Greater Thermostability Research Articles

Influence of conditioning temperature on the postpellet recovery and efficacy of 2 microbial phytases for broiler chicks

SUMMARY The effect of conditioning temperature on phytase efficacy for growth performance and bone mineralization was evaluated using 432 male Ross broilers. Birds were fed a wheat-soy-based diet that was formulated to be adequate in all nutrients with the exception of available P. Diets were conditioned at 75, 80, 85, or 90°C and fed with (1,000 phytase units/kg) or without 1 of 2 microbial phytases in a 3 × 4 factorial design. Phytase A was found to have greater thermostability than phytase B (78.3 vs. 57.5%) as an average across all conditioning temperatures. Phytase increased (P < 0.05) BW gain from d 0 to 21 and birds fed diets containing phytase A were significantly heavier than birds offered diets with phytase B. Increasing conditioning temperature decreased (P < 0.01) BW gain. Supplementing diets with phytase improved (P < 0.05) FE and birds fed diets containing phytase A were more efficient (P < 0.05) than broilers fed diets containing phytase B. Tibia ash concentrations were improved (P < 0.05) by approximately 10% with phytase A and B compared with birds fed diets without phytase. Birds fed phytase A diets had higher (P < 0.05) bone ash concentrations than birds that received the diet containing phytase B. In conclusion, the inclusion of an intrinsically thermostable exogenous phytase before pelleting would be of benefit to bird performance and bone mineralization in broilers.

Understanding Thermostability Factors of Aspergillus niger PhyA Phytase: A Molecular Dynamics Study

Molecular dynamics simulation was used to study the dynamic differences between native Aspergillus niger PhyA phytase and a mutant with 20% greater thermostability. Atomic root mean square deviation, radius of gyration, and number of hydrogen bonds and salt bridges are examined to determine thermostability factors. The results suggest that, among secondary structure elements, loops have the most impact on the thermal stability of A. niger phytase. In addition, the location rather than the number of hydrogen bonds is found to have an important contribution to thermostability. The results also show that salt bridges may have stabilizing or destabilizing effect on the enzyme and influence its thermostability accordingly.

The Preparation of a Hyper-Thermostable Whole-Cell Biocatalyst and its Application for Biosynthesis of Biodiesel

In this study, a hyper-thermostable lipase whole-cell biocatalyst has been developed. The hydrolytic activity of the codon-optimized ROL displayed on the yeast cell surface was 19.5 U /g dried cells cultured in SD medium, while the biomass was 13 g/L. Moreover, the whole-cell biocatalyst presented great thermostability. After 10 min incubation at 90 °C, 95 °C and 100 °C, the residual activity of lipase still remained 89%, 86% and 68%, respectively. Therefore, the whole-cell biocatalyst was applied in bioconversion of FAME and biodiesel. In our study, 50 mM oleic acid was most reasonable for bioconversion of FAME, and 50 mM soy oil was most reasonable for bioconversion of biodiesel, when the reaction were carried out in hexane as the solvent, and the molar ratio of oleic acid (or soy oil) and methanol was 1:4. Reactions were catalyzed by 0.5 g lyophilized bioimprinted whole-cell biocatalyst and incubated at 45 °C for 12 h with shake.

Comparative analysis of the catalytic components in the archaeal dye-linked l-proline dehydrogenase complexes

Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (α4β4 and αβγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the β subunit (PDHβ) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHβs from α4β4- and αβγδ-type enzyme complexes, eight PDHβs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α4β4-type PDHβs showed greater thermostability than most of the αβγδ-type PDHβs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α4β4-type PDHβs utilized ferricyanide as the most preferable electron acceptor, whereas αβγδ-type PDHβs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHβs likely reflect whether they were from an αβγδ- or α4β4-type complex, though the wider divergence observed within αβγδ-type PDHβs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHβs did not reflect the complex type. Interestingly, the k cat value for free α4β4-type PDHβ from P. horikoshii was much larger than the value for the same subunit within the α4β4-complex. This indicates that the isolated PDHβ could be a useful element for an electrochemical system for detection of L-proline.

Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.

Isolation and Characterization of Laccase Activity in a Novel &lt;i&gt;Bacillus amyloliquefaciens&lt;/i&gt; LC02

Bacterial strains exhibiting laccase activity were isolated from the forest soil. A strain LC02 with syringaldazine oxidation ability was obtained using enrichment medium supplemented with copper ions. The isolated