To investigate some of the properties of immobilised enzymes.

Introduction

The four-session practical exercise is designed to give some insight into the preparation and properties of immobilised enzymes. 

It consists of three parts:

• Preparation and properties of covalently-immobilised a-amylase. 
• Preparation and properties of non-covalently-immobilised a-amylase.
• The use of a packed-bed and stirred tank reactor.

Before starting work, read through the Methods and Results sections. This practical is very demanding and must be approached with thought and care. It will be necessary to retain samples of the soluble enzyme before and after coupling (as well as the immobilised enzyme, of course) for protein and activity assay (see Appendix A) in order to determine the amount of enzyme coupled. You are expected to work in teams of three with a named team leader. You must plan and organise your experiments carefully, for which marks will be awarded. a-amylase is an enzyme produced and purified from Bacillus. It hydrolyses the a-1-4 links in starch randomly along its structure (i.e. it is an endo-glycosidase). It cannot hydrolyse a-1-6 links. Complete hydrolysis of starch by a-amylase produces a mixture of short glucose oligomers (e.g. maltose, maltotriose), some limit dextrin containing a-1-6 links but relatively little glucose. The quality of hydrolysed starches is given in terms of its dextrose equivalent (DE), which equals the percentage of the starch that is hydrolysed. (’Dextrose’ is another word for glucose). 

a-Amylase is assayed by the creation of new reducing (terminal; equivalent in reducing power to glucose) sugar by the catalysed hydrolysis of soluble starch.

In this practical, a-amylase is immobilised by means of covalent and non-covalent binding to solid supports. The amount of enzyme attached to the supports is determined and the activities of the immobilised enzymes are compared to that of the free (non-immobilised) enzyme. Packed bed reactors containing the immobilised enzymes are prepared and their ability to hydrolyse starch compared. 

Plan
During week 1 you should

• Prepare covalently-immobilised a-amylase
• Prepare non-covalently-immobilised a-amylase
• Construct standard curves for protein and reducing sugar (see Appendix A for details). Draw these before week 2 when they will be required.

During week 2 you should 

• Wash the covalently-immobilised a-amylase
• Wash the non-covalently-immobilised a-amylase
• Assay samples from the preparations; see ‘Assays’ later

During weeks 3 and 4 you should run the stirred tank and packed bed reactors and analyse their products.

Covalent Immobilisation of a-amylase (Methods Enzymol. 44, pp. 98-99)

Week 1. Weigh out 1.0 g Enzacryl AA gel. (This is a gel based on a polyacrylamide matrix with aromatic amino groups present as the reactive moieties). Add to 50 ml ICE-COLD 2 M HCl, stir at 0°C, and gradually add 40 ml ICE-COLD 2% sodium nitrite solution (Together these produce nitrous acid, HNO₂).

NaNO₂ + HCl HNO₂ + NaCl

Keep the beaker surrounded by ice during addition. This should take about 10 – 15 minutes. (This creates the reactive diazonium groups from the aromatic amino groups. If these are allowed to warm up, they decompose to give nitrogen gas with the loss of their specific reactivity)

Stir for another 15 minutes, then filter the gel on a filter paper disc on a Buchner funnel, by suction. Wash with 200 ml ICE-COLD 20 mM phosphate buffer, pH 7.0, 0.1 mM CaCl₂ (a-amylase buffer). Add the buffer in 25 ml batches, and KEEP IT COLD. At this stage, the amino groups of the gel matrix should be diazotized. Quickly scrape gel off the filter into a test tube. Add 5 ml of an ICE-COLD solution of a-amylase (2 mg/ml in phosphate buffer). Cap, label (’Cov‘) swirl in an ice bath for about an hour and leave in the fridge until next week. (This allows the diazo groups to covalently couple to the tyrosine phenolic groups on the enzyme)

Non-Covalent Immobilisation of a-amylase

Week 1. Weigh out 0.6 g dry phenolic resin (invented and patented by M. F. Chaplin, J Chem Soc., Perkin 1, 1979, pp 2144-2153), suspend in 20 ml 20 mM K phosphate pH 7.0, 0.1 mM CaCl2 (a-amylase buffer) for 10 minutes. Filter and re-suspend in 5 ml of 2 mg/ml a-amylase in the 20 mM phosphate buffer. Cap, label (’Non‘) swirl for 30 min and leave in the fridge until next week

N.B.: Keep a solution of the free enzyme (0.5 ml 2 mg/ml) similarly capped in the fridge as a comparison for both ‘Cov‘ and ‘Non‘ above (label ‘Enz‘).

