Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.     

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.     

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.     

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.     

定点突变提高里氏木霉木聚糖酶 (XYN II) 的稳定性

通过定点突变的方法,在来源于里氏木霉Trichderma reesei的木聚糖酶XYN II的N-末端两个β折叠片层间添加二硫键,以提高木聚糖酶的稳定性。原酶XYN-OU和突变酶XYN-HA12 (T2C、T28C和S156F) 分别在毕赤酵母中分泌表达,突变酶与原酶纯化后进行酶学性质比较。结果表明：突变酶最适反应温度由50℃提高到60℃;在70℃的半衰期由1 min提高到14 min;最适反应pH为5.0,与原酶保持一致,但是在50℃、30 min条件下的pH稳定范围由4.0~9.0扩展到3.0~10.0。对木聚糖酶分子改良的结果反映出在β片层间添加二硫键可以有效改善酶在较高温度下三维结构的刚性,提高热稳定性。     

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.     

The role of the site 342 in catalytic efficiency and pH optima of endoglucanase II from Trichoderma reesei as probed by saturation mutagenesis

The role of site 342 of endoglucanase II from Trichoderma reesei in catalytic efficiency and pH optima was investigated by site saturation mutagenesis. The mutations identified in this study can be divided into three separate classes according to their amino acid features. When Asn342 was substituted by hydrophobic and non-polar amino acids, most variants exhibited an up-shift in pH optimum and their catalytic efficiency was similar to that of the wild-type at their optimal pH. N342R variant had a pH optimum at 6.2. N342K variant did not give an up-shift in pH optimum, although K and R are both amino acids carrying positive charges. Molecular modelling indicated that residue 342 was located at the C-terminus of one of the α-helices near two catalytic residues. Hydrophobic side chains and more H-bonds would make the helix more rigid, which might affect the stability and activity of the enzyme at higher pH.     

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.     

碱与微波联合处理蔗渣对里氏木霉发酵产纤维素酶的影响

以蔗渣为原料,采用碱和微波辐射联合处理后用于里氏木霉纤维素酶的液态发酵。采用单因素试验与正交试验确定了最佳的处理条件为:0.30 mol/L的NaOH溶液浸泡,微波功率160 W,处理5 min。在此条件下得到的单位能耗的酶活净增值最高。后续发酵结束后,酶活较未经处理的蔗渣发酵后所得酶活有显著提高。其中,β-葡萄糖苷酶活、滤纸酶(FPase)活、羧甲基纤维素酶(CMCase)活分别提高了81.3%,88.2%,154.5%。     

Enzymatic properties of a neutral endo-1,3(4)-beta-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C. Xyl II was stable at 40 degrees C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1). Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.     

Cold adaptation of a mesophilic cellulase,EG Ⅲ from Trichoderma reesei,by directed evolution

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold-adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG Ⅲ with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG Ⅲ calculated from Arrhenius equation are 13.3 kJ· mol-1 and 26.2 kJ ·mol-1, respectively. Therefore, the increased specific activity of w-3 at lower tempera     

Purification and characteristic of endo-(1--4)-beta-xylanase from Geotrichum candidum 3C

A method of purification of endo-(1-->4)-beta-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid of Geotrichum candidum 3C, grown for three days, is described. The enzyme purified 23-fold had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30-45 degrees C for 1 h. With xylan from beach wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50 degrees C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10, 12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0–pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60–70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k _cat /K _m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Carbon Source Control of Cellobiohydrolase I and II Formation by Trichoderma reesei

Regulation of the formation and secretion of two cellulase components from Trichoderma reesei QM 9414, cellobiohydrolases I and II (CBH I and CBH II, respectively), by the carbon source was investigated. With monoclonal antibodies against CBH I and CBH II it was found that during cultivation on carbon sources which enable fast growth (glucose, glycerol, and fructose), no formation of CBH I occurred, whereas low levels of CBH II were formed. Lactose and cellulose, which allow comparably slower growth, promoted the formation of both CBH I and CBH II. However, noncarbohydrate carbon sources as citrate or acetate, which also enable only slow growth, did not promote the formation of CBH I or CBH II. The addition of glucose or glycerol to lactose- or cellulose-pregrown mycelia, on the other hand, only partially reduced the formation of CBH I. This reduction was also achieved by several other metabolizable and nonmetabolizable carbon compounds, e.g., fructose, galactose, β-methylglucoside, 2-deoxyglucose, and rhamnose, as well as by transfer to no carbon source at all. This result indicates that the control of CBH I synthesis by the carbon source is due to induction and not to repression. The use of cycloheximide and 5-fluorouracil as inhibitors at and before translation, respectively, revealed a half-life for CBH I mRNA of at least several hours, which may, at least in part, account for the prolonged synthesis of some CBH I under these conditions. Northern (RNA) hybridization with full copies of cbh1 and cbh2 genes indicated that the control of CBH I and CBH II biosyntheses by the carbon source operates mainly at the pretranslational level. We conclude that the low rate of cellulase synthesis on glucose and some other carbon sources is due to the lack of an inducer and not to carbon source repression.     

Properties of a chimeric glucose dehydrogenase improved by site directed mutagenesis

Glucose dehydrogenase, a membrane bound enzyme oxidizing glucose to gluconic acid in the periplasmic space of Gram-negative bacteria plays a key role in mineral phosphate solubilization and is also an industrially important enzyme, being used as a glucose biosensor. A chimeric glucose dehydrogenase (ES chimera) encoding the N-terminal transmembrane domain from Escherichia coli and the C-terminal periplasmic domain from Serratia marcescens was constructed and the expression was studied on MacConkey glucose medium. The phosphate solubilizing ability of the chimeric GDH was also evaluated, substantiating the role of GDH in mineral phosphate solubilization (MPS). Four mutants of ES chimeric GDH were generated by site directed mutagenesis and the enzyme properties studied. Though the substrate affinity was unaltered for E742K and Y771M, the affinity of H775A and EYH/KMA to glucose and galactose decreased marginally and the affinity to maltose increased. Though Y771M showed a decreased GDH activity there was an increase in the heat tolerance. All the mutants showed an increase in the EDTA tolerance. The triple mutant EYH/KMA showed improved heat and EDTA tolerance and also an increase in affinity to maltose over the ES chimeric GDH.     

Increased affinity and stability of an anti-HIV-1 envelope immunotoxin by structure-based mutagenesis

HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env). To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env. Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V(H) increased the potency of the immunotoxin by approximately 10-fold in a cell-killing assay. Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin. Conversion to a disulfide-linked two-chain format further stabilized the protein. N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.     

Study of substrate specificity of human aromatase by site directed mutagenesis.

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.     

Efficient site directed in vitro mutagenesis using ampicillin selection.

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.