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| Biochemistry, 2005 Jan 11, 44(1), 387 - 397 Biochemical Characterization and Identification of the Catalytic Residues of a Family 43 beta-d-Xylosidase from Geobacillus stearothermophilus T-6; Shallom D et al.; beta-d-Xylosidases are hemilcellulases that hydrolyze short xylooligosaccharides into xylose units . Here, we describe the characterization and kinetic analysis of a family 43 beta-xylosidase from Geobacillus stearothermophilus T-6 (XynB3) . Enzymes in this family use an inverting single-displacement mechanism with two conserved carboxylic acids, a general acid, and a general base . XynB3 was most active at 65 degrees C and pH 6.5, with clear preference to xylose-based substrates . Products analysis indicated that XynB3 is an exoglycosidase that cleaves single xylose units from the nonreducing end of xylooligomers . On the basis of sequence homology, amino acids Asp15 and Glu187 were suggested to act as the general-base and general-acid catalytic residues, respectively . Kinetic analysis with substrates bearing different leaving groups showed that, for the wild-type enzyme, the k(cat) and k(cat)/K(m) values were only marginally affected by the leaving-group reactivity, whereas for the E187G mutant, both values exhibited significantly greater dependency on the pK(a) of the leaving group . The pH-dependence activity profile of the putative general-acid mutant (E187G) revealed that the protonated catalytic residue was removed . Addition of the exogenous nucleophile azide did not affect the activities of the wild type or the E187G mutant but rescued the activity of the D15G mutant . On the basis of thin-layer chromatography and (1)H NMR analyses, xylose and not xylose azide was the only product of the accelerated reaction, suggesting that the azide ion does not attack the anomeric carbon directly but presumably activates a water molecule . Together, these results confirm the suggested catalytic role of Glu187 and Asp15 in XynB3 and provide the first unequivocal evidence regarding the exact roles of the catalytic residues in an inverting GH43 glycosidase. Mol Microbiol, 2005 Jan, 55(1), 197 - 205 The three S-layer-like homology motifs of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer; Huber C et al.; Summary The S-layer protein SbpA of Bacillus sphaericus CCM 2177 recognizes a pyruvylated secondary cell wall polymer (SCWP) as anchoring structure to the peptidoglycan-containing layer . Data analysis from surface plasmon resonance (SPR) spectroscopy revealed the existence of three different binding sites with high, medium and low affinity for rSbpA on SCWP immobilized to the sensor chip . The shortest C-terminal truncation with specific affinity to SCWP was rSbpA(31-318) . Surprisingly, rSbpA(31-202) comprising the three S-layer-like homology (SLH) motifs did not bind at all . Analysis of the SbpA sequence revealed a 58-amino-acid-long SLH-like motif starting 11 amino acids after the third SLH motif . The importance of this motif for reconstituting the functional SCWP-binding domain was further demonstrated by construction of a chimaeric protein consisting of the SLH domain of SbsB, the S-layer protein of Geobacillus stearothermophilus PV72/p2 and the C-terminal part of SbpA . In contrast to SbsB or its SLH domain which did not recognize SCWP of B . sphaericus CCM 2177 as binding site, the chimaeric protein showed specific affinity . Deletion of 213 C-terminal amino acids of SbpA had no impact on the square (p4) lattice structure, whereas deletion of 350 amino acids was linked to a change in lattice type from square to oblique (p1). J Appl Microbiol, 2004, 97(5), 1015 - 20 Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone; Uma Maheswar Rao JL et al.; AIM: Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220 . METHODS AND RESULTS: The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C . The bacterial pellets were obtained after growing G . thermoleovorans at different temperatures, and used in determining total PA . The cell-free culture filtrates were used in alpha-amylase assays . During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually . The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures . Enzyme titre enhanced up to 70 degrees C, and thereafter declined . Extracellular enzyme and protein levels declined in the presence of exogenously added PA . The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm) . Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it . CONCLUSIONS: The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase . Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm . SIGNIFICANCE AND IMPACT OF THE STUDY: A substantial increase in the synthesis and secretion of alpha-amylase was attained in G . thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG. J Bacteriol, 2004 Oct, 186(20), 6928 - 37 Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases; Shallom D et al.; The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability . alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes . Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A) . Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability . Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization . Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily . It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies . To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N) . The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme . Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C . It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity . Structural and mechanistic explanations for these effects are discussed. Biochimie, 2004 Jul, 86(7), 481 - 5 Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V; Saavedra CP et al.; The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E . coli and the recombinant protein was purified and characterized . The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes . UV-visible spectra showed absorption bands at 279 and 410 nm . The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan . The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein . Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site . The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C . At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia . At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed. Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11275 - 80 Epub 2004 Jul 26. Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry; Zolotnitsky G et al.; Relating thermodynamic parameters to structural and biochemical data allows a better understanding of substrate binding and its contribution to catalysis . The analysis of the binding of carbohydrates to proteins or enzymes is a special challenge because of the multiple interactions and forces involved . Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (DeltaHa) and allows the determination of the binding constant (free energy), entropy, and stoichiometry . In this study, we used ITC to elucidate the binding thermodynamics of xylosaccharides for two xylanases of family 10 isolated from Geobacillus stearothermophilus T-6 . The change in the heat capacity of binding (DeltaCp = DeltaH/DeltaT) for xylosaccharides differing in one sugar unit was determined by using ITC measurements at different temperatures . Because hydrophobic stacking interactions are associated with negative DeltaCp, the data allow us to predict the substrate binding preference in the binding subsites based on the crystal structure of the enzyme . The proposed positional binding preference was consistent with mutants lacking aromatic binding residues at different subsites and was also supported by tryptophan fluorescence analysis. Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1461 - 3 Epub 2004 Jul 21. Crystallization and preliminary crystallographic analysis of a thermostable family 52 beta-D-xylosidase from Geobacillus stearothermophilus T-6; Czjzek M et al.; Beta-D-xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units . In this study, the first crystallization and preliminary X-ray analysis of a family 52 glycoside hydrolase, the beta-D-xylosidase (XynB2) from Geobacillus stearothermophilus T-6, is described . XynB2 is a dimeric protein consisting of two identical subunits of 705 amino acids with a calculated molecular weight of 79 894 Da . XynB2 was crystallized by the hanging-drop vapour-diffusion method and the crystals were found to belong to space group P1, with unit-cell parameters a = 80.6, b = 97.5, c = 107.2 A, alpha = 107.4, beta = 98.2, gamma = 106.6 degrees . The native crystals diffracted X-rays to a resolution of 2.0 A. J Bacteriol, 2004 Aug, 186(15), 4894 - 902 A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101; Kitagawa W et al.; p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides . To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited . In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized . The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp . strain BA-5-17 (76%), respectively . The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC . Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment . The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol . Furthermore, the crude cell extract of E . coli containing pETnpcC converted hydroxyquinol to maleylacetate . These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase . The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source . These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R . opacus SAO101. Anal Biochem, 2004 Aug 1, 331(1), 106 - 14 Identification of biogenic organotellurides in Escherichia coli K-12 headspace gases using solid-phase microextraction and gas chromatography; Swearingen JW Jr et al.; Escherichia coli JM109 cells, expressing the genes encoded in a 3.8-kb chromosomal DNA fragment from Geobacillus stearothermophilus V, produced volatile organotellurium compounds which were released into the headspace gas above liquid cultures when amended with tellurite anions in micromolar amounts . Headspace sampling was achieved using gas-syringe extraction or solid-phase microextraction using carboxen-polydimethysiloxane fibers . In addition to dimethyl telluride and dimethyl ditelluride, two new organometalloidal compounds were detected using gas chromatograph with mass spectrometric or fluorine-induced chemiluminescence detection . These compounds are methanetellurol and dimethyl tellurenyl sulfide . The significance of these findings with regard to the current knowledge about bacterial tellurite resistance is discussed. J Appl Microbiol, 2004, 97(2), 402 - 9 An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range; Kida N et al.; AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures . METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N . Kida, Y . Mochizuki and F . Taguchi, Microbiology and Immunology 2003; 47: 279-283) . The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3 . The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature . The KMT reagent had many practical advantages, i.e . activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures . The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen . CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores. PDA J Pharm Sci Technol, 2004 May-Jun, 58(3), 130 - 46 Development of a sterilizing in-place application for a production machine using Vaporized Hydrogen Peroxide; Mau T et al.; The use of steam in sterilization processes is limited by the implementation of heat-sensitive components inside the machines to be sterilized . Alternative low-temperature sterilization methods need to be found and their suitability evaluated . Vaporized Hydrogen Peroxide (VHP) technology was adapted for a production machine consisting of highly sensitive pressure sensors and thermo-labile air tube systems . This new kind of "cold" surface sterilization, known from the Barrier Isolator Technology, is based on the controlled release of hydrogen peroxide vapour into sealed enclosures . A mobile VHP generator was used to generate the hydrogen peroxide vapour . The unit was combined with the air conduction system of the production machine . Terminal vacuum pumps were installed to distribute the gas within the production machine and for its elimination . In order to control the sterilization process, different physical process monitors were incorporated . The validation of the process was based on biological indicators (Geobacillus stearothermophilus) . The Limited Spearman Karber Method (LSKM) was used to statistically evaluate the sterilization process . The results show that it is possible to sterilize surfaces in a complex tube system with the use of gaseous hydrogen peroxide . A total microbial reduction of 6 log units was reached. Protein Expr Purif, 2004 Jul, 36(1), 150 - 6 Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli; Jeong KJ et al.; High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated . For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used . To develop an optimal host-vector system, several different recombinant E . coli strains were compared for leptin production . In flask cultures, E . coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins) . By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin . For the large-scale production of human leptin, fed-batch cultures of recombinant E . coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions . Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively . These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner. J Immunol, 2004 Jun 1, 172(11), 6642 - 8 A novel approach to specific allergy treatment: the recombinant fusion protein of a bacterial cell surface (S-layer) protein and the major birch pollen allergen Bet v 1 (rSbsC-Bet v 1) combines reduced allergenicity with immunomodulating capacity; Bohle B et al.; Counterregulating the disease-eliciting Th2-like immune response of allergen-specific Th lymphocytes by fostering an allergen-specific Th1-like response is a promising concept for future immunotherapy of type I allergy . The use of recombinant allergens combined with more functional adjuvants has been proposed . In this respect, we present a novel approach . The gene sequence encoding the major birch pollen allergen, Bet v 1, was fused with the gene encoding the bacterial cell surface (S-layer) protein of Geobacillus stearothermophilus, resulting in the recombinant protein, rSbsC-Bet v 1 . rSbsC-Bet v 1 contained all relevant Bet v 1-specific B and T cell epitopes, but was significantly less efficient to release histamine than rBet v 1 . In cells of birch pollen-allergic individuals, rSbsC-Bet v 1 induced IFN-gamma along with IL-10, but no Th2-like response, as observed after stimulation with Bet v 1 . Intracellular cytokine staining revealed that rSbsC-Bet v 1 promoted IFN-gamma-producing Th cells . Moreover, rSbsC-Bet v 1 induced IFN-gamma synthesis in Bet v 1-specific Th2 cell clones, and importantly, increased IL-10 production in these cells . In conclusion, genetic fusion of an allergen to S-layer proteins combined reduced allergenicity with immunomodulatory capacity . The strategy described in this work may be generally applied to design vaccines for specific immunotherapy of type I allergy with improved efficacy and safety. Gene, 2004 Mar 31, 329, 187 - 95 Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus; Ewis HE et al.; Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones . The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison . The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da . Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli . The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30 . The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol) . Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively . With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1) . Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family . The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries. J Bacteriol, 2004 Mar, 186(6), 1758 - 68 Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology; Mader C et al.; The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology . The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid . For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used . Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding . The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized . Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites . The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall Kd of 7.7 x 10(-7) M . Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (Kd = 2.6 x 10(-5) M), medium (Kd = 6.1 x 10(-8) M), and high (Kd = 6.7 x 10(-11) M) affinities . Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH . Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated. Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 583 - 5 Epub 2004 Feb 25. Crystallization and preliminary X-ray analysis of family 39 beta-D-xylosidase from Geobacillus stearothermophilus T-6; Czjzek M et al.; beta-D-Xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units . In this study, the crystallization and preliminary X-ray analysis of the beta-D-xylosidase (XynB1) from Geobacillus stearothermophilus T-6, a family 39 glycoside hydrolase, are described . XynB1 is a tetrameric protein consisting of four identical subunits of 503 amino acids and with a calculated molecular weight of 58 001 Da . Both the native and the selenomethionine-containing XynB1 were crystallized by the hanging-drop vapour-diffusion method and the crystals were found to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 92.7, b = 165.7, c = 311.0 A . The native crystals diffracted X-rays to a resolution of 2.1 A. Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 545 - 9 Epub 2004 Feb 25. A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolution; Bar M et al.; Xylanases (1,4-beta-D-xylan xylanhydrolases; EC 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans . The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity . An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized . Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution . A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics . The new crystals belong to a C-centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1 A, beta = 119.7 degrees . These crystals diffract X-rays to better than 1.5 A resolution, showing a very clear diffraction pattern of relatively high quality . The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas . A full diffraction data set to 1.45 A resolution (94.1% completeness, R(merge) = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95 K using synchrotron radiation . Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6 . They were used for a full measurement of 1.8 A resolution diffraction data at 100 K (90.9% completeness; R(merge) = 5.0%) . These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability. Biosci Biotechnol Biochem, 2004 Jan, 68(1), 96 - 103 High level expression of thermostable lipase from Geobacillus sp . strain T1; Leow TC et al.; A thermostable extracellular lipase of Geobacillus sp . strain T1 was cloned in a prokaryotic system . Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues . The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids . Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly . Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively . Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization . The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively . It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11) . This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
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