Covalent Immobilisation of a-amylase

Filter the gel (’Cov‘), using a fluted filter paper, into a test tube; use 5 ml of 20 mM K phosphate buffer pH 7.0 to aid this process. Keep about 2 ml of the filtrate for assay of unbound protein and activity, (Label it ‘Cov-supernatant‘)

Wash gels to remove any free enzyme, using 3 x 20 ml batches of 20 mM potassium phosphate/500 mM NaCl pH 7.0 (’high salt buffer’). Let the gel damp-dry briefly between each 20 ml portion of buffer. Wash once more in the same buffer without NaCl, and re-suspend the gel in 5 ml in 20 mM K phosphate (pH 7.0). Label it ‘Cov-immobilised‘ and refrigerate until next week.

Non-Covalent Immobilisation of a-amylase

Filter the gel (’Non‘), using a fluted filter paper, into a test tube; use a further 5 ml of 20 mM K phosphate buffer pH 7.0 to aid this process. Keep about 2 ml of the filtrate for assay of unbound protein and activity, (Label it ‘Non-supernatant‘), .

Wash gels to remove any free enzyme, using 3 x 20 ml batches of 20 mM K phosphate buffer pH 7.0. Re-suspend in 5 ml of this buffer. Label it ‘Non-immobilised‘ and refrigerate until next week.

At this stage During Week 2 you should have the following samples:

• Cov-supernatant: unbound a-amylase; left over from covalent immobilisation (2 ml of not more than 1.0 mg/ml)
• Cov-immobilised: covalently bound a-amylase; 1 g dry gel containing not more than 10 mg enzyme.
• Non-supernatant: unbound a-amylase; left over from non-covalent immobilisation (2 ml, not more than 1.0 mg/ml).
• Non-immobilised: non-covalently bound a-amylase; 0.6 g dry gel containing not more than 10 mg enzyme.
• Enz: stored free a-amylase (0.5 ml of 2.0 mg/ml). Some of this should be refrigerated for next week.

Assays (See Appendix for details of the procedures)

Dilute samples ‘cov-supernatant‘ and ‘non-supernatant‘ 1:20 v/v and sample ‘enz‘ 1:40 v/v. Assay these diluted samples of ‘cov-supernatant‘, ‘non-supernatant‘ and ‘enz‘ for protein content (Assay 1) and a-amylase activity by production of reducing equivalents (Assay 3). Ensure that you record how you dilute these samples in your notebook.

Although you know the concentration of protein in sample ‘enz‘ (2 mg/ml), you will probably get a different value as determined in the Dye-binding assays due to the different standard protein (bovine serum albumin not a-amylase) used. Use this value to ‘correct’ the protein concentrations (i.e. if the apparent Dye-binding concentration of sample ‘enz‘ is 1.5 mg/ml then all final protein concentrations as determined by the Dye-binding method should be multiplied by the factor 2.0/ 1.5).

Tabulate the protein concentration (mg/ml, uncorrected and corrected) and activity (mmol reducing sugar released/min/ml and mmol reducing sugar released/min/mg) of the diluted and original undiluted samples cov-supernatant, non-supernatant and enz. By allowing for the volumes of solutions used in the binding (5 ml) and filtering (another 5 ml), tabulate also the total protein content of the supernatants. 

This Table will allow you to calculate:

1. the (corrected) weight of a-amylase protein not bound to each immobilisation matrix from the protein concentrations in the unbound residual enzyme samples cov-supernatant and non-supernatant. Make sure that you allow for the dilutions and the final volume of the wash solution (10 ml) and the correction for the use of the bovine serum albumin standard.
2. the weight of protein bound to each immobilisation matrix, by subtracting the (corrected weight of) protein not bound (from above) from the amount added (5 ml x 2 mg/ml = 10 mg).
3.  the percentage of the enzyme protein that was added that is bound to each immobilisation matrix.
4. the specific activity of the original a-amylase solution used (sample ‘enz‘); Note that the specific activity equals the activity of one mg a-amylase protein. the units are in mmol reducing sugar released per min per mg of protein).
5. the specific activity of the unbound a-amylase solutions left after each of the immobilisation processes. Note that these would be expected to be identical to the specific activity of the original a-amylase solution used (sample ‘enz‘) unless some denaturation occurred in the immobilisation process.

