Rennin is a proteolytic enzyme found principally in the tissue of the fourth stomach of young calves, but also in some other ruminant mammals. Extracts from the fourth stomach of calves (vells) contain both active Rennin and also a zymogen prorennin which is activated to Rennin by exposure to pH 3.6 at 4°C for 1 to 2 days (Methods of Enzymatic Analysis, Bergmeyer, H.U., ed. Vol 2, 69, 1955, Academic Press, New York). The crude extract from vells used in cheese and known as Rennet may be purified to a stage where it is relatively easy to obtain crystalline Rennin.

Crystalline Rennin is a heterogeneous protein and can be separated chromatographically into two components Rennin A and Rennin B. These two components can also be obtained by activation of the two separate zymogens, prorennin A and prorennin B (Foltmann, B.,C.R. Lab. Carlsberg. Ser. Chim., 32, 425, 1962).

Rennin is most stable between pH 5.5 and 6.0 and is relatively unstable above pH 6.5 (Foltmann, B, Acta Chem. Scand., 13, 1927, 1959). Its optimum activity on hemoglobin is at pH 3.7 and it is relatively inactive above pH 4.9. The amino acid constitution has been determined and this show many similarities to pepsin (Foltmann, B & B.S. Hartley, Biochem. J., 104, 1064,1967). The molecular weight of Rennin has been given as 40,000 and the isoelectric point is at pH 4.5 (Schwander, H., et al, Helv. Chim. Acta, 35, 553, 1952).

1. Substrate: 100 ml of cow's milk (or a solution of dried milk powder of equivalent consistency) and add 1 ml CaCl2 (1.0 M).
2. Standard sol'n.: Dilute Rennet 1:100 with distilled water.
3. Enzyme sol'n.: Dilute appropriately so that the clotting time is 0.75 to 1.5 times that of the standard Rennet

1. Set up water bath at 30°C.
2. Into 50 ml test tubes add 10 ml lots of substrate and incubate in water bath at 30°C for at least 45 minutes.
3. At zero time add 1 ml diluted standard Rennet into the tube of substrate. Mix gently in the water bath and allow a film of liquid to form on the side of the tube and record time (t1) when clotting commences.
4. Repeat step 3. Clotting times should agree within 0 to 5 seconds.
5. Repeat step 3 using enzyme solution. Record clotting time (t2).
6. Calculate activity as shown below.