Cloning of a gene encoding cinnamoyl ester hydrolase from the ruminai bacterium Butyrivibrio fibrisolvens E14 by a novel method

Abstract

A gene (cinI) encoding a cinnamoyl ester hydrolase (CEH) has been isolated from the ruminal bacterium, Butyrivibrio fibrisolvens E14, using a model substrate, MUTMAC [4-methylumbelliferoyl (p-trimethylammonium cinnamate chloride)]. CinI has significant amino-acid similarities with members of a large and diverse family of hydrolases with a serine/aspartic acid/histidine catalytic triad. Our analyses identified two previously unclassified amino acid sequences, the amino-terminal domain of unknown function in XynZ from Clostridium thermocellum and XynC, an acetylxylan esterase from Caldicellulosiruptor saccharolyticus, as members of the same family of hydrolases. A previously described esterase with CEH activity, XylD from Pseudomonas fluorescens ssp. cellulosa, is not similar to CinI. CinI was expressed at high levels in the periplasmic fraction of E. coli TOPP2 and released ferulic acid from Fara [5-O-(trans-feruloyl)-arabinofuranose] prepared from wheat bran.

Expression of a Butyrivibrio fibrisolvens E14 gene (cinB) encoding an enzyme with cinnamoyl ester hydrolase activity is negatively regulated by the product of an adjacent gene (cinR)

“…were prepared as described previously (Dalrymple et al, 1996). MUTMAC, lactose, cellobiose, methylated pectin, a-D-galacturonic acid, p-coumaric acid and L-arabinose were obtained from Sigma.…”

“…Prior to ligation, DNA samples were purified using the Magic DNA preparation kit (Promega). Colonies containing inserts were screened with MUTMAC overlays as described previously (Dalrymple et al, 1996). The nucleotide sequences of subcloned restriction fragments were determined by the dideoxynucleotide chaintermination procedure using the Sequenase sequencing kit (Amersham).…”

“…Uninduced and induced cultures were harvested by centrifugation after a further 16 h incubation at 37 "C. Cells were resuspended in PBS (Sambrook et al, 1989) and lysed by sonication, or fractionated as described by Hsiung et al (1986). Culture supernatants, sucrose fractions, periplasm fractions and sonicated cells were assayed for enzyme activity by spotting samples at the appropriate dilution and overlaying with MUTMAC-containing agarose as previously described (Dalrymple et al, 1996). Proteins were separated by SDS-PAGE by the method of Laemmli (1970).…”

“…High-titre stocks of bacteriophage genomic clones were diluted to obtain either plate lysates or well-separated plaques on lawns of E. coli KW251 cells containing the appropriate plasmid by using standard methods described by Sambrook et al (1989), except that the top agarose contained 10% clarified and autoclaved rumen fluid (see Dalrymple et al, 1996). T o recover proteins from the plate lysates, 3 ml of 100 mM NaC1,17 mM MgCl,, 50 mM Tris/HCl, p H 7.5, was poured onto the surface and the plates rocked at room temperature for 2-3 h. CEH activity was measured as described previously (Dalrymple et al, 1996). On plates with separated plaques, CinB activity was visualized using MUTMAC as described previously (Dalrymple et al, 1996).…”

“…described (Dalrymple et al, 1996). Although predicted to be an intracellular enzyme, CinI (here renamed CinA) exhibited significant cleavage of Fara [5-0-(transferuloyl) -arabinofuranose] derived from wheat bran, demonstrating the utility of MUTMAC for screening for enzymes with activity against plant-derived cinnamoyl esters.…”