Enzyme activity assay

In this simulator or virtual laboratory we can explore how the activity of an enzyme varies depending on the conditions of its environment. The enzyme we will study is 6-O-α-L-ramnosyl-D-glucosidase, EC 3.2.1.168, from Actinoplanes missouriensis, included in the sample for the assay.

The objective is to find optimal pH and temperature conditions for this enzyme.

1. Slide the controls to pick a pH and a temperature for the assay.
2. Press, in order, the buttons to add components of the reaction mixture into the cuvette.
   The “add substrate” button will first empty the cuvette. The “add sample” button will start the incubation.
3. Once the colour has developed, press the button in the spectrophotometer to measure absorbance, and write down its value.
4. Systematically change either pH or temperature and write down each absorbance value in your lab notebook, or in the table on the right.

pH 7 T 50°C

buffer and substrate add sample add incubate for 1h ✅ DNS add and 10 min at 100°C ✅

540nm

A =

Lab notebook:
(write down the measurements)

add another row

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Suggestion: prepare two graphs that will show variation of absorbance (which reflects the enzyme activity) depending, respectively, on temperature and pH. Obtain from them an estimate of the optimal temperature and the optimal pH for this enzyme.
Data in the table on the right may be selected, copied and pasted to a spreadsheet, or exported to a text file (comma-delimited or tab-delimited) so they can later be imported to a spreadsheet or graph program.

Fundamentals of the assay (details of the reaction)

Assay for enzyme activity of ramnosyl glucosidase

The enzyme 6-O-α-L-ramnosyl-D-glucosidase (EC 3.2.1.168) hydrolises the glycosidic bond in a substrate that we add, hesperidin, a natural glycoside formed by the combination of rutinose and hesperetin. Rutinose that gets released is the disaccharide 6-O-α-L-ramnosyl-D-glucose and has reducing character (on the C1 of glucose).

hesperidin		rutinose		hesperetin
	EC 3.2.1.168		+

Then an assay is applied that detects reducing sugars (in this case, rutinose); this consists on its reaction with dinitrosalicylate (DNS). DNS, of a yellow colour in alkaline medium, is reduced to brick-red coloured 3-amino-5-nitrosalicylate:

(the aldehyde of the reducing sugar gets oxidised to an acid) The red colour of the product is measured at 540 nm.

Based on experimental data from Bárbara Neher; doi:10.​1007/​s00253-015-7088-x ; PMID:26549237

Version 1.3, September 2022 Disponible también en español