A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.     

Increased alkali stability in Trichoderma reesei endo-1, 4-beta-xylanase II by site directed mutagenesis

A number of engineered Trichoderma reesei endo-beta-1,4-xylanase (Xyn II) mutants were created and activity tests were performed for increased stability. The stability of the earlier characterized mutant Y5 (T2C, T28C, K58R, +191D) was further increased by the mutations creating the constructs P9 (N97R+F93W+H144K), P12 (H144C+N92C), P15 (F180Q+H144C+N92C) and P21 (H22K+F180Q+H144C+N92C). The resistance towards thermal inactivation at alkaline pH was increased in all of the mutants. Residual activity T(50%) was increased 4-5 degrees C for P9 at pH 9. The performance of the P9 mutant in sulphate pulp bleaching was also tested and was shown to increase brightness markedly compared to the reference. The bleaching results showed the industrial potential of the obtained mutant.     

Functional conformational changes of endo-1,4-xylanase II from Trichoderma reesei: A molecular dynamics study

Recent crystallographic studies have revealed a range of structural changes in the three-dimensional structure of endo-1,4-xylanase (XYNII) from Trichoderma reesei. The observed conformational changes can be described as snapshots of an open-close movement of the active site of XYNII. These structures were further analyzed in this study. In addition, a total of four 1 ns molecular dynamics (MD) simulations were performed representing different states of the enzyme. A comparison of the global and local changes found in the X-ray structures and the MD runs suggested that the simulations reproduced a similar kind of active site opening and closing as predicted by the crystal structures. The open-close movement was characterized by the use of distance difference matrixes and the Hingefind program (Wriggers and Schulten, Proteins 29:1–14, 1997) to be a ‘hinge-bending’ motion involving two large rigidly-moving regions and an extended hinge. This conformational feature is probably inherent to this molecular architecture and probably plays a role in the function of XYNII. Proteins 31:434–444, 1998. © 1998 Wiley-Liss, Inc.     

Irreversible thermal denaturation of Trichoderma reesei endo-1,4-beta-xylanase II and its three disulfide mutants characterized by differential scanning calorimetry

Kinetic as well as energetic aspects of the thermal denaturation of Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) and its three thermostable disulfide mutants were characterized by means of differential scanning calorimetry (DSC) in different solution conditions. The calorimetric transitions were strongly scan-rate dependent, characteristic for an irreversible, kinetically controlled protein denaturation. The DSC-determined T*-values (the temperature at which the denaturation rate constant equals 1min(-1)), and the activation free energies for the transitions are consistent with the apparent transition temperatures of TRX II determined earlier by mass spectrometry. Protein aggregation, connected with the irreversibility of the transitions, was present in all cases but was less pronounced with the mutants as well as highly dependent on experimental conditions.     

The active site of an acidic endo-1,4-beta-xylanase of Aspergillus niger

The substrate binding site of an acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Bond cleavage frequencies and V/Km parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293-316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xylosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger beta-xylanase appears to be an endo-1,4-beta-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate.     

Expression of endo-1, 4-beta-xylanase from Trichoderma reesei in Pichia pastoris and functional characterization of the produced enzyme

Background  

In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast Pichia pastoris can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes.     

Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH

We studied the effects of increase in the number of surface arginines on the enzyme activity and stability of Trichoderma reesei endo-1,4-beta-xylanase II. The number of arginines was increased in two mutant series. The first set contained six arginines on different sides of the protein surface. These arginines had no significant effect on the thermostability. However, the optimal pH region became narrower. Another series of five arginines was engineered into the 'Ser/Thr surface', formed of part of the double-layered beta-sheet located on one side of the 'right-hand-like' xylanase. These mutations shifted the activity profile to the alkaline region by approximately 0.5-1.0 pH units. In addition, the arginines on the Ser/Thr surface increased the enzyme activity at high temperature, although the enzyme stability in the absence of substrate decreased significantly at 50-55 degrees C. In the presence of the substrate, the thermostability increased 4-5-fold at 60-65 degrees C. Thus, the substrate neutralized the destabilizing effect of Ser/Thr surface arginines and revealed a stabilizing effect of the same mutations. The stabilizing effect of arginines at high temperatures was seen clearly only when five arginines were introduced into the Ser/Thr surface.     

A symbiont-independent endo-1,4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita

Substituted xylan polymers constitute a major part of the hemicellulose fraction of plant cell walls, especially in monocotyledons. Endo-1,4-beta-xylanases (EC 3.2.1.8) are capable of hydrolyzing substituted xylan polymers into fragments of random size. Many herbivorous animals have evolved intimate relationships with endosymbionts to exploit their enzyme complexes for the degradation of xylan. Here, we report the first finding of a functional endo-1,4-beta-xylanase gene from an animal. The gene (Mi-xyl1) was found in the obligate plant-parasitic root-knot nematode Meloidogyne incognita, and encodes a protein that is classified as a member of glycosyl hydrolase family 5. The expression of Mi-xyl1 is localized in the subventral esophageal gland cells of the nematode. Previous studies have shown that M. incognita has the ability to degrade cellulose and pectic polysaccharides in plant cell walls independent of endosymbionts. Including our current data on Mi-xyl1, we show that the endogenous enzyme complex in root-knot nematode secretions targets essentially all major cell wall carbohydrates to facilitate a stealthy intercellular migration in the host plant.     