Note: you have to dilute the samples until they contain about 50 mg/ml protein so that your determinations are in the right range for the assays. Do not forget to allow for these dilutions when you determine the protein content and a-amylase activity of the original solutions.

Prepare two small packed bed reactors containing all of the covalently and non-covalently immobilised a-amylase (’cov-immobilised‘ and ‘non-immobilised‘). Do not allow them to run dry. (Note that if they are allowed to develop an air lock, they will not flow and must be repacked) Run 5 ml of 1% starch in 20 mM K phosphate pH 7.0, 0.1 mM CaCl₂. through each column.

 Remove the gels (’cov-immobilised‘ and ‘non-immobilised‘) from the columns. Using all of the samples of immobilised enzymes determine (separately) their activity in a stirred reactors (beaker) containing 50 ml of 1.0% w/v starch in 20 mM K phosphate pH 7.0, 0.1 mM CaCl2. Withdraw samples at intervals (e.g. 1 min, 5 min, 10 min, etc.) to determine their reducing sugar content. 

From the assay of the stirred tank and packed bed reactors you should calculate :

1. the concentration of reducing equivalent in the partially-hydrolysed starch samples (mmoles of reducing equivalent produced per ml per reactor).
2. the productivity of the reactors (mmoles of reducing equivalent produced per minute per reactor), 
3. the fractional conversions, X (X = moles of reducing equivalents produced/moles of potential glucose units in the starch solution); Note that to calculate the number of moles of potential glucose in the 1% starch, solution, the apparent M.Wt of potential glucose is 180 – 18 = 162, as water is necessary to release the glucose; the complete hydrolysis of 162 g of starch produces one mole (180 g) of glucose, Also remember that the number of moles in a sample is the weight in grams divided by the weight of one mole (i.e. weight/ M.Wt). Also, note that because a-amylase cannot hydrolyse limit dextrins, maltose, maltotriose or maltotetraose, the highest value expected for the fractional conversion, X, is about 0.2.
4. the dextrose equivalent DE of the products (in this case the fractional conversion, X, expressed as a percentage), 
5. the activity of the immobilised enzymes (mmoles of reducing equivalent produced per minute per g resin).
6.  the specific activity of the immobilised enzymes (mmoles of reducing equivalent produced per minute per mg enzyme) by using the known amount of protein immobilised (determined previously).
7. the effectiveness factors for the immobilised enzymes. Note that the effectiveness factor is the specific activity of the immobilised enzyme divided by the specific activity of an equal quantity of the free enzyme (calculated previously).

Assay samples ‘cov-immobilised‘, ‘non-immobilised‘ and ‘enz‘ for a-amylase activity by loss of iodine reactive material (Assay 4). This reaction may be very rapid with excess free enzyme. For the free and immobilised enzymes estimate the % digestion of the starch when the iodine reactive material has been used up, by comparing these results with your specific activity results from the production of reducing equivalents.

1. determine the relative specific activities of the immobilised enzymes compared with the free a-amylase. Use the reciprocals of the times needed to decolourise the blue starch-iodide divided by the amounts of a-amylase protein present.
2. compare these specific activities with those calculated earlier. Explain your results on the basis that starch molecules, once next to an immobilised a-amylase, have difficulty diffusing away due to their bulk. Thus, immobilised a-amylase is expected to produce some starch that is completely hydrolysed before other starch molecules are hydrolysed at all, whereas free a-amylase hydrolyses all starch molecules roughly equally.

 Practical: Appendices

Assays
Note that all assays should be done in duplicate, where possible. Ensure all the cuvettes are clean by checking their absorption against each other at the assay wavelength before use. 

1 Dye-binding Protein Assay 
1.5 ml of protein sample solution (0 – 50 mg/ml) is mixed with 1.5 ml Coomassie blue reagent (0.6% dye in dilute perchloric acid). Use 1.5 ml distilled water plus 1.5 ml Coomassie blue reagent as blank to zero the spectrophotometer. Read the absorbency at 620 nm. 

