A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes.

BACKGROUND: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. RESULTS: In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate-phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. CONCLUSIONS: We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis.

Characterization of the arabinoxylan-degrading machinery of the thermophilic bacterium Herbinix hemicellulosilytica-Six new xylanases, three arabinofuranosidases and one xylosidase.

Herbinix hemicellulosilytica is a newly isolated, gram-positive, anaerobic bacterium with extensive hemicellulose-degrading capabilities obtained from a thermophilic biogas reactor. In order to exploit its potential as a source for new industrial arabinoxylan-degrading enzymes, six new thermophilic xylanases, four from glycoside hydrolase family 10 (GH10) and two from GH11, three arabinofuranosidases (1x GH43, 2x GH51) and one ß-xylosidase (GH43) were selected. The recombinantly produced enzymes were purified and characterized. All enzymes were active on different xylan-based polysaccharides and most of them showed temperature-vs-activity profiles with maxima around 55-65°C. HPAEC-PAD analysis of the hydrolysates of wheat arabinoxylan and of various purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) was used to investigate their substrate and product specificities: among the GH10 xylanases, XynB showed a different product pattern when hydrolysing AXOS compared to XynA, XynC, and XynD. None of the GH11 xylanases was able to degrade any of the tested AXOS. All three arabinofuranosidases, ArfA, ArfB and ArfC, were classified as type AXH-m,d enzymes. None of the arabinofuranosidases was able to degrade the double-arabinosylated xylooligosaccharides XA2+3XX. ß-Xylosidase XylA (GH43) was able to degrade unsubstituted XOS, but showed limited activity to degrade AXOS.

Differences in biomass degradation between newly isolated environmental strains of Clostridium thermocellum and heterogeneity in the size of the cellulosomal scaffoldin.

Cellulolytic bacterial strains with high activity were isolated from cellulose degrading enrichment cultures derived from thermophilic biogas plants and environmental samples. The 16S rRNA gene sequences of the strains revealed >99.8% sequence identity and affiliation with the species Clostridium thermocellum. The strains differed in their ability to degrade crystalline cellulose, especially at an elevated temperature of up to 67 °C and at relatively low pH values (pH 6.5). To evaluate the influence of amino acid sequences on the discrepancies in cellulose degradation efficacy, the gene for the major cellulosomal component CelR was sequenced for all strains. The sequences were found to be almost identical (>99%). In contrast, the cellulosomal scaffoldin gene cipA showed more differences in the amino acid sequence and contained 8 or 9 cohesin modules, which indicated a different size of the cellulosome depending on the isolate. Based on MALDI-TOF MS analysis the relative abundance of important cellulosomal enzyme classes was determined. The strains with better biomass degradation properties (BC1 and NB2) had a significantly higher fraction of xylanases.

The consequence of an additional NADH dehydrogenase paralog on the growth of Gluconobacter oxydans DSM3504.

Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O2 and CO2 transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

Rapid access to genes of biotechnologically useful enzymes by partial genome sequencing: the thermoalkaliphile Anaerobranca gottschalkii.

Anaerobranca gottschalkii strain LBS3 T is an extremophile living at high temperature (up to 65 degrees C) and in alkaline environments (up to pH 10.5). An assembly of 696 DNA contigs representing about 96% of the 2.26-Mbp genome of A. gottschalkii has been generated with a low-sequence-coverage shotgun-sequencing strategy. The chosen sequencing strategy provided rapid and economical access to genes encoding key enzymes of the mono- and polysaccharide metabolism, without dilution of spare resources for extensive sequencing of genes lacking potential economical value. Five of these amylolytic enzymes of considerable commercial interest for biotechnological applications have been expressed and characterized in more detail after identification of their genes in the partial genome sequence: type I pullulanase, cyclodextrin glycosyltransferase (CGTase), two alpha-amylases (AmyA and AmyB), and an alpha-1,4-glucan-branching enzyme.

Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea.

Thermoacidophiles are prokaryotic microorganisms with the stunning capability to survive and multiply at extremely low pH and simultaneously at high temperatures. The mechanisms by which these organisms, exclusively members of the Archaea, cope with their harsh surroundings are poorly understood. The genome sequences of several representatives of the thermoacidophilic genera Picrophilus, Thermoplasma and Sulfolobus have recently become available. Genome-wide comparison has revealed a number of features as possible facets of the overall acidophilic survival strategy of the most thermoacidophilic organisms known, such as a high ratio of secondary over primary transport systems, the composition of the respiratory chain, and the frequent genetic input via lateral gene transfer (LGT) during evolution.

Genome sequence of Picrophilus torridus and its implications for life around pH 0.

The euryarchaea Picrophilus torridus and Picrophilus oshimae are able to grow around pH 0 at up to 65 degrees C, thus they represent the most thermoacidophilic organisms known. Several features that may contribute to the thermoacidophilic survival strategy of P. torridus were deduced from analysis of its 1.55-megabase genome. P. torridus has the smallest genome among nonparasitic aerobic microorganisms growing on organic substrates and simultaneously the highest coding density among thermoacidophiles. An exceptionally high ratio of secondary over ATP-consuming primary transport systems demonstrates that the high proton concentration in the surrounding medium is extensively used for transport processes. Certain genes that may be particularly supportive for the extreme lifestyle of P. torridus appear to have been internalized into the genome of the Picrophilus lineage by horizontal gene transfer from crenarchaea and bacteria. Finally, it is noteworthy that the thermoacidophiles from phylogenetically distant branches of the Archaea apparently share an unexpectedly large pool of genes.

[Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: molekuliarnaia struktura lokusa.

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from Thermotoga maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the E. coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the alpha-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the Thermotoga maritima cofactor-dependent alpha-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.

