Molecular cloning of genes from Ruminococcus flavefaciens encoding xylanase and beta(1-3,1-4)glucanase activities

Abstract

Clones expressing activity against xylan or 0(1-3,1-4)glucan (lichenan) were isolated from a library of Ruminococcusflavefaciens 17 DNA made in bacteriophage A EMBL3. Hybridization analyses indicated the recovery of four separate genes encoding xylanases that showed no detectable associated carboxylmethylcellulase activity. One of these genes was associated with clones that also expressed P(1-3,1-4)glucanase and I-xylosidase activities. Ruminococcus flavefaciens is an important plant cell wall-degrading bacterium found in the rumen and hind gut of mammals (3). Many strains are known to be capable of causing extensive degradation of highly crystalline forms of cellulose, and this has led to interest in the enzymology (6, 17, 18) and genetic determination (1) of cellulases in this species. In addition, strains of R. flavefaciens are capable of extensive degradation of the noncellulosic polysaccharide components of plant cell walls (15) because they produce a variety of other polysaccharidases, including xylanase and pectinase (17, 21). These activities are likely to contribute substantially to the overall rate and extent of cell wall digestion even though the degradation products are often not utilized for growth. This report describes the analysis of genomic DNA clones from recently isolated R. flavefaciens 17 that express activities against two noncellulosic plant cell wall polysaccharides, xylan and 3(1-3,1-4)glucan. MATERIALS AND METHODS Strains. R. flavefaciens 17 is a recent isolate obtained nonselectively from the rumen of a fistulated cow that was receiving a diet of grass cubes, hay, and concentrates. Fermentation products, cell morphology and Gram stain, colony pigmentation, and the range of substrates fermented all agree with accepted characteristics for the species (3). Anaerobic methods. The M2 medium of Hobson (8) containing 0.2% cellobiose as the energy source was used to grow R. flavefaciens by the methods of Bryant (2). DL-Threonine (10 mM) was included in cultures used for DNA extraction. Library screening and molecular biology procedures. Chromosomal DNA was extracted from R. flavefaciens 17 by standard methods (19), but mutanolysin was used to achieve lysis. Size-fractionated Sau3A-cut DNA (average length,

Directed evolution of a β‐1,3‐1,4‐glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics

“…Genes encoding b-1,3-1,4-glucanase have been cloned from many strains [5][6][7][8][9][10][11][12][13][14][15][16][17][18]. However, a major disadvantage of these enzymes is their lack of thermal stability when used in industrial processes [40].…”

“…b-1,3-1,4-glucanase gene has been cloned from a variety of Bacillus, including B. amyloliquefaciens [5], B. macerans [6], B. polymyxa [7], B. brevis [8], B. circulans [9], B. licheniformis [10], B. subtilis [11] and B. halodurans [12]. In addition, this enzyme could be cloned from other bacteria, such as Ruminococcus flavefaciens [13], Clostridium thermocellum [14], Streptococcus bovis [15] and Bacteroides succinogenes [16]. The fungi including Talaromyces emersonii [17] and Orpinomyces [18] could produce b-1,3-1,4-glucanase.…”

Cellulosome gene cluster analysis for gauging the diversity of the ruminal cellulolytic bacteriumRuminococcus flavefaciens

“…The previously published sca gene cluster sequences from R. flavefaciens strain FD-1 and 17 (Jindou et al, 2006) were obtained from the GenBank database (accession number: AJ278969 for strain 17, AM262974 for strain FD-1). (Flint et al, 1989). The scaffoldin gene cluster from this strain was compared with that of strain FD-1 (Jindou et al, 2006) C94 (ATCC 19208) Isolated by M.P.…”

The hydrolysis of lucerne cell‐wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria

“…The cellulolytic and hemicellulolytic rumen bacterial species Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus Javefaciens, Butyrivabrio jbrasolvens and Bacteroides ruminicola are recognized as the predominant species grown on and degrading leguminous hays (Dehority & Scott 1967;Collings & Yokoyama 1980;Cheng et al 1983;Russell 1984;Miron & Yokoyama 1990). Recently much progress has been achieved in the identification and characterization of the cellulases and hemicellulases complexes of these bacterial species (Hespell et al 1987;Howard & White 1988;Irvin & Teather 1988;Flint et al 1989;Gong et a/. 1989;Sewell et al 1989;Whitehead & Hespell 1989;McDermid et a/.…”