A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 +/- 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0 degrees-20 degrees C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant toelectrophilic attack of active-site carboxyl residues, glycosidehydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. Theapparent inactivation and association constants (k(i), 1/K(i)) are one order of magnitude higher for thexylobiose and xylotriose derivatives. The effects of the aglycone chainlength can clearly be described. Xylobiose and n-alkyl beta-D-xylopyranosides are competitive ligands and provide protectionagainst inactivation. MS measurements showed 1:1 stoichiometries inmost labelling experiments. Electrospray ionization MS/MS analysisrevealed the nucleophile Glu(86) as the modified residue inthe T. lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyranoside was used, whereas the acid/base catalyst Glu(178) was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu(86) and Glu(177) in T. reesei Xyn II are similarly modified, confirming earlier X-raycrystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996)Biochemistry 35, 9617-9624]. The inability of the omega-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10enzymes is discussed in terms of different ligand-subsiteinteractions.

The highest beta-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified beta-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 degrees C and pH 4.0. It was stable for 30 min at 70 degrees C and has a broad pH stability (2.0-12.0). ManIII showed K (m), V (max), and K (cat) values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s(-1) with locust bean gum as substrate, respectively. It was inhibited by mannose with a K (I) of 0.610(-3) mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.

No abstract

A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant toelectrophilic attack of active-site carboxyl residues, glycosidehydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. Theapparent inactivation and association constants (k(i), 1/K(i)) are one order of magnitude higher for thexylobiose and xylotriose derivatives. The effects of the aglycone chainlength can clearly be described. Xylobiose and n-alkyl beta-D-xylopyranosides are competitive ligands and provide protectionagainst inactivation. MS measurements showed 1:1 stoichiometries inmost labelling experiments. Electrospray ionization MS/MS analysisrevealed the nucleophile Glu(86) as the modified residue inthe T. lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyranoside was used, whereas the acid/base catalyst Glu(178) was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu(86) and Glu(177) in T. reesei Xyn II are similarly modified, confirming earlier X-raycrystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996)Biochemistry 35, 9617-9624]. The inability of the omega-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10enzymes is discussed in terms of different ligand-subsiteinteractions.