A standard curve is prepared by using the stock solution of bovine serum albumin (BSA, 50 mg/ml) using at least four data points in duplicate. e.g. 0.4 ml stock + 1.1 ml water (= 0.4/1.5 x 50 mg/ml = 13.3 mg/ml), 0.8 ml stock + 0.7 ml water, etc. N.B. only the concentration within the 1.5 ml ’sample’ solution is relevant; the (constant) amount of reagent added is not relevant for sample concentration calculations

2 Reducing Sugar Assay 
2.0 ml of DNS reagent (ready prepared; 3,5-dinitrosalicylic acid and sodium potassium tartrate dissolved in dilute sodium hydroxide) is added to sample (200 ml, 0.2 ml), containing 0 – 2 mg reducing sugar (i.e. 0 – 10 mg/ml). The tube is placed in a boiling water bath and the solution heated at 100°C for 5 minutes. Rapidly cool in ice to room temperature. Use 0.2 ml distilled water plus 2.0 ml DNS reagent, heated as above, as blank to zero the spectrophotometer. Read absorbency at 570 nm. A standard curve is prepared by using the stock solution of maltose (10 mg/ml) using at least four data points in duplicate. e.g. 0.05 ml stock + 0.15 ml water (= 0.05/0.2 x 10 mg/ml = 2.5 mg/ml), 0.1 ml stock + 0.1 ml water, etc. N.B. only the concentration within the 0.2 ml ’sample’ solution is relevant; the (constant) amount of reagent added is not relevant for sample concentration calculations. You are reminded that the M.Wt. of maltose is 342 and maltose contains a single reducing group (i.e. 342 g maltose contains one mole of reducing group/equivalent). For your graphs, you must calculate the molar concentration of reducing groups in the standard maltose solutions.

3 Assay of a-amylase by production of reducing equivalents 
Add 0.8 ml 20 mM K phosphate (a-amylase buffer) to 0.2 ml soluble enzyme in phosphate buffer (containing about 10 mg amylase). Note that the enzyme solutions must be diluted before they are assayed). Pre-incubate for about 4 minutes at 37°C. Add 1.0 ml, 1% starch in phosphate buffer (pre-warmed to 37°C). Incubate for exactly 5 minutes at 37°C. Stop the reaction by removing 0.2 ml of the incubated mixture and adding this to 2 ml of DNS reagent. 

The tube should be placed in a boiling water bath and the solution heated at 100°C for 5 minutes to develop the reducing sugar assay colour. Rapidly cool in ice to room temperature and read absorbency at 570 nm. Use 0.1 ml buffer plus 0.1 ml starch plus 2.0 ml DNS reagent, heated as above, as a blank to zero the spectrophotometer. Note that the reducing sugars in only 0.2 ml of the 2.0 ml in the 37°C incubation mixture is used in the reducing sugar assay and allowance should be made for this when calculating the amount of reducing sugar produced by the enzyme in the 0.2 ml original sample.

4 Assay of a-amylase by loss of iodine reactive material 
Make a mixture of 0.1 ml buffer plus 0.1 ml starch for use as blank. Add one drop to one drop of K phosphate containing 0.05% iodine. A blue coloration will be observed. 

Free enzyme assay: Add 0.8 ml K phosphate (20 mM, pH 7, ‘a-amylase buffer’) to 0.2 ml enzyme (containing about 10 µg free amylase). Incubate for 4 minutes at 37°C. Add 1 ml 1% starch (pre-warmed to 37°C) and incubate at 37°C. At known times (e.g. 0, 30 s, 1, 2, 5, 10 min etc), remove 1 drop and drop into 1 ml K phosphate containing 0.05% iodine. 

Immobilised enzyme assay: Add 1.0 ml K phosphate (20 mM, pH 7, ‘a-amylase buffer’) to half the immobilised enzyme. Incubate for 4 minutes at 37°C as above. Add 1 ml 1% starch. Keep the immobilised enzymes agitated. At known times (e.g. 0, 30 s, 1, 2, 5, 10 min etc), remove one drop and drop into one drop of K phosphate containing 0.05% iodine. In both assays, blue coloration will be observed while macromolecular starch is still present. The enzyme activity is inversely proportional to the time taken. If no blue colour is observed in the first samples, repeat the assay as either (1) the reaction has already occurred at to rapid a pace, or (2) you forgot to add the enzyme/ iodine/starch/etc.

