Alginate-modifying Enzymes in Australian Marine Algae

Twelve Australian marine algae from Sydney coastal waters, Ecklonia radiata, Hormosira banksii, a species of Sargassum, a species of Cystophora, Padinafraseri, Dictoyota dichotoma, Endarachne binghamiae, Splanchnidium rugosum, Colpomenia sinuosa and C. peregrina, Acinetospora crinata and a species of Zonaria were tested for alginate lyase and alginate epimerase activity. Easily soluble protein (ca 140 mg/kg wet algae) was prepared from acetone powders (3-15% of wet algae) at low temperatures (ca 0 °C) in tris(hydroxymethyl)aminomethane HC1 buffer (pH 7.8) containing 4% (w/v) polyvinylpyrolidone, 1.5 ΙΉΜ dithiothreitol and 200 mM NaCl. A species of Zonaria, Ecklonia radiata and Acinetospora crinata had highest lyase activity while the Zonaria species, the two Colpomenia species and A. crinata had highest epimerase activity. There were significant variations in enzyme activity between species, seasons and tissues. A 2 —8 fold purification was found for the epimerase from a single ammonium sulphate precipitation but the lyase gave only a l — 3 fold improvement. Partial acid hydrolysis demonstrated that epimerised low mannuronic acid alginate gave ten to twenty per cent conversion to guluronic acid and the mannuronic/guluronic acid mixed segments showed the greatest degree of epimerisation. Introduction Alginic acid, the major polysaccharide of the Phaeophyta, has manifold industrial applications where there is a requirement for fine control of water function in processes and products. One important commercial aspect of the chemical composition of alginate is the relative abundance of the monomers, mannuronic and guluronic acid. High guluronic acid alginate has particular use industrially where high gel strength is required. It has been demonstrated that mannuronic acid, in the intact polymer, can be enzymically epimerised to guluronic acid (Haug and Larsen 1969, 1971). Most studies of this unusual biological catalysis have been conducted with enzymes prepared from the bacterial source Azotobacter vinelandii (Larsen 1981, Skjak-Braek and Larsen 1985). However problems exist in using microbial fermentation to produce alginate or its enzymes on a large scale. The inherently high viscosities encountered in reactor growth limit oxygen transport and lead to poor productivity. The enzymes of alginate modification have also been located and partially purified in the brown marine algae (Madgwick et al. 1973 a, b, 1978, Shiraiwa et al. 1975, Ishikawa and Nisizawa 1981). The biotechnical objective of the current research programm, is, therefore, to derive these enzymes directly from the alginate forming algae. Apart from the technical value of algal epimerase, the algal lytic enzymes have uses s reagents of fundamental research into the structural complexities of the alginate molecule (Boyd and Turvey 1978). In the present work some twelve previously unexamined species of Australian brown seaweeds have been surveyed to identify convenient sources of polymannuronate-5-epimerase and alginate lyase. Botanica Marina / Vol. XXIX / 1986 / Fase. 4 Copyright © 1986 Walter de Gruyter · Berlin · New York 330 Greene and Madgwick: Alginate-modifying enzymes in Australian marine algae Materials and Methods Algol material Algae were collected from the eastern and south-facing shores of NSW near Sydney in 1984. The harvested plants were cleaned of epiphytes and frozen immediately in dry ice. Preliminary taxonomic identiflcation was made by Dr R. J. King, School of Botany, University of New South Wales, by reference to Christianson et al. (1981) and Borowitzka, King and Larkum (1982). Harvest details are given in Table I. Acetone powder preparation This procedure has been described in detail in Madgwick et al. (1973 a). The algal tissue was mechanically disintegrated after freezing in a dry-ice (40 — 50 wet grams algae/200 —300 ml acetone) acetone bath. The resulting fine particulate brei was then allowed to thaw to approximately —10 °C whereupon the powder was dehydrated by several replacements of cold acetone. The acetone was drawn off by rapid filtration or by centrifugation at —10 °C. Ethyl ether was then drawn through the powder to complete the drying process leaving a buff coloured to off-white, fluffy residue. This acetone powder will remain äs a stable source of enzymes for 1—2 days if stored over blue silica gel at 0 °C, but it is preTerable to treat it with the extraction solvent immediately. Extraction of enzymes This technique was essentially the same äs described in Madgwick et al. (1978) except that dithiothreitöl (DTT) (Cleland's reagent) and polyvinylpyrolidone (PVP) were increased 1.5 and 2 respectively. The acetone powder was triturated with a solution containing 4% PVP, 1.5 mM DTT, 200 mM NaCl in 0.1 M tris(hydroxymethyl)aminomethane HC1 buffer at pH 7.8 at 0 °C for two hours (l gram acetone powder/20 ml extraction solvent). This solution, removed by filtration or centrifugation from the powder, contained active enzymes and was used äs soon äs possible in assay procedures. Table L Harvest details. Algae Ecklonia radiata (C. Agardh) J. Agardh Hormosira banksii (Turner) Decaione Sargassum sp. Cystophora sp. Padina fraseri (Greville)Greville Dictyota dichotoma (Hudson) Lamouroux Endarachne binghamiae J. Agardh Splanchnidium rugoswn (Linnaeus) Greville Colpomenia sinuosa (Roth) Derbes et Solier Acinetospora crinata (Carmichael ex Harvey in J. D. Hooker) Kornmann Colpomenia peregrina (Sauvageau) Hamel Zonaria sp. Month of harvest