Galactomanana de Caesalpinea pulcherrima: biossíntese, estrutura e aplicação em matrizes cromatográficas

Abstract

The behavior of the endospermic gum from Caesalpinia pulcherrima seed and its chemical composition was investigated during the maturation process. The flowers appeared 150 days after germination. The pods were collected in 2 days intervals until complete maturation and separated in three parts: seed coat, endosperm and cotyledons. Caesalpinia pulcherrima seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weigh and chemical constituents were low. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage lipids, protein and carbohydrate were accumulated in large quantities with carbohydrate accounting for approximately 96% of endosperm dry matter. The other observations was that starch is transiently accumulated in the seed coat of developing seed (25th -35th daf). During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed and storage compound synthesis ended. Galactomannans are located in the endospermic tissues, where they are deposited in sites of considerable enlargement of cell walls and sometimes fully replace the live protoplast of the cell. Endosperm can account for up to 40-46% of the seed weight, and this percentage is directly correlated to the contents of galactomannan (81% of the seed dry weight). The C. pulcherrima endospermic gum was purified and the galactomannan isolated were analyzed by gel permeation chromatography. In all samples it was observed a single relatively broad peak, which would result from a polydisperse population of molecules with a high molecular size in solution. The abrupt increase of molecular weight (MW) was observed at 29th to 35th daf (2.8 x 104 — 1.8 x 105 g/mol). This can be justified due the greater galactomannan synthesis that occurs at the early of seed maturation and between 39th to 49th daf a increase in galactomannan molecular weight was discreet. During the seed maturation the [i] and KH increase while the degree of polydispersion (MW/Mn) decreases, indicating that in the early stages (25th daf) many polysaccharides are formed with different molecular masses and later the polysaccharides present a homogeneous and a high molecular mass and, consequently, a high degree of aggregation. The galactomannan at the earliest stages of deposition had a Man/Gal ratio of about 3. During late deposition this ratio increases, rapidly, stabilizing at about 3.7, the characteristic ratio of the mature seed. The levels of a-galactosidase in the developing endosperms was low during early galactomannan deposition, but increased subsequently, peaking during late galactomannan deposition (49th daf).The developmental patterns of the a-galactosidase activity and of the increase in Man/Gal ratio of the C. pulcherrima galactomannan were closely similar, indicating a cause-and effect relationship. Probably, the final Man/Gal ratio in the mature seed is, to an appreciable extent, the result of galactose removal from the primary biosynthetic product by an a-galactosidase activity which is present in the endosperm during late galactomannan deposition. Affinity chromatography were realized in different C. pulcherrima gum columns, treated with epichlorydrin for the formation of cross linkages between the polysaccharides chains. Then, they were tested for the isolation of galactose specific lectins and to investigate possible modifications on fine structure that occurs during galactomannan deposition Affinity columns obtained at 25th, 29th and 35th daf showed a higher capacity of lectin-binding when compared with the columns of 39th, 45th and 49th daf. This may be due the increase in Man/Gal ratio observed in this period that possibly affect the galactomannan fine structure and thus the interaction lectin-carbohydrate was altered too.