No abstract

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.

Endoxylanase (xynA) and P-xylosidase (xynB) genes from Thevmoanaerobacterium saccharofyticum were subcloned from a cosmid clone (pXDM1) to generate pXPH3. The nucleotide sequence of a PstI-Hind111 fragment in pXPH3 that contained xynB revealed an open reading frame (ORF) of 1500 bp encoding a 55 kDa protein.Another open reading frame (ORFl) of unknown function was found 21 bp downstream from the first stop codon of xynB. xynB, ORFl and xynA had the same direction of transcription. xynB from T. saccharolyticum strain B6A-RI exhibited 45 YO amino acid similarity, with 18 % amino acid identity to xynA of T. saccharofyticum strain B6A-RI, and 61 YO similarity and 37 YO identity with the j?-xylosidase gene from Cafdoceffum saccharofyticum. Recombinant j?-xylosidase was purified from E. coli (pXPH3) cells. The enzyme was a monomer with a molecular mass of 55 kDa. The specific activity and pH and temperature optima for hydrolysis of p-nitrophenyl-P-Dxylopyranoside (pNPX) were 5-53 U mg-', 5.5 and 70 "C, respectively. The P-xylosidase was stable at 65 "C, but lost activity at 85 "C. The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.

An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined. The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase. The beta-amylase was enriched in both acidic and hydrophobic amino acids. The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5. The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition. The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins. The enzyme displayed kcat. and Km values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively. The enzyme was antigenically distinct from plant beta-amylases.

No abstract