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Essential carboxy groups in xylanase A.

ABSTRACT: An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.

SUBMITTER: Bray MR

PROVIDER: S-EPMC1131682 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Project description:Water-insoluble cationic poly(vinyl alcohol) (PVA) films were fabricated using a mixed aqueous solution of PVA and poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC)-co-methacrylic acid (MAAc)-co-5-methacrylamido-1,2-benzoxaborole (MAAmBO)) copolymer (3D). The surface of the PVA film is typically negatively charged, and simple fabrication methods for water-insoluble PVA films with cationic surface charges are required to expand their application fields. METAC, which has a permanent positive charge owing to the presence of a quaternary ammonium cation, was selected as the cationic unit. The MAAc and MAAmBO units were used as two types of cross-linking structures for the thermal cross-linking of the hydroxy and carboxy groups of the MAAc unit (covalent bonding) as well as the diol and benzoxaborole groups of the MAAmBO unit (dynamic covalent bonding). The films were thermally cross-linked at 135 °C for 4 h without the addition of materials. After immersion in surplus water at 80 °C for 3 h, the cross-linked PVA/3D films retained almost 100% of their weights. The ζ-potential of the water-insoluble PVA/3D film was 9.4 ± 0.8 mV. The PVA/3D film was strongly dyed using anionic acid red 1 (AR1) because of its positively charged surface. Interestingly, it could also be slightly dyed using cationic methylene blue (MB) and became transparent (original state) after immersion in water for 2 days. These results suggested that positive and negative charges coexisted in the PVA/3D film, and the surface properties were positively inclined. Moreover, the degree of hemolysis of the PVA/3D films was similar to that of the negative control, which showed high blood compatibility. To our knowledge, this is the first report on the fabrication of water-insoluble cationic PVA films using two types of cross-linking structures containing carboxy and benzoxaborole groups. The cross-linked PVA films were analyzed using Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and contact angle (CA) and ζ-potential measurement, as well as by determining the mechanical properties, adsorption of charged molecules, and biocompatibility. These readily fabricated water-insoluble PVA films with positive charges can show potential applications in sensors, adsorption systems, and antimicrobial materials.

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Essential carboxy groups in xylanase A.

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