Water → H-Asp-Arg-Val-Tyr-Val-His-Pro-Ala-OH

Participants

Reaction Steps

A 25 μl aliquot of the enzyme suspension was added to the above reaction-broth mixture and incubated at 37° C. for 30 minutes. The enzyme reaction was quenched by placing the mixture in a boiling water bath for 10 minutes. A blank was prepared by adding the enzyme aliquot to the reaction mixture after the boiling water treatment. A positive control consisted of a 10 μl mixture of methanol and water (1:1). A 1.0 ml portion of 0.2M K2HPO4 --KH2PO4 buffer (pH=8.3) was added to each boiled mixture and then 0.5 ml of 3% 2,4,6-trichloro-S-triazine in dioxane (3% TT) was added and the mixture vortexed. The solutions were centrifuged and the supernatant from each solution was measured for optical density (O.D.) at 382 nm. The percentage of inhibition of the angiotensin I-converting enzyme was defined by the equation: ##EQU1## Broth samples from medium 3 (see Table I) showing at least 90% inhibition were deemed positive. For the other media listed, a minimum inhibition percentage of 80% indicated a positive result. A positive assay result indicated that the cultured broth was producing the desired compound, 176.

Provenance & Evidence