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.

• Ingestion (eetin’ an’ swallowin’ it)
• Peristalsis (movin’ it along)
• Digestion (smashin’ it up)
• Absorption (mekin’ it part of you)
• Egestion (defaecation or “shitting”)

The wall of the alimentary canal consists of:

• a lumen through which the food travels
• a mucosa, consisting of epithelium, connective tissue and muscles in the ileum which cause the movement of microvilli
• a submucosa
• circular muscle which contracts behind, and relaxes in front of the bolus. This widens in places to form sphincter muscles, like the cardiac and pyloric sphincter, which keep the food in the stomach for as long as is needed and enables the irregular feeding of carnivores. The anal sphincter keeps faeces in your body until a suitable time; otherwise life could be quite awkward!
• longitudinal muscle which relaxes behind, and contracts in front of the bolus.
• a serosa on the outside which protects the gut from friction. The epithelium surrounding it is called the mesentery and joins the various organs of the gut together.

The buccal cavity (gob) has a tongue (no…!), which rolls food into a bolus so that it passes easily down the oesophagus.
The stomach has cardiac and pyloric sphincters which keep food in and regulate it’s flow into the small intestine in small squirts. It is surrounded by muscle which helps to churn and mix the contents.
The small intestine (duodenum, jujenum, ileum) is long thin and folded, and therefore it has a large area over which to digest food. It has villi and microvilli, which further increase the available surface area. Its cells produce mucous, which lubricates the passage of food and reduce damage and friction.
The large intestine (colon) has the job of re-absorbing all the fluids secreted and ingested into the alimentary canal, and compacting the undigested remains for storing in the rectum. It is fairly wide. Bacteria exist here which use some of the undigested remains (fibre) and synthesise vitamins such as K, B₁₂ and riboflavin.

Digestion of protein, starch and lipids (walk-through) and control of secretions

Physical digestion begins in the mouth with mastication. This process breaks the food up and increases the surface area for enzymes to work on. Saliva from the salivary gland contains amylase, which begins the digestion of starch to maltose. Stimulation of salivary glands is by the nervous system in response to real or imagined food. Saliva also lubricates the movement of the food, which is then rolled into a bolus by the tongue and travels down the oesophagus to the stomach. Here it is mixed with gastric juice (pH 2), which is produced in gastric pits/glands in the stomach epithelium. Gastric juice is released in response to the presence of food in the buccal cavity. As the stimulation by the nervous system wears off, the presence of food stimulates cells in the stomach lining to produce gastrin. This passes into the bloodstream and stimulates the release of gastric juice for several hours. These consist of:
 

• Chief cells, which secrete pepsinogen, which is converted to its active enzyme by hydrochloric acid and pepsin itself. Producing the inactive form first prevents it from digesting the cell cytoplasm contents.
• Oxyntic cells, which produce hydrochloric acid. This converts pepsinogen to its active form as mentioned above, provides optimum pH for pepsin to work in, and kills most bacteria. The stomach epithelium is protected from ulceration by a thick layer of mucous produced by goblet cells.

Gastric juice therefore contains pepsin and pepsinogen, as well as HCl, and chemical digestion begins. Both pepsin and HCl catalyse hydrolysis. Pepsin is an endopeptidase: it is an enzyme which catalyses the hydrolysis of proteins to polypeptides. It does this by hydrolysing the inside of the protein molecule, thus increasing the number of ends for the exopeptidases to work on. The pyloric sphincter opens when the chyme (the mixture of food and gastric juice) is of the correct consistency, and releases the food into the small intestine. Lipid stimulates the production of entergasterone by the stomach mucosa, which has an antagonistic effect on gastrin. Therefore, the more fat the food contains, the longer it remains in the stomach. The first section of this is the duodenum, which runs nearly horizontally across your body below your stomach. When chyme enters the duodenum, its lining releases a number of hormones. One of these is secretin, which stimulates the production of the sodium hydrogencarbonate portion of the pancreatic juice. Cholecystokinin-pancreozymin (CCK-PZ) stimulates the gall bladder to contract, and the release of digestive enzymes from the pancreas. At the beginning of this, ducts running from the gall bladder (bile duct) and the pancreas (pancreatic duct) form the common duct and secrete bile and pancreatic juice into the duodenum. Pancreatic juice

• Is rich in sodium hydrogencarbonate, which neutralises the acidic chyme from the stomach, raising the pH to about 7.
• Is also rich in enzymes. They are summarised in the table below. The bits in bold must be learnt.

Bile

• Is stored in the gall bladder
• Contains no active enzymes
• Contains bile salts which emulsify fats, increasing the surface area for the action of lipase  

The duodenum contains deep folds between its villi, which have Brunner’s glands below them. The latter secrete a viscous fluid containing water, hydrogencarbonate ions and mucoprotein, and serves to protect the wall of the duodenum from the pepsin and chyme. Both extracellular digestion and intracellular digestion occur in the small intestine. The cells of the epithelium contain enzymes in their cytoplasm and embedded within their cell surface membrane. Digestion therefore occurs outside, on the way into, and inside the cytoplasm of, these cells. Resulting from the digestion in the lumen of the small intestine, carbohydrates have been hydrolysed to disaccharides and proteins to dipeptides and tripeptides. Their further digestion takes place in the cell surface membrane of the microvilli of the intestinal mucosa. Disaccharidases break disaccharides down into their constituent monosaccharides. Most of these are released back into the lumen of the gut. A similar process occurs with dipeptides and tripeptides, which are taken up by the microvilli and broken down into their respective amino acids. Some also diffuse into the cytoplasm of the cells of the villi, where they are digested.