15

minutes.

| Non-chlorinated waste
Lugol’s            Iodine Solution| Causes skin irritation / Causes serious eye irritation / May cause respiratory irritation. May cause allergy or asthma symptoms or breathing difficulties if inhaled.  In  case  of  inadequate

ventilation,    wear    respiratory

| Chlorinated waste
 | protection. Avoid breathing dust or mist. Wear suitable protective clothing, gloves and eye / face protection. Rinse eyes immediately and copiously with clean water for 15

minutes.

|
---|---|---

Equipment Required:

• Boiling water from a kettle (caution, adult supervision required).
• Stop watch / timer.
• You are strongly advised to wear personal protective equipment (PPE), including gloves, laboratory coat and safety googles.
• Optional: Potatoes.

Potato Starch Instructions

1. Weigh out 12 g of Potato Starch (4x non-heaped spoons) into the 500 mL beaker.

2. Add 50 mL cold tap water and stir.
   Observation – generate a white slurry without any lumps.

3. Boil 150 mL tap water in a kettle.
   Caution – boiling water, adult supervision required!

4. Pour 150 mL boiling water into the beaker and stir solution for 2 minutes.
   Observation – thick and viscous solution which is hard to stir.

5. Using a Pasteur pipette, extract 1 mL and place position 1 of the sample plate (this the control well for future reference).

6. Using a Pasteur pipette, drop 2 drops on to the refractometer and record the BRIX value.

7.  Using a clean Pasteur pipette, add 3x drops of Amylase enzyme to the beaker, start your stopwatch, and stir for 10 seconds.
   Observation – solution should turn less viscous as the fast action amylase digests starches into oligosaccharides and simple sugars

8. After 2 minutes, using the Pasteur pipette, extract 1 mL solution and place in position 2 of the sampling well. Next, add 3 drops of 1% Lugol’s Iodine solution to the well and record the colour.

9. Using a Pasteur pipette, drop 2 drops on the refractometer chamber and record the BRIX value.

10. Repeat steps 9-11 four more times after 4, 6, 8 and 10 minutes, each time recording the colour and BRIX value.
    Observation – the iodine solution should turn from dark blue, to light blue, then brown and finally yellow at the final timepoint.
    Observation – the BRIX value should start around 2 and end around 5 at the final timepoint

Potato Starch Expected Results

Figure 5. Expected Results from potato starch digestion experiment. Top) 6-well sampling plate changing in colour using Lugol’s Iodine solution, #1 = starch only black, then turning to brown, orange and finally yellow as the digestion reaction progresses. Bottom) Corresponding plot of time against BRIX diffractometer readings, graph generated using spreadsheet software.

Crushed Malt Instructions

1. Weigh out 20 g of Crushed Malt (10x non-heaped spoons) into the 500 mL beaker.

2. Boil 200 mL tap water in a kettle.
   Caution – boiling water, adult supervision required!

3. Pour 200 mL boiling water into the beaker and stir solution for 2 minutes.
   Observation – thick and viscous solution which is hard to stir.

4. Using a Pasteur pipette, extract 1 mL and place position 1 of the sample plate (this the control well for future reference).

5. Using a Pasteur pipette, drop 2 drops on to the refractometer and record the BRIX value.

6. Using a clean Pasteur pipette, add 3x drops of Amylase enzyme to the beaker, start your stopwatch, and stir for 10 seconds.
   Observation – solution should turn less viscous as the fast action amylase digests starches into oligosaccharides and simple sugars

7. After 2 minutes, using the Pasteur pipette, extract 1 mL solution and place in position 2 of the sampling well. Next, add 3 drops of 1% Lugol’s Iodine solution to the well and record the colour.

8. Using a Pasteur pipette, drop 2 drops on the refractometer chamber and record the BRIX value.

9. Repeat steps 9-11 four more times after 4, 6, 8 and 10 minutes, each time recording the colour and BRIX value.

   • Observation – the iodine solution should turn from dark blue, to light blue, then brown and finally yellow at the final timepoint.
   • Observation – the BRIX value should start around 2 and end around 5 at the final timepoint.

Crushed Malt expected results

Figure 6. Expected Results from Crushed Malt Digestion Experiment. Top) 6-well sampling plate changing in colour using Lugol’s Iodine solution, #1 = starch only black, then turning to brown, orange and finally yellow as the digestion reaction proceeds. Bottom) Corresponding plot of time against BRIX diffractometer readings, graph generated using spreadsheet software

Additional Experiments:

In addition to the above, try repeating the experiments to build up more trends and insights into the world of enzymes. A list of additional experiments include:

• Repeat the experiments using more / less starting material.
• Repeat the experiments using more / less enzyme.
• Repeat the experiment at higher / lower temperatures.
• Repeat the experiment using macerated potatoes.
• Try adding baker’s yeast (5% mass / volume) to the end solution to make alcohol. Fermentation will take 6-12 hours and will cause bubbles (CO2 gas) and froth to appear.

Contact

• Scientific and Technical Limited
• John Eccles House OX4 4GP England
• Registered in England and Wales 15611682
• Email: Sales@carboncapture-instruments.com
• Website: www.carboncapture-instruments.com

References

• Computer Instruments | Home
• Carbon Capture Instruments
• Carbon Capture Instruments

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