Thermostabilization of carboxymethylcellulase from Aspergillus niger by carboxyl group modification

The carboxyl groups of purified carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 were modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). The half-lives of GAM15 at different temperatures were significantly enhanced whereas those of GAM75 were reduced as compared with the native CMCase. The activation energies of denaturation of native, GAM15 and GAM75 were 40, 35 and 59kJ mol respectively. Native CMCase and GAM15 showed no compensation effect, whereas native and GAM75 gave temperature of compensation of 44¡C. Gibb's free energy of activation for denaturation (DG*) of GAM15 was increased as compared with native CMCase. Surprisingly the entropies (DS*) of activation for denaturation were negative for native and GAM75 and decreased further for GAM15 between the temperature range of 45 to 65¡C. A possible explanation for the thermal inactivation of native and increased thermal stability of GAM15 is also discussed.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Kinetics of improved production and thermostability of an intracellular β-glucosidase from a mutant-derivative of Cellulomonas biazotea

The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism. The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)). The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme.     

Enhancement of the viscometric endocellulase activity of Polyporus arcularius CMCase IIIa by cellobiose and cellooligosaccharides

Purification and viscometric characterization of three CMCases from Polyporus arcularius were carried out. The three CMCases, I, II, and IIIa, were estimated to have molecular masses of 39.1 kDa, 36.3 kDa, and 24.3 kDa, respectively. The addition of cellobiose and cellooligosaccharides to the reaction mixtures of CMCase I and II inhibited viscometric endocellulase activity. Following the addition of 20 mM cellobiose, CMCase I and II activities fell to about 30%–36% of their activity in the absence of cellobiose. CMCase IIIa activity, on the other hand, increased in proportion to the increase in cellobiose or cellooligo-saccharide concentration. Maximal enhancement of CMCase IIIa activity was observed following the addition of cellobiose, whereas less enhancement was observed with cellooligosaccharides spanning more than two glucoside units. The addition of 20 mM cellobiose resulted in an increase greater than 500% in CMCase IIIa activity. Inhibition of CMCase I and II by cellobiose and cellooligosaccharides may be the result of competition between the substrate and the reaction products. One of the reaction products of CMCase IIIa may bind to a site other than the active site of the enzyme, thus enhancing CMCase IIIa activity.     

Stopped-flow kinetic studies of the cellulase-catalyzed conversion of cellulose to glucose

The kinetics of the cellulase-catalyzed conversion of soluble cellulose into glucose have been studied over a range of substrate concentrations and temperatures, and at pH values ranging from 4.75 to 7.0. Lineweaver-Burk plots were linear and led to V = 6.2muM/s and K(m) = 13.1 mM at pH 5.8 and 25.0 degrees C. The pK values corresponding to the free enzyme are 4.8 and 6.8 and are consistent with carboxyl and imidazole groups as the active ionizing species. These pK values were little changed in the enzyme-substrate intermediate that reacts in the ratedetermining step, suggesting that the ionizing groups are still free in this intermediate. The activation energy corresponding to V/K(m) is 80.6 kJ/mol, and that corresponding to V is 38.7 kJ/mol. The corresponding entropies of activation are 21 J K(-1) mol(-1) and -157 J K(-1) mol(-1), respectively.     

Gas Chromatographic Determination of Optical Purity of a Chiral Derivatization Reagent,N-Acyl-l-prolyl Chloride

Two endo-type cellulases, tentatively called carboxymethyl cellulases (CMCases) I and II, were purified by gel filtration, ion-exchange chromatography, affinity chromatography, and chromato-focusing from a culture supernatant of Penicillium purpurogenum. Their homogeneity was verified by analytical polyacrylamide gel electrophoresis. The molecular weights of CMCases I and II, estimated by gel filtration, were 72,000 and 50,000, respectively. CMCases I and II contained about 12% and 8% carbohydrate, and had isoelectric points of 4.3 and 3.9, respectively. CMCase I produced cellobiose, glucose, and a trace amount of cellotriose from H₃PO₄-swollen cellulose and Avicel (microcrystalline cellulose), while CMCase II produced cellobiose and cellotriose with a small amount of glucose and cellotetraose. The products from reduced cellopentaose by both enzymes were released predominantly in the β-configuration. CMCase II appeared to act in more random fashion than I against carboxymethyl cellulose. These results suggest that both enzymes attack insoluble cellulose randomly, although there are some differences in the mode of hydrolytic action.     

