The enzyme lactase hydrolyses the disaccharide lactose to glucose and galactose. This enzyme is produced in the small intestine of mammals and by some fungi and bacteria. Fig. 1.1 shows this reaction. [Figure 1.1] The Michaelis-Menten constant, Km, shows the affinity of an enzyme for its substrate. The lower the Km the greater the affinity. A student carried out an investigation to find the Km of the enzyme lactase at different pH values commonly found in organisms. The student: • made 1 dm³ of 0.15moldm⁻³ solution of lactose, molar mass 342.3 g mol⁻¹ • used proportional dilution to make a total of five lactose solutions from 0.15moldm⁻³ stock solution, each of 500cm³ • used 1 g per 100 cm³ lactase solution • used a glucose biosensor to measure the initial rate of reaction at each pH. A glucose biosensor measures glucose in mmoldm⁻³. Fig. 1.2 shows the experimental set-up. [Figure 1.2]

1. (a)(i)Describe how the student could make 1 dm³ of 0.15moldm⁻³ solution of lactose.[2]
2. (a)(ii)Describe how the student made a further four solutions of lactose by proportional dilution.[2]
3. (b)(i)Identify the independent and dependent variables in this investigation. independent dependent[2]
4. (b)(ii)Suggest a suitable control for this investigation.[1]
5. (b)(iii)Describe a method that the student could use to find the Km of lactase at different pH values. The solutions were made as described in (a) (i) and (a) (ii) and the apparatus shown in Fig. 1.2 was used. Your method should be set out in a logical way and be detailed enough to let another person follow it. You should not include details of how to make the lactose or lactase solutions.[6]
6. (c)(i)Complete Fig. 1.3 by sketching a graph to show the effect of substrate concentration at one pH on the initial rate of reaction. Indicate on your graph how the student could find the Km value of lactase at that pH.[4]
7. (c)(ii)Table 1.1 shows the results that the student obtained for Km at different pH values. [Table 1.1: pH (A, B, C, D, E), Km/arbitrary units (0.216, 0.112, 0.166, 0.178, 0.324)] State which of these pH values, A to E, is closest to the likely optimum pH of lactase. Explain your answer.[2]

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