Isolation and Properties of Napier Grass B-Amylase

Abstract

Studies of the forage plant, Napier grass, Pennisetum purpureum, have been started in an effort to clarify its exceptional water-utilizing capability. Hydrolytic enzymes are given primary attention. The present paper concerns a ß-amylase from immature internode tissues. A much-studied amylase from sugarcane leaves served as a reference point for comparison of properties. A standard assay for the amylase was established with soluble potato starch acting as substrate. The assay is suitable for large numbers of crude or semi-purified Napier grass extracts. Amylase was heavily concentrated in meristematic and immature internodal tissues. Moderate amounts were present in leaf blades. Only traces were found in leaf sheaths and mature stem tissues. It was estimated that two to three times more amylase potential is present in Napier grass than in sugarcane. Over 90 percent of the amylase was precipitated from clarified water extracts between 40- and 60-percent saturation with ammonium sulfate. Optimum pH was 5.5, and temperature 46° C. The enzyme was heat stable up to 52° C. Napier grass amylase remained stable for at least 2 weeks under refrigeration and for 90 hours at room temperature. Velocity relationships with time, protein concentration and substrate level were studied.  K_m for Napier grass amylase was virtually identical to that of sugarcane amylase. Gel filtration experiments yielded amylase in a homogenous mass partially resolved from noncatalytic protein. Contrary to sugarcane, no evidence of a secondary, α-amylase was present. Prolonged dialysis did not inhibit. Cyanide, lead, iodide, and meta-silicate did cause inhibition. Activation was not achieved with manganese or traces of sugar additives. No evidence was found indicating that the Napier grass amylase was a protein-carbohydrate complex, as is the case with sugarcane amylase. Paper chromatography revealed that maltose was the lone primary product. After 4 hours the maltose was in turn hydrolyzed to free glucose by a maltase believed to be latent within the amylase preparation. The products maltose and glucose were obtained with potato starch, corn starch, amylose and amylopectin. The preparation was inactive against inulin. Paper electrophoresis revealed four distinct amylase peaks. Three possessed a net negative charge and one a positive charge. Four peaks are also obtained with sugarcane amylase, but their net charges are opposite those of the Napier grass enzyme. This is taken as further evidence that, unlike sugarcane amylase, the Napier grass enzyme is not a protein-carbohydrate complex. The biochemical significance of Napier grass amylase is discussed at length.