The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity.

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from alpha-1,4-linked glucans or malto-oligosaccharides to other alpha-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (beta/alpha)(8) barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (beta/alpha)(8) barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites -2 and -1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the -2 subsite by the side-chain of Lys151 and the stretch of residues 314-317, providing an explanation for the strict transfer specificity of MTase.

Crystallization and preliminary X-ray crystallographic studies on 4-alpha-glucanotransferase from Thermotoga maritima.

Thermotoga maritima 4-alpha-glucanotransferase (GTase), a 52 kDa molecular-weight amylolytic enzyme, has been crystallized by the hanging-drop vapour-diffusion method using PEG monomethylether 5000 as a precipitating agent. A complete data set has been collected to 2.6 A resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 92.6, b = 180.3, c = 199.2 A.

Thermophilic adaptation of proteins.

Hyperthermophilic organisms optimally grow close to the boiling point of water. As a consequence, their macromolecules must be much more thermostable than those from mesophilic species. Here, proteins from hyperthermophiles and mesophiles are compared with respect to their thermodynamic and kinetic stabilities. The known differences in amino acid sequences and three-dimensional structures between intrinsically thermostable and thermolabile proteins will be summarized, and the crucial role of electrostatic interactions for protein stability at high temperatures will be highlighted. Successful attempts to increase the thermostability of proteins, which were either based on rational design or on directed evolution, are presented. The relationship between high thermo-stability of enzymes from hyperthermophiles and their low catalytic activity at room temperature is discussed. Not all proteins from hyperthermophiles are thermostable enough to retain their structures and functions at the high physiological temperatures. It will be shown how this shortcoming can be surpassed by extrinsic factors such as large molecular chaperones and small compatible solutes. Finally, the potential of thermostable enzymes for biotechnology is discussed.

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Molecular characterization of the Sinorhizobium meliloti nlpD gene.

The Sinorhizobium meliloti nlpD gene consists of 1,539 nucleotides and codes for 512 amino acids. Expression of the nlpD gene as a histidine-tagged protein in Escherichia coli resulted in the production of a 57-kDa protein. The deduced polypeptide sequence of NlpD contains one unusual hexamer repeat (KVQRGQ), one tetramer (TVTV) and two direct and inverted trimer repeats (KAA, AAK). The N-terminal amino acid residues displayed similarity with signal peptides of secreted bacterial lipoproteins. Mutations of the S. meliloti nlpD gene caused decreased survival of cells in the stationary phase.

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent alpha-glucosidase.

The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.

Crystallization and preliminary X-ray crystallographic studies on maltosyltransferase from Thermotoga maritima.

Thermotoga maritima maltosyltransferase (MTase) is a 73.7 kDa molecular weight amylolytic enzyme which catalyzes the transfer of maltosyl units from maltodextrins or starch to suitable acceptors. Crystals of recombinant MTase have been obtained by the hanging-drop vapour-diffusion method using ammonium phosphate as a precipitating agent. The crystals belong to space group P4(1)22 or its enantiomorph P4(3)22, with unit-cell parameters a = b = 148.7, c = 106.7 A. The asymmetric unit appears to contain one subunit, corresponding to a very low packing density of 4.0 A(3) Da(-1). The crystals diffract X-rays to at least 2.4 A resolution on a synchrotron-radiation source.

The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage beta-1,3/beta-1, 4-glucan.

Thermotoga maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1-C2). Its catalytic domain (-B-) is flanked by duplicated non-catalytic domains. The C-terminal repeated domains represent cellulose-binding domains (CBDs). Xylanase domains related to the N-terminal domains of XynA (A1-A2) are called thermostabilizing domains because their deletion normally leads to increased thermosensitivity of the enzymes. It was found that a glutathione-S-transferase (GST) hybrid protein (GST-A1A2) containing both A-domains of XynA can interact with various soluble xylan preparations and with mixed-linkage beta-1,3/beta-1,4-glucans. GST-A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST-C2, which contains the C-terminal CBD of XynA, did not interact with soluble xylan. Another hybrid protein, GST-A2, displayed the same binding properties as GST-A1A2, indicating that A2 alone can also promote xylan binding. The dissociation constants for the binding of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose by GST-A2, as determined at 20 degrees C by fluorescence quench experiments, were 8.1 x 10(-3) M, 2.3 x 10(-4) M, 2.3 x 10(-5) M, 2.5 x 10(-6)M and 1.1 x 10(-6) M respectively. The A-domains of XynA, which are designated as xylan binding domains (XBD), are, from the structural as well as the functional point of view, prototypes of a novel class of binding domains. More than 50 related protein segments with hitherto unknown function were detected in about 30 other multidomain beta-glycanases, among them putative plant (Arabidopsis thaliana) xylanases. It is argued that polysaccharide binding and not thermostabilization is the main function of A-like domains.

Pullulanase from the hyperthermophilic bacterium Thermotoga maritima: purification by beta-cyclodextrin affinity chromatography.

This is the first report about the isolation of a type I pullulanase from a hyperthermophilic bacterium, Thermotoga maritima strain MSB8. Purification of the enzyme from a cleared cell-free extract was achieved by anion-exchange chromatography and beta-cyclodextrin affinity chromatography. Using this convenient two-step method we have purified the pullulanase 406-fold with a 26% yield. The purified enzyme displayed maximum pullulan hydrolysis at pH 5.9 and 90 degrees C (15-min assay) and was remarkably resistant against thermoinactivation, having a half-life at 90 degrees C of about 3.5 h. To our knowledge, the T. maritima pullulanase is the most thermostable type I pullulanase known to date. The affinity-based purification protocol described here may be useful for the efficient isolation of other pullulanases.

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties.

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.