Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate

Abstract

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS FA,Ac ) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOS FA,Ac , a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose.Hemicelluloses comprise a heterogeneous, highly branched mixture of complex polysaccharides. In plant cell walls, these polysaccharides form intimate associations with cellulose, pectin, and lignin and markedly reduce the degradability of plant fiber (20). In a microbial ecosystem such as the rumen, fiberdegrading microorganisms meet this challenge by operating as a consortium. Several hemicellulolytic rumen species, especially the ones belonging to Prevotella and Butyrivibrio, have been shown to synergistically enhance the rate and extent of plant cell wall hydrolysis during co-or sequential culturing with cellulolytic bacteria (12,18,31,32).The hemicelluloses present in the cell walls of grasses, cereals, and hardwoods largely comprise xylan, a polymer consisting of linear chains of D-xylopyranosyl residues linked through ␤-(134) glycosidic linkages. For grasses and cereals, the xylan backbone is heavily substitu...

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

“…Another example, Clostridium thermocellum XynY [54], contains xylanase and feruloyl esterase domains (clustering with Type D FAEs) [45]. However, based on preliminary modelling analyses, the multifunctional capability of EstG34 appears to be catalysed by a single catalytic domain, with no obvious carbohydrate binding domains (data not shown).…”

“…Ferulated polysaccharides are essential in directing cell wall cross-linkages and serve as a defensive mechanism against invasion by plant pathogens [54].…”

Identification and Crystallographic Analysis of a New Carbohydrate Acetylesterase (SmAcE1) from Sinorhizobium meliloti

“…Among these proteins, carbohydrate esterases (CEs) play key roles in making other CAZymes access their carbohydrate substrates by removing acetyl groups from substituted saccharides with various chain lengths [5,6]. In the physiological aspect, CEs are considered to be involved in the formation of cell walls, interactions between host and microbes, and energy metabolism pathways [7][8][9][10][11][12][13][14][15]. CEs are also important for biotechnological application, as modifications of carbohydrates are necessary for the preparation of biofuel, drug carriers, and cosmetics [16][17][18].…”

Dinâmica do microbioma ruminal de ovinos (<i>Ovis aries</i>) e sua relação com a degradação de biomassa

“…Asteriscos indicam diferenças significativas entre a dieta controle e bagaço dentro de cada coleta (*P<5% Prevotella é fortemente associado a dietas ricas em carboidratos . Alguns membros deste gênero possuem enzimas hemicelulolíticas e proteolíticas (MATSUI et al, 2000), além disso, podem degradar xilana, pectina e amido (KRAUSE et al, 2003;KABEL et al, 2011).…”

“…Alguns membros deste gênero possuem enzimas com alta atividade hemicelulolítica e proteolítica (MATSUI et al, 2000), os quais poderiam degradar xilana, pectina e amido (KABEL et al, 2011).…”