Efficient Extraction of Ferulic Acid from Sugar Beet Pulp Using the Culture Supernatant of Penicillium chrysogenum

Abstract

Ferulic acids have been found in the cell walls of many plant products including maize bran, 1) sugarcane baga-sse, 2) wheat bran, 3) sugar beet pulp, 4,5) and spinach. 6) In the three former tissues, ferulic acid is esterified to the C5 of arabinose in arabinoxylan, a 1,4Dxylan to which L arabinofuranosyl residues are attached at position 2 or 3. In contrast, ferulic acids mainly attach to the C2 position of 1,5linked arabinofuranose residues or the C6 of 1,4linked galactopyranose residues in the hairy region of pectins of sugar beet and spinach. 7,8) Recently, Levigne et al. demonstrated that ferulic acid is also linked to the C5 position of arabinofuranose residues in sugar beet pectin by analysis of the structures of feruloylated oligosaccha-rides obtained by treating sugar beets with a commercial enzyme preparation (Driselase). 9) Furthermore, some feru-lic acids exist as dehydrodimers (e.g. 55dehydrodiferulic acid) in the cell walls of several plant species, which are formed through an oxidation reaction catalyzed by peroxi-dases. 10,11) Possible roles of ferulic acid in plant cell walls are to decrease the digestibility of the cell wall by microorganisms 12) and to regulate cell growth 13) by crosslinking cell wall polysaccharides. Ferulic acid esterases (FAEs) are enzymes that catalyze the hydrolysis of ester linkages between ferulic acids and sugars or alcohols. The number of studies concerning microbial FAEs has been increasing in the last ten years and many FAEs have been isolated and characterized. In the utilization of agroindustrial wastes such as sugar beet pulp, effective degradation of plant cell walls requires FAEs for the release of ferulic acid as well as polysaccha-ridases such as xylanases 14) and arabinanases. 15) Moreover, ferulic acid has several potential applications: it may be useful as an antioxidizing agent, 16,17) an antiinflammatory drug, 18,19) and a food preservative that inhibits microbial growth. 20,21) Enzymatic extraction of ferulic acid from industrial wastes has been studied. Penicillium funiculosum FAEB releases 98% of esterified ferulic acid from wheat bran in the presence of xylanase and releases 35% of es-terified ferulic acid from sugar beet pulp in the presence of a mixture of endoarabinanase and Larabinofurano-sidase. 22) To the best of our knowledge, that releases more ferulic acid from sugar beet pulp than any other enzyme. In this paper we describe the screening of microorganisms that can achieve high yield extraction of free ferulic acid from sugar beet pulp. In addition, we describe the isolation and some of the characteristics of a novel FAE, termed FAE1, produced by P. chrysogenum 31B. MATERIALS AND METHODS Chemicals and reagents. ResourceQ 6 mL, ResourceS 6 mL, MonoQ HR 55, MonoS HR 55 and HiLoad 1660 Superdex 75 columns were purchased from Amer-sham Biosciences. All other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka) unless otherwise stated, and were of certified reagent grade. Organism and cultivation conditions. The microorganisms were obtained from the Institute for Fermentation, Osaka (IFO) or isolated from rotten sugar beet, and main-J. Appl. Glycosci., 52, 115120 (2005) ! C Abstract: We found a microorganism, Penicillium chrysogenum 31B, that has high ability to release ferulic acid from sugar beet pulp. Approximately 85% of alkaline-extractable ferulic acid in sugar beet pulp could be released using the culture supernatant of P. chrysogenum 31B. However, the culture supernatant did not efficiently extract ferulic acid from wheat bran, peel of corn seed, or sugar-cane bagasse. A ferulic acid esterase (FAE-1) was purified from the culture filtrate of P. chrysogenum 31B. The molecular mass of the enzyme was determined to be 62 kDa by SDSPAGE. Optimum conditions for enzyme activity were 50 C and pH 67. The enzyme showed activity towards methyl esters of hydroxycinnamic acids including ferulic acid, p-coumaric acid, and caffeic acid, but was not active on methyl sinapinate or 3,4-dimethoxy cinnamate. The lack of activity of FAE-1 toward these substrates appears to be due to the presence of two methoxy groups on the benzene ring. The substrate specificity of FAE-1 seemed to be similar to that of ferulic acid esterase (CinnAE) of As-pergillus niger. However, there was a difference between FAE-1 and CinnAE in respect to activity towards methyl vanillate. It is remarkable that FAE-1 hydrolyzed methyl vanillate, which, to our knowledge, is the first report of a ferulic acid esterase hydrolyzing a hydroxybenzoic acid methyl ester.