A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain

Abstract

The 5' regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5' region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5' 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5' 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.

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

“…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. 4).…”

X4 Modules Represent a New Family of Carbohydrate-binding Modules That Display Novel Properties

“…All three enzymes contain an identical N-terminal region that comprises a typical family 2a CBM joined via a serine-rich linker to the X4 module (19,20). As the X4 modules in these Cellvibrio enzymes are shown to bind polysaccharides (see below), henceforth these sequences will be designated as a new family of CBMs (family 35, CBM35).…”

“…This is exemplified by three plant cell wall-degrading enzymes from Cellvibrio japonicus, Xyn10B (xylanase), Abf62A (arabinofuranosidase), and Est1A (acetyl xylan esterase) that contain an identical ϳ150-amino acid module, termed X4, whose role in enzyme function is unclear (19,20). This X4 module is also present in a mannanase (Man5C) from the same organism (21).…”

Purification and characterization of a ferulic acid esterase (FAE-III) from Aspergillus niger: specificity for the phenolic moiety and binding to microcrystalline cellulose

“…This enzyme did not bind to xylan. The cellulose binding domain exhibited almost complete homology with the corresponding regions of a xylanase and an arabinofuranosidase, and is related to the cellulose binding domain of the cellulases from the same organism (Ferreira et al, 1991(Ferreira et al, , 1993. Fungal cellobiohydrolases also contain a cellulose binding domain (Tomme et al, 1988 ;Ong e t al., 1989) but there have been no reports of non-cellulase enzymes containing a cellulose binding domain in fungi, unlike the bacterial ferulic acid esterase of P. fltlorescens.…”

“…Only a few species have been reported to produce esterases which cleave the phenolic compounds from the sugar moiety, leaving the remainder of the polysaccharide open for further hydrolytic attack by other enzymes. Recently, these esterases have been purified and partially characterized from Streptomyces olivochromogenes (Faulds & Williamson, 1991, 1993a, Neocallimastix (Borneman e t al., 1991(Borneman e t al., , 1992, Pseudomonas jtlorescens (Ferreira et al, 1993), Penicillium pinophilum (Castanares e t al., 1992) and several from Aspergillm (Tenkanen et a/., 1991 ;Faulds & Williamson, 1993b). The specificities of the enzymes for methyl esters of cinnamic acids, acting as model substrates, show that the Pseudomonas esterase and two isoforms from Aspergillus niger (Faulds & Williamson, 1993b) are specific for compounds with different substitutions on the phenolic ring of the cinnamic acid.…”