Genetic organization, sequence and biochemical characterization of recombinant  -xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI

Abstract

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.

Stereochemical Course and Reaction Products of the Action of β‐Xylosidase from Thermoanaerobacterium saccharolyticum Strain B6A‐RI

“…When pxylosidase activity is monitored by measuring the amount of pnitrophenol released from p-nitrophenyl p-D-xyloside, the present high transglycosylating activity cannot be detected. The presence of xylotriose in the early stages of the hydrolysis of xylobiose by T. snccharolyticum /i-xylosidase has been detected by TLC (Lee et al, 1993). Transglycosylating activity is a property shared by a number of glycosyl hydrolases such as p-galactosidases, cellulases, P-glucosidases, etc.…”

“…p-Nitrophenyl p-D-xylopyranoside was used for this study since earlier experiments had shown it to be the best substrate for the enzyme (Lee and Zeikus, 1993). Fig.…”

“…The families are defined as in Henrissat and Bairoch (1993 Preparation of cell extracts. 7: saccharolyticum B6A-RT pxylosidase was purified as a recombinant enzyme from Escherichia coli Sure (e14-(mcrA), sbcC,recB,recJ,umuC: :Tn5(kunr),uvrC,supE44,lac,gyrA96,relAI,endAl,[F' proAB,laclqZMI.5, TnlO, ( t e f ) ] ) (Stratagene) harboring plasmid pXHP3 (Lee and Zeikus, 1993). Cells were harvested from an overnight culture in .5 1 Luria-Bertani medium, supplemented with 100 pg/ml ampicillin.…”

Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity

“…Putative prokaryotic-sequence-like Ϫ10 (TATAGA) and Ϫ35 (TATGTG) promoter sequences as well as a Shine-Dalgarno (GGGGG) site were noted. Screening of protein and nucleic acid data banks revealed that the xylB gene is a homolog of the previously described xynB gene isolated from Thermoanaerobacterium sacharrolyticum (20), the products having 98% amino acid similarity and 91% identity by Bestfit analysis. The differences between the peptide sequences of these two genes are, for the most part, conservative.…”

“…While a number of ␤-xylosidase genes have been isolated from a variety of fungal and mesophilic bacterial sources, few have been isolated from thermophilic bacteria (2,20,23). In contrast to the situation for xylanases and xylosidases, for which microbiological and biochemical information is available, very few genes encoding acetyl xylan esterases have been verified (12,23,24,32), the xynD gene from Pseudomonas fluorescens being the only bacterial representative which has been expressed in Escherichia coli and characterized (12).…”