A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module

Abstract

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.Keywords: Feruloyl esterase; cinnamoyl esterase; carbohydrate binding module; Penicillium funiculosum; ferulic acid.Plant cell walls are comprised of a heterogeneous mixture of polymers including cellulose and hemicellulose (e.g. arabinoxylans, pectins, galactomannans, b-glucans) and their biodegradation is a key component in the global cycling of photosynthetically fixed carbon. Hydroxycinnamates are increasingly recognized as important components in determining the physical and chemical properties of plant cell walls. Ferulates and p-coumarates are linked to cell wall polysaccharides through ester bonds (135 to arabinofuranose residues in heteroxylans of Graminaceous monocots such as wheat, barley, maize; 132 to arabinofuranose residues or 136 to galactopyranose residues in the pectins of Caryophylaceous dicots such as sugar beet and spinach). There is evidence to suggest that both the monomeric forms (e.g. ferulate and p-coumarate) and the various dehydrodimers (e.g. 5±5 diferulate) may act as nucleation sites during lignification in the latter stages of wall development [1], and it has been established that ferulates and p-coumarates are linked to lignin through ester and/or ether linkages [1±4]. There is also speculation that hydroxycinnamates serve to link cell wall proteins with other structural polymers. The presence of ferulates and dehydrod...

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

“…The N terminal amino acid sequence of FAE 1 was determined to be AFQSKXVEFGAQIDIPNVKVNF, where X indicates an undetermined residue. The sequence was compared with those of A. niger FAEA, 24) A. niger FAEB, 25) P. funiculosum FAEB, 22) Talaromyces stipitatus FAEC 26) and Pseudomonas fluorescens XYLD. 27) FAE 1 showed relatively high homologies to A. niger FAEB (50%) and T. stipitatus FAEC (41%) (Fig.…”

“…Penicillium funiculosum FAEB releases 98% of esterified ferulic acid from wheat bran in the presence of xylanase and releases 35% of esterified ferulic acid from sugar beet pulp in the presence of a mixture of endo arabinanase and L arabinofuranosidase. 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 FAE 1, produced by P. chrysogenum 31B.…”

“…Penicillium funiculosum FAEB releases 98% of esterified ferulic acid from wheat bran in the presence of xylanase and releases 35% of esterified ferulic acid from sugar beet pulp in the presence of a mixture of endo arabinanase and L arabinofuranosidase. 22) To the best of our knowledge, that releases more ferulic acid from sugar beet pulp than any other enzyme.…”

The Neisseria meningitidis Serogroup A Capsular Polysaccharide O-3 and O-4 Acetyltransferase

“…These proteins share with MynC a semiconserved motif (GSSKGG) in the N-terminal region. Typically, serine esterases contain an N-terminal conserved GSSSG motif (assumed to be the catalytic motif), where the center serine residue (underlined) is the active-site nucleophile (55). However, purified MynC did not show acetylesterase activity when tested with the synthetic substrate p-nitrophenyl acetate used in other in vitro esterase assays (data not shown) (56).…”

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

“…The first fungal feruloyl esterase with a carbohydrate-binding module (CBM) from Penicillium funiculosum was reported by Kroon et al 12) At first, this enzyme was exhibited the presence of types A and B FAEs; these two proteins are nearly identical by amino acid homology. Types A and B FAEs should show similar functional behavior since both catalytic domains are highly conserved.…”