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 (XOSFA,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 XOSFA,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.

[1]  J. Brückner.  Estimation of monosaccharides by the orcinol-sulphuric acid reaction. , 1955 .

[2]  M. P. Bryant,et al.  PEPTIDES AND OTHER NITROGEN SOURCES FOR GROWTH OF BACTEROIDES RUMINICOLA , 1964, Journal of bacteriology.

[3]  B. A. Dehority,et al.  Extent of Cellulose and Hemicellulose Digestion in Various Forages by Pure Cultures of Rumen Bacteria , 1967 .

[4]  J. Joseleau,et al.  Isolation, purification, and characterization of a complex heteroxylan from industrial wheat bran. , 1982, Journal of Agricultural and Food Chemistry.

[5]  P. Gorin,et al.  Location of O-acetyl groups in the acidic d-xylan of mimosa scabrella (bracatinga). A study of O-acetyl group migration , 1984 .

[6]  N. Shibuya,et al.  Structural features of rice bran hemicellulose , 1985 .

[7]  C. MacKenzie,et al.  Cooperativity of Esterases and Xylanases in the Enzymatic Degradation of Acetyl Xylan , 1986, Bio/Technology.

[8]  A. Voragen,et al.  Determination of the degree of methylation and acetylation of pectins by h.p.l.c. , 1986 .

[9]  J. Costerton,et al.  Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose. , 1987, Canadian Journal of Microbiology (print).

[10]  R. B. Hespell,et al.  Esterase activities in Butyrivibrio fibrisolvens strains , 1988, Applied and environmental microbiology.

[11]  M. Coughlan.  Enzyme systems for lignocellulose degradation. , 1989 .

[12]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[13]  A. Voragen,et al.  Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process , 1990 .

[14]  J. Miron.  The hydrolysis of lucerne cell-wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria. , 1991, The Journal of applied bacteriology.

[15]  A. Voragen,et al.  Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods. , 1992, Analytical biochemistry.

[16]  T. Lam,et al.  Covalent Cross-Links in the Cell Wall , 1994, Plant physiology.

[17]  J. Vigouroux,et al.  Isolation and partial characterization of feruloylated oligosaccharides from maize bran. , 1995, Carbohydrate research.

[18]  D. Weisleder,et al.  Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates. , 1996, Biochimica et biophysica acta.

[19]  崇 韮澤.  ＭＡＬＤＩ-ＴＯＦ-ＭＳ , 1996 .

[20]  M. Fondevila,et al.  Interactions between Fibrobacter succinogenes, Prevotella ruminicola, and Ruminococcus flavefaciens in the digestion of cellulose from forages. , 1996, Journal of animal science.

[21]  N. Carpita.  STRUCTURE AND BIOGENESIS OF THE CELL WALLS OF GRASSES. , 1996, Annual review of plant physiology and plant molecular biology.

[22]  E. Simonne,et al.  Could the Dumas Method Replace the Kjeldahl Digestion for Nitrogen and Crude Protein Determinations in Foods , 1997 .

[23]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..

[24]  A. A. Shatalov,et al.  (2-O-α-d-Galactopyranosyl-4-O-methyl-α-d-glucurono)-d-xylan from Eucalyptus globulus Labill , 1999 .

[25]  J. Thibault,et al.  Ferulic acid and diferulic acids as components of sugar‐beet pectins and maize bran heteroxylans , 1999 .

[26]  A. Voragen,et al.  Glucuronoarabinoxylans from maize kernel cell walls are more complex than those from sorghum kernel cell walls. , 2000 .

[27]  A. Voragen,et al.  Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium , 2000 .

[28]  V. Venturi,et al.  The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity. , 2000, Microbiology.

[29]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[30]  Alphons G. J. Voragen,et al.  Handbook of Food Enzymology , 2002 .

[31]  M. Tenkanen,et al.  Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech. , 2002, Carbohydrate research.

[32]  J. Parajó,et al.  Hydrothermally treated xylan rich by-products yield different classes of xylo-oligosaccharides , 2002 .

[33]  A. Voragen,et al.  Complex xylo-oligosaccharides identified from hydrothermally treated Eucalyptus wood and brewery's spent grain , 2002 .

[34]  J. Eyzaguirre,et al.  Comparison of catalytic properties of acetyl xylan esterases from three carbohydrate esterase families , 2003 .

[35]  H. Gilbert,et al.  Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9A resolution. , 2003, Journal of molecular biology.

[36]  P. de Waard,et al.  Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood. , 2003, Carbohydrate research.

[37]  D. Evtuguin,et al.  Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill. , 2003, Carbohydrate research.

[38]  A. Travis,et al.  16S rDNA library-based analysis of ruminal bacterial diversity , 2004, Antonie van Leeuwenhoek.

[39]  K. Poutanen,et al.  Deacetylation of xylans by acetyl esterases of Trichoderma reesei , 1990, Applied Microbiology and Biotechnology.

[40]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[41]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[42]  J. Grabber.  How Do Lignin Composition, Structure, and Cross‐Linking Affect Degradability? A Review of Cell Wall Model Studies , 2005 .

[43]  Rafael A. Irizarry,et al.  Bioinformatics and Computational Biology Solutions using R and Bioconductor , 2005 .

[44]  A. Voragen,et al.  Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides. , 2006, Journal of chromatography. A.

[45]  F. Guillon,et al.  Wheat arabinoxylans : Exploiting variation in amount and composition to develop enhanced varieties , 2007 .

[46]  H.F.A. Van den Borne,et al.  Structural differences of xylans affect their interaction with cellulose , 2007 .

[47]  P. Weimer,et al.  Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR , 2007, Applied Microbiology and Biotechnology.

[48]  H. R. Sørensen,et al.  Enzymatic hydrolysis of wheat arabinoxylan by a recombinant "minimal" enzyme cocktail containing beta-xylosidase and novel endo-1,4-beta-xylanase and alpha-l-arabinofuranosidase activities. , 2007, Biotechnology progress.

[49]  C. Faulds,et al.  Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides. , 2007, Journal of biotechnology.

[50]  H. R. Sørensen,et al.  Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant “Minimal” Enzyme Cocktail Containing β‐Xylosidase and Novel endo‐1,4‐β‐Xylanase and α‐l‐Arabinofuranosidase Activities , 2007 .

[51]  K. Chou,et al.  Gpos-PLoc: an ensemble classifier for predicting subcellular localization of Gram-positive bacterial proteins. , 2007, Protein engineering, design & selection : PEDS.

[52]  K. Chou,et al.  Cell-PLoc: a package of Web servers for predicting subcellular localization of proteins in various organisms , 2008, Nature Protocols.

[53]  Robert D. Finn,et al.  InterPro: the integrative protein signature database , 2008, Nucleic Acids Res..

[54]  C. Abbas,et al.  Biochemical Analysis of a β-d-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23 , 2009, Journal of bacteriology.

[55]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[56]  Michael Levitt,et al.  Protein segment finder: an online search engine for segment motifs in the PDB , 2008, Nucleic Acids Res..

[57]  K. Nelson,et al.  Comparative Genome Analysis of Prevotella ruminicola and Prevotella bryantii: Insights into Their Environmental Niche , 2010, Microbial Ecology.