The experiment was set up to be as fair as possible. Then the potato chips were cut into 15 cylinders using a cork cutter and then cut to a length of exactly 4cm each with a scalpel. Then three were weighed at one time on a top pan balance to find an average mass, this was done because it did not involve confusion over which potato cylinder was which after the experiment. Once these averages were found and recorded the potato chips were placed into their test tube solutions of 0.0M(distilled water), 0.25M, 0.5M, 0.75M and 1.0M solutions of sucrose. Then the contents, each containing three potato cylinders were left for 50minutes. When the time was over I removed the potato with a pair tweezers and placed them on a paper towel to try and dry of any excess water. Then I weighed the potato again together in their threes on a top pan balance and found the average mass.
 

Prediction
 

 I believe from my past knowledge of a similar experiment involving raw eggs that the potato cylinders placed in the distilled water will be hyperosmotic, therefore the water will diffuse by osmosis into the potato from an area of high concentration down the concentration gradient. This will result in a gain of mass for the potato.
 

    However in the 1.0M solution I think that the opposite will occur as there will be a higher concentration of water within the potato than the solution, containing lots of solute, this will make the potato hpo-osmotic and it will lose its water content as the water diffuses out.Finally, a difference between the egg experiment and the potatoes that the cells will not reach equilibrium (become iso-osmotic). This is because plants have a cellulose cell wall which cannot expand beyond a certain point as it is turgid, therefore the contents will never be able to be equal to the outside or it will not be able to give out all its water as it has a rigid structure. It is the pressure created by the cell wall which stops the cell reaching equilibrium. I think that the contents of the cell will be about 0.3M concentration as it will be in between 0.25M and 0.5MThe experiment has yielded a graph, (see attached) this graph gives me an excellent set of data. The graph shows that in the distilled water the potato gains in mass by approximently 7.4% then it gains 4.4% in the 0.25M solution. In the 0.5 M solution there is a change, the gradient of the graph decreases steeply and the potato loses mass to 4.4% then in the 0.75M solution the gradient decreases as it loses yet more mass to 10.4%.
 

 The first observation I made was after the potato cylinders were placed in their solutions I could see a difference. The ones in the 0.0M and 0.25M solutions were floating and the potatoes in the 0.5M, 0.75M and 1.0M solutions were at the bottom of the test tube, this lead me to drawing my next conclusion.
 

 The graph shows that the potato in the 0.0M solution and in the 0.25M is hyperosmotic, as I said in my prediction, this means that there is a higher water potential in the distilled water and 0.25M solution than in the potato, which has a high concentration of solutes. Therefore this is why the water diffuses by osmosis down the concentration gradient from high concentration to low concentration, resulting in the potato gaining mass.
 

 The opposite occurs in the solutions where the molarity is higher 0.5M, 0.75M and 1.0M, the potato in these is hypo-osmotic because there is a higher concentration of water inside the potato than in the solutions, therefore the water diffused out of the potatoes by osmosis, down the concentration gradient and into the solutions outside, this resulted in the loss of mass.
 

 Another important fact is that the graph makes a shape that will result in the potato not being able to take in any more water or lose any more. Like I said in my prediction the cell wall causes the pressure that prevents this from happening, therefore the cell will never become iso-osmotic like an egg.
 

 On my graph I have marked the point where the graph line crosses the place on the axis where the potato neither loses mass nor gains mass. This happens at 0.36M, therefore the contents of the potato cells in molar strength is 0.36M.
 

 This result is very close to my prediction of 0.3M and it shows that the contents of the cell are between 0.25M and 0.5M which is why in these concentrations they gained mass and lost mass.
 

  I think that the experiment went very well, there were no odd results and they produced a good graph. However there were a few areas where there could be improvement. Firstly, when I dried off the excess water on the potato cylinders after the experiment and before I weighed them, I used a paper towel. This might have either taken some water out of the potato or it might of left some excess water on the potato. This part of the experiment is difficult to     come up with an accurate and fair method, as other ways would also lead to some slight mistakes.
 

     Also the potato itself was not definitely from the same potato and was not exactly the same size, although I did try to cut them to 4cm each, this could have effected the amount of water gained or lost.
 

     Another way of improving the results would have been to leave the experiment running longer, this would have enabled me to find the saturation point (when the potato can no longer take in any more water) and dehydration point (when the potato cannot lose any more water) and therefore get a more accurate result.
 

     Finally, I could extend the experiment to a more exact level by looking at the potato cylinders under a microscope, then I would be able to see the cells in greater detail and draw some more observational results.