Preparation and properties of immobilised a-amylase

Practical: week 1

Enzymes and Enzyme Technology
Preparation and properties of immobilised a-amylase

Introduction to the practical exercise

The two-session practical exercise is designed to give some insight into the preparation and properties of immobilised enzymes. Note that the preparation of the immobilised enzymes should have been accomplished during the ‘Biosensor’ practical exercise. Also note that the write-up for this experiment should contain twice the words in each section outlined earlier and the marks awarded will total to a maximum of 300.

It consists of two parts:
1. Preparation and properties of covalently-immobilised a-amylase.
2. Preparation and properties of non-covalently-immobilised a-amylase.

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 Assays) 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).

Amylopectin (alpha-linked 1,4-D-glucose with alpha-linked 1,6 branchpoints) is hydrolysed randomly by bacillus alpha-amylase to give limit dextrin. It does not hydrolyse alpha 1,6 links or their neighbouring alpha 1,4 links

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.

During the ‘Biosensors’ practical exercise you should
• Prepare covalently-immobilised a-amylase
• Prepare non-covalently-immobilised a-amylase

During week 1 you should
• Wash the covalently-immobilised a-amylase
• Wash the 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
• Assay samples from the preparations; see ‘Assays’ later

Covalent Immobilisation of a-amylase (Methods Enzymol. 44, pp. 98-99)
Safety note: The following makes use of nitrous acid. This gives off toxic brown fumes of nitrogen oxides, if warmed. It is important from both a safety and experimental point of view that it is kept ICE-COLD.

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 forward arrow 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)

aromatic amine + HNO2 --> diazonium salt --> couples to enzyme tyrosine groups

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
Safety note: The following makes use of a fine powder. Treat it with care and do not allow this to form a dust cloud Clean all spillages with slightly damp tissue.

Resin structure

Complex poly phenol with some quinone groups

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').