Ferulic acid esterase activity was detected in extracts of barley malt using an assay employing a novel artificial substrate, mono-feruloyl glycerol. Mono-feruloyl glycerol has been synthesized and analysed to determine its degree of substitution and purity. It consists of a mixture of the two isomers 1-feruloyl glycerol and 2-feruloyl glycerol. The extraction offerulic acid esterase and its assay conditions have been optimised. The presence of both a detergent and reduced glutathione in the extraction medium increased the amount of enzyme extracted. ApH of 7.5 was optimal for enzyme activity. The enzyme in solution was only stable up to 30°C. The crude extract containing the enzyme releasedfree ferulic acidfrom both soluble and insoluble cell wall materials. After extraction of the soluble enzyme, insoluble enzyme, capable of releasing free ferulic acid from feruloyl glycerol, was detected in the residual grain solids.

The extraction and the assay conditions for acetic acid esterase from barley malt have been optimised. An assay method was developed using diacetin as a substrate. The presence of reduced glutathione and the detergent Triton‐X‐100 in the extraction medium improved the yield of enzyme. The optimal pH for extraction was 8.0. When malt extracts were held at various temperatures acetic acid esterase was denatured at temperatures above 30°C. The optimum pH when measuring activity was 7.0. The crude enzyme extract released acetic acid from both soluble and insoluble cell wall materials. An insoluble, active form of the enzyme remained in the residual grain solids after the soluble form had been extracted.

A partial purification of ferulic acid esterase, which degrades feruloyl glycerol, has been achieved from barley malt in small yields. Coloured and viscous materials were removed from the malt extract using batch-elution anion exchange chromatography. Further steps included gradient elution anion exchange chromatography and gel filtration chromatography. Estimations of the molecular weight varied greatly from 22KDa to 158 KDa, possibly because the protein interacted with the matrix of the gel exclusion chromatography column and because multiple forms of the enzyme were present. The partially purified feruloyl esterase had an apparent Km of 0.46% feruloyl glycerol. However more than one enzyme may be present and the substrate contains two isomers and so Michelis-Menten kinetics may not be appropriate.

Partial purifications (84, 114 and 390 fold) of a soluble acetic acid esterase from barley malt that degrades diacetin have been achieved. Enzyme recoveries were 20%, 20% and 25% in three consecutive runs. Initial problems of high viscosity and colour in the extract were overcome with the use of batch-elution anion exchange chromatography. This was followed by gradient elution anion exchange chromatography and gel filtration chromatography, which gave the greatest purification, but which gave erratic estimates of the molecular weight of the enzyme (40, 48 and 84 kDa). The apparent Km value of the acetyl esterase was tentatively estimated to be 25mM diacetin.