Characterization of endo-1,4-beta-D-glucanases purified from Trichoderma viride

Four electrophoretically distinct endo-1,4-beta-D-glucanases (EC 3.2.1.4) from Trichoderma viride have been identified and named as isozymes, Endoglucanases I, II, III and IV, according to their electrophoretic mobilities on polyacrylamide gels. Endoglucanases II, III and IV, the homogeneity of each of which was established by discontinuous gel electrophoresis and ultracentrifugation, had specific activities on CM-cellulose of 1010, 60 and 250 specific fluidity units/mg protein, respectively. These enzymes have similar pH optima (pH 4.0-4.5) and are labile at pH values greater than 8.0. The endoglucanases are high in acidic and hydroxylated amino acids and glycine, but low in basic amino acids. Values of 12.0, 10.3 and 13.1 have been determined for the epsilon 1%280 of purified Endoglucanases II, III and IV, respectively. Sedimentation equilibrium analysis has established the molecular weights of Endoglucanases II, III and IV to be 37 200, 52 000 and 49 500, respectively. The three endoglucanases contain mannose, galactose, glucose and glucosamine. Mannose is the principal neutral sugar in each enzyme. Endoglucanase II is distinguished by its low carbohydrate content, 4.5% (w/w), compared to Endoglucanases III and IV which contain 15.0% and 15.2% carbohydrate, respectively.     

Enzymic activities of endo-1,4-beta-D-glucanases purified from Trichoderma viride

Endoglucanases II, III and IV (EC 3.2.1.4) from Trichoderma viride are highly active in degrading CM-cellulose or phosphoric acid swollen cellulose, and only slightly active on Avicel. The specific activities of the endoglucanases increase with the length of the cellooligosaccharide substrates. By rate and product analyses using high pressure liquid chromatography the mode of action of Endoglucanase III was differentiated from that of Endoglucanases II and IV. Endoglucanase III has a low affinity for cellobiose, reacts rapidly with cellotriose, and gradually increases in reactivity with cellooligosaccharides as degree of polymerization increases from four to six. In addition to cleaving internal glycosidic bonds of polymeric substrates, it preferentially cleaves cellobiosyl units from the non-reducing end of oligosaccharides. The cellobiosyl units are often, under initial reaction conditions, transferred to the substrate-acceptor. Endoglucanases II and IV show a preference for internal glycosidic bonds of cellooligosaccharides. The soluble products from the initial action of Endoglucanases II and IV on swollen cellulose are glucose, cellobiose, and cellotriose, which are slowly converted to glucose and some cellobiose.     

Cloning of an endo-1,4-beta-xylanase gene from Penicillium canescens and construction of multicopy strains

The complete gene xylA that encodes endo-1,4-beta-xylanase secreted by Penicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven-eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30-50% of the total secreted protein.     

Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism

Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.     

Insight of Endo-1,4-Xylanase II from Trichoderma reesei: Conserved Water-Mediated H-Bond and Ion Pairs Interactions

Endo-1,4-Xylanase II is an enzyme which degrades the linear polysaccharide beta-1,4-xylan into xylose. This enzyme shows highest enzyme activity around 55 °C, even without being stabilized by the disulphide bridges. A set of nine high resolution crystal structures of Xylanase II (1.11–1.80 Å) from Trichoderma reesei were selected and analyzed in order to identify the invariant water molecules, ion pairs and water-mediated ionic interactions. The crystal structure (PDB-id: 2DFB) solved at highest resolution (1.11 Å) was chosen as the reference and the remaining structures were treated as mobile molecules. These structures were then superimposed with the reference molecule to observe the invariant water molecules using 3-dimensional structural superposition server. A total of 37 water molecules were identified to be invariant molecules in all the crystal structures, of which 26 invariant molecules have hydrogen bond interactions with the back bone of residues and 21 invariant water molecules have interactions with side chain residues. The structural and functional roles of these water molecules and ion pairs have been discussed. The results show that the invariant water molecules and ion pairs may be involved in maintaining the structural architecture, dynamics and function of the Endo-1,4-Xylanase II.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

The titration of the active centers of cellobiohydrolase from Trichoderma reesei

A novel approach has been developed for the titration of enzyme active centers and for the determination of the molecular activity of enzymes. It is based on the simultaneous use of a nonspecific chromogenic substrate and a specific ligand (a substrate or an inhibitor), the latter being tightly bound with the enzyme's active center. The approach is demonstrated using the titration (that is, the determination of the molar concentration of the enzyme active centers) of purified cellobiohydrolase I (CBH I) (EC 3.2.1.91) of the fungus Trichoderma reesei. p-Nitrophenyl-beta-D-lactoside was used as a reference substrate (Km = 0.5 mM), and cellobiose and CM-cellulose as specific ligands. The molecular weight of CBH I as it was determined by the titration with cellobiose was 42,000 +/- 3,000. The inhibition constant by cellobiose was (6 +/- 1) X 10(-6) M. The value of the catalytic constant for the hydrolysis of p-nitrophenyl-beta-D-lactoside calculated from the titration data was equal to 0.063 s-1. CM-cellulose turned out to be more efficient titration agent for cellobiohydrolase than cellobiose, and might be used for the titration of the enzyme in concentrations of the latter of 0.008-0.02 mg/ml. The titration data showed that the inhibition constant of CM-cellulose toward CBH I was equal to (1.0 +/- 0.2) X 10(-7) M.     

Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei.

Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.     

Induction of endo-1,4-beta-xylanase and beta-galactosidase in the original and recombinant strains of the fungus Penicillium canescens

The induction of the synthesis of secreted enzymes endo-1,4-beta-xylanase (EC 3.2.1.8) and beta-galactosidase (EC 3.2.1.23) in the original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for the induction: the synthesis of beta-galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4-beta-xylanase was observed at 5 to 10 mM. An increase in the number of endo-1,4-beta-xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of beta-galactosidase; the synthesis of endo-1,4-beta-xylanase in the high-copy-number recombinant producing beta-galactosidase was affected to a lesser extent. The amount of the enzymes synthesized did not depend on the saccharide used as a sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.