Breakdown kinetics of the tri-chromium(III) oxo acetate cluster ([Cr(3)O(OAc)(6)]+) with some ligands of biological interest

Kinetics for the breakdown of the trinuclear chromium acetate cluster, [Cr(3)O(OAc)(6)](+), with a series of monoprotic and diprotic ligands in weakly acidic aqueous media (pH approximately 4 or approximately 5) have been investigated spectrophotometrically at 40-60 degrees C. The results point to an ion-pair equilibrium as the first step followed by associative interchange mechanism forming the mononuclear product of the reaction. Pseudo-first-order rates were determined from absorbance data and associated activation parameters were calculated using the Eyring equation. Enthalpy and entropy terms of the reactions (e.g., histidine, DeltaH(double dagger) = 75 +/- 15 kJ mol(-1), DeltaS(double dagger) = -130 +/- 25 J K(-1) mol(-1); lactic acid, DeltaH(double dagger) = 66 +/- 13 kJ mol(-1), DeltaS(double dagger) = -155 +/- 30 J K(-1) mol(-1); glycine, DeltaH(double dagger) = 31 +/- 6 kJ mol(-1), DeltaS(double dagger) = -225 +/- 45 J K(-1) mol(-1)) are consistent with an associative interchange (I(a)) mechanism, and produce a linear isokinetic plot (slope = 50 degrees C). Rates and activation parameters are comparable to those of substitution reactions of the chromium(III) hexaaqua cation. Other ligands studied included malonic acid and the amino acid, aspartic acid. Observed rates are faster than water exchange rates, but typically slower than anion substitution rates, and indicate that trinuclear chromium(III) clusters are expected to be kinetically stable in neutral to slightly acidic conditions.     

The cellular location of Prevotella ruminicola beta-1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.     

Purification and some properties of a carboxymethyl cellulase from Favolus arcularius

Favolus arcularius, a wood-rotting basidiomycete, produced carboxymethyl cellulose-hydrolyzing enzymes (CMCases) in culture media. Three main peaks of CMCase activity were separated as CMCase I, II and III at pHs from 4.4 to 5.2 by isoelectric focusing. Further, CMCase IIIa was purified from the CMCase III fraction. The molecular weight of CMCase IIIa was determined to be about 28,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was not active on avicel, cellobiose and laminarin, but could randomly hydrolyze cellooligosaccharides to form G₁ and G₂ units as the end products. The apparent K_m value of the enzyme against CMC was 0.28%.     

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.     

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Partial characterization of the extracellular carboxymethylcellulase activity produced by the rumen bacterium Bacteroides succinogenes

In cultures of Bacteroides succinogenes, in which cellulose was the source of carbohydrate, from 70 to 80% of the carboxymethylcellulase (CMCase) activity was present in the culture fluid. The crude extracellular enzyme readily hydrolyzed acid-swollen cellulose with the production of glucose and cellobiose. Of this extracellular CMCase, 50-62% was associated with sedimentable membrane fragments, 9-13% with nonsedimentable material with a molecular weight greater than 4 X 10(6), and 28-38% with molecules having a molecular weight of approximately 45 000. Polyacrylamide gel electrophoresis (PAGE), in the presence of sodium dodecyl sulfate, revealed that both the nonsedimentable and the sedimentable fraction had complex protein compositions. The nonsedimentable and sedimentable CMCase fractions, after treatment with Triton X-100, were subjected to PAGE in the presence of 0.2% (w/v) Triton X-100. The results indicated the presence of fast- and slow-migrating CMCases in the former, and of a slow-migrating CMCase in the latter. An apparently uncharged CMCase, which probably corresponded to the slow-migrating component by PAGE, was partially purified from the concentrated culture supernate by solubilization in Triton X-100 and chromatography on DEAE--Sepharose, CM--Sepharose, and Phenyl--Sepharose. The partially purified CMCase had a pH optimum of 5.6-6.6 and a temperature optimum of 50 degrees C.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with iota-carrageenan

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.     

Heat capacity effects in protein folding and ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics

Large "anomalous" heat capacity (DeltaC(p)) effects are a common feature of the thermodynamics of biomolecular interactions in aqueous solution and, as a result of the improved facility for direct calorimetric measurements, there is a growing body of experimental data for such effects in protein folding, protein-protein and protein-ligand interactions. Conventionally such heat capacity effects have been ascribed to hydrophobic interactions, and there are some remarkably convincing demonstrations of the usefulness of this concept. Nonetheless, there is also increasing evidence that hydrophobic interactions are not the only possible source of such effects. Here we re-evaluate the possible contributions of other interactions to the heat capacity changes to be expected for cooperative biomolecular folding and binding processes, with particular reference to the role of hydrogen bonding and solvent water interactions. Simple models based on the hydrogen-bonding propensity of water as a function of temperature give quantitative estimates of DeltaC(p) that compare well with experimental observations for both protein folding and ligand binding. The thermodynamic contribution of bound waters in protein complexes is also estimated. The prediction from simple lattice models is that trapping of water in a complex should give more exothermic binding (DeltaDeltaH-6 to -12 kJ mol(-1)) with lower entropy (DeltaDeltaS(0) approximately -11 J mol(-1) K(-1)) and more negative DeltaC(p) (by about -75 J mol(-1) K(-1)) per water molecule. More generally, it is clear that significant DeltaC(p) effects are to be expected for any macromolecular process involving a multiplicity of cooperative weak interactions of whatever kind.     

Thermodynamic characterization of ligand-induced conformational changes in UDP-N-acetylglucosamine enolpyruvyl transferase

The binding of UDP-N-acetylglucosamine (UDPNAG) to the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) was studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDPNAG and also in its absence as demonstrated by MALDI mass spectrometry. The covalent attachment of fosfomycin affects the thermodynamic parameters of UDPNAG binding significantly: In the absence of fosfomycin the binding of UDPNAG is enthalpically driven (DeltaH = -35.5 kJ mol(-1) at 15 degrees C) and opposed by an unfavorable entropy change (DeltaS = -25 J mol(-1) K(-1)). In the presence of covalently attached fosfomycin the binding of UDPNAG is entropically driven (DeltaS = 187 J mol(-1)K(-1) at 15 degrees C) and associated with unfavorable changes in enthalpy (DeltaH = 28.8 kJ mol(-1)). Heat capacities for UDPNAG binding in the absence or presence of fosfomycin were -1.87 and -2.74 kJ mol(-1) K(-1), respectively, indicating that most ( approximately 70%) of the conformational changes take place upon formation of the UDPNAG-MurA binary complex. The major contribution to the heat capacity of ligand binding is thought to be due to changes in the solvent-accessible surface area. However, associated conformational changes, if any, also contribute to the experimentally measured magnitude of the heat capacity. The changes in solvent-accessible surface area were calculated from available 3D structures, yielding a DeltaC(p) of -1.3 kJ mol(-1) K(-1); i.e., the experimentally determined heat capacity exceeds the calculated one. This implies that other thermodynamic factors exert a large influence on the heat capacity of protein-ligand interactions.     

Two-electron reduction of quinones by rat liver NAD(P)H:quinone oxidoreductase: quantitative structure-activity relationships

Mammalian NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) catalyzes the two-electron reduction of quinones and plays one of the main roles in the bioactivation of quinoidal drugs. In order to understand the enzyme substrate specificity, we have examined the reactions of rat NQO1 with a number of quinones with available potentials of single-electron (E(1)(7)) reduction and pK(a) of their semiquinones. The hydride transfer potentials (E(7)(H(-))) were calculated from the midpoint potentials of quinones and pK(a) of hydroquinones. Our findings imply that benzo- and naphthoquinones with a van der Waals volume (VdWvol) < or = 200 A(3) are much more reactive than glutathionyl-substituted naphthoquinones, polycyclic quinones, and FMN (VdWvol>200 A(3)) with the same reduction potentials. The entropies of activation (DeltaS(not equal)) in the reduction of "fast" oxidants are equal to -84 to -76 J mol(-1) K(-1), whereas in the reduction of "slow" oxidants Delta S(not equal)=-36 to -11 J mol(-1) K(-1). The large negative Delta S(not equal) in the reduction of fast oxidants may be explained by their better electronic coupling with reduced FAD or the formation of charge-transfer complexes, since fast oxidants bind at the dicumarol binding site, whereas the binding of some slow oxidants outside it has been demonstrated. The reactivity of quinones may be equally well described in terms of the three-step (e(-),H(+),e(-)) hydride transfer, using E(1)(7), pK(a)(QH*), and VdWvol as correlation parameters, or in terms of single-step (H(-)) hydride transfer, using E(7)(H(-)) and VdWvol in the correlation. The analysis of NQO1 reactions with single-electron acceptors and quinones using an "outer-sphere" electron transfer model points to the possibility of a three-step hydride transfer.     

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(?)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.     

Studies on the isothermal crystallization of D-glucose and cellulose oligosaccharides by differential scanning calorimetry.

Isothermal crystallization from the glassy state of D-glucose and cellulose oligosaccharides (e.g., cellobiose, cellotriose, and cellotetraose) has been studied by differential scanning calorimetry. The crystallization of amorphous D-glucose and oligosaccharides was very difficult in the absence of traces of water. Amorphous cellobiose and cellotetraose crystallized far more rapidly than amorphous D-glucose and cellotriose. The activation energy for the crystallization of cellobiose and cellotetraose was approximately 10-12 kJ. mol(-1), while that for D-glucose and cellotriose was approximately 1-2 kJ. mol(-1). An odd-even effect seemed to be associated with the crystallization process of these saccharides.