Biochemical Analyses of Multiple Endoxylanases from the Rumen Bacterium Ruminococcus albus 8 and Their Synergistic Activities with Accessory Hemicellulose-Degrading Enzymes

Abstract

Ruminococcus albus 8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of R. albus 8 was generated. The genomic data depicted a bacterium endowed with multiple forms of plant cell wall-degrading enzymes. The endoxylanases of R. albus 8 exhibited diverse modular architectures, including incorporation of a catalytic module, a carbohydrate binding module, and a carbohydrate esterase module in a single polypeptide. The accessory enzymes of xylan degradation were a ␤-xylosidase, an ␣-L-arabinofuranosidase, and an ␣-glucuronidase. We hypothesized that due to the chemical complexity of the hemicellulose encountered in the rumen, the bacterium uses multiple endoxylanases, with subtle differences in substrate specificities, to attack the substrate, while the accessory enzymes hydrolyze the products to simple sugars for metabolism. To test this hypothesis, the genes encoding the predicted endoxylanases were expressed, and the proteins were biochemically characterized either alone or in combination with accessory enzymes. The different endoxylanase families exhibited different patterns of product release, with the family 11 endoxylanases releasing more products in synergy with the accessory enzymes from the more complex substrates. Aside from the insights into hemicellulose degradation by R. albus 8, this report should enhance our knowledge on designing effective enzyme cocktails for release of fermentable sugars in the biofuel industry.

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

“…Glucose, cello-oligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose), mannose, and manno-oligosaccharides (mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose), each at a final concentration of 1 mg/ml, were incubated with 0.1 M CbCel9B/Man5A wild type, CbCel9B/Man5ATM1, and CbCel9B/ Man5ATM5 in a citrate buffer (10 mM sodium citrate, 150 mM NaCl [pH 5.5]) at 75°C for 14 h. The total reaction volume was 40 l. The reaction products were dried using a SpeedVac concentrator (Thermo Fisher Scientific, Pittsburgh, PA) and dissolved in 3.5 l of H 2 O, and 1 l of the products was analyzed by thin-layer chromatography (TLC) using a 250 m thick Whatman silica gel 60A (Maidstone, England). The TLC method was the same as described in our earlier report (39). Briefly, reaction products were spotted on a 10-cm-by-20-cm TLC plate and kept at room temperature for 15 min until the plate was dry.…”

“…A portion (2.5 mg) of PASC/ml was incubated with 0.5 M CbCel9B/Man5A WT, TM1, and TM5 at 75°C. At different time intervals (0 min, 2 min, 10 min, 60 min, 4 h, and 24 h), aliquots were taken and subjected to high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis to detect end products as described earlier (39).…”

“…1A) were amplified from the genomic DNA of C. bescii DSM 6725 by PCR using PrimeSTAR HS DNA polymerase (Takara, Shiga, Japan) (see Table S1 in the supplemental material for primer sequences). The PCR products were cloned into the pET-46 Ek/LIC vector according to the protocols described by the manufacturer (Merck, Darmstadt, Germany) and also described in our previous report (39 Gene expression and protein purification. The recombinant pET-46 Ek/LIC plasmids harboring sequences coding for either the CbCel9B/ Man5A wild type or one of its truncation variants were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on a lysogeny broth (LB) agar plate supplemented with 100 g of ampicillin/ml and 50 g of chloramphenicol/ml.…”

“…To completely hydrolyze plant cell wall polysaccharides into simple sugars, a suite of cellulases and hemicellulases are required to operate in synergy (25,39). Thermostable cellulases and hemicellulases are appealing to the biofuel industry due to their perceived advantages over their mesophilic counterparts.…”

Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase

“…Ruminococcus flavefaciens and Ruminococcus albus are among the most important plant cell wall-degrading bacteria in the rumen. These two species produce all required enzymes for hydrolyzing the plant cell wall polysaccharides, cellulose and hemicellulose (5,6).…”