Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI

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.

Cloning and Characterization of a β-1,4-Mannanase 5C Possessing a Family 27 Carbohydrate-Binding Module from a Marine Bacterium,Vibriosp. Strain MA-138

“…31) Some xylan-binding modules classified in family 22 were originally identified as thermostabilizing modules, because their removal from thermophilic enzymes resulted in decreases in the thermostability and/or optimal temperature of the enzymes. [50][51][52] Removal of the N-terminal CBM27 of Man26 of C. saccharolyticus strain Rt8B.4 resulted in a decrease in the optimal temperature of the enzyme. 53) However, removal of the C-terminal CBM27 region from Man5C of strain MA-138 did not affect its optimal temperature (data not shown).…”

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain

“…The duplicated C-terminal domain belongs to a novel type of cellulose-binding domains (CBDs), also present in modular xylanases from Thermoanaerobacterium saccharolyticurn (Lee et al, 1993). Clostridium thermocellurn (M.Y.…”

Functions of Family-22 Carbohydrate-Binding Modules inClostridium josuiXyn10A

“…Family-22 CBMs were originally identified as thermostabilizing modules, i.e., shifts (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) C) in optimum temperature upon artificial removal of family-22 CBMs from thermophilic modular xylanases were observed in C. cellulovorans XynA (from 90 to 70 C), 8) C. stercorarium Xyn10B (75 to 60 C), 15) C. thermocellum Xyn10C (80 to 60 C), 16) XynX (70 to 60 C), 20) XynY (75 to 60 C), 10) and T. saccharolyticum XynA (75 to 65 C) 13) in the presence of the substrate. Furthermore, an increase in thermostability in the absence of the substrate was observed in C. cellulovorans XynA, C. thermocellum XynX, and C. thermocellum XynX and XynY.…”

“…10,13,14) Recently, furthermore, we have found that a truncated derivative of C. stercorarium Xyn10B, composed of two family-22 CBMs and a CM, preferred -1,3-1,4-glucan, such as barley -glucan or lichenan, to xylan as a substrate, and removal of the CBMs from this enzyme drastically reduced catalytic activity toward -1,3-1,4-glucan but not toward xylan, suggesting that the family-22 CBMs in C. stercorarium Xyn10B are a determinant factor for -1,3-1,4-glucanase activity. 15) By contrast, C. thermocellum Xyn10C is less active toward -1,3-1,4-glucan regardless of the presence or absence of a family-22 CBM.…”