Study of an Enzyme Activity in Extracts of Ginkgo biloba Leaves

Abstract

Biocatalyst is a strong tool for preparation of enantiopure compounds through kinetic resolution or direct synthesis. However, enzymes do not always satisfy the needs of researchers for this purpose because they often do not have enough activity or selectivity toward unnatural substrates. To solve the problems, it is important to find new enzymes from nature, although researchers have tried to change enzyme activity or selectivity by mutagenesis such as random mutagenesis and rational design. Researchers have isolated a variety of enzymes from various sources such as bacteria, fungi, mammals, and plants. Diabetic enzymes such as lipases and proteases from bacteria and mammals are well characterized and utilized successfully in academic and industrial areas. The enzymes involved in mechanisms for protecting plants from insect or fungal attack are also useful in organic synthesis. Such enzymes generally catalyze reactions to produce toxic chemicals in the last step of secondary metabolism to kill predators. As an example, hydroxynitrilases from cyanogenic plants catalyze the biodegradation of cyanogenic glycosides to release hydrogen cyanide. The reverse reaction is interesting in organic synthesis because hydroxynitrilases catalyze formation of enantiopure cyanohydrins from aldehydes or ketones and hydrogen cyanide. Searching enzymes involved in the secondary metabolism of plants may provide useful biocatalysts in organic synthesis because the enzymes have a key role for synthesis of many biologically active compounds. Ginkgo biloba leaves have high anti-insect activity and biological activity. Thus, they might contain an interesting enzyme involved in the secondary metabolism and the enzyme can be useful for organic synthesis. We have chosen Ginkgo biloba leaves for finding a new enzyme because Ginkgo biloba leaves potentially contain enzymes involved in the secondary metabolism and also the study of the enzymatic activity of extracts from Ginkgo leaves has not been focused while the chemicals in them have been well characterized. Herein, we represent an enzyme activity in Ginkgo biloba leaves. Most enzymes are classified into six groups, such as oxidoreductase, transferases, hydrolases, lyases, isomerases, and ligases. Activities for oxidoreductase, hydrolase, and lyase were chosen for screening enzyme activities in Ginkgo biloba leaves because these enzymes are important in organic synthesis. We assayed the activities of peroxidase for oxidoreductase, β-amylase, nitrilase, and esterase for hydrolase, and hydroxynitrilase for lyase. Ginkgo biloba leaves were ground by a homogenizer and washed with ethyl acetate to remove organic components. Ginkgo powders were extracted with the reaction buffers for activity screening. Under the assay condition tested, Ginkgo extracts showed only the peroxidase activity (Table 1). In the assay condition, peroxidases oxidize pyrogallol to purpurogallin using hydrogenperoxide (eq. 1). The absorbance change can be monitored at 420 nm. The reaction coordinate showed clear difference between the reaction by Ginkgo extracts and the blank reaction (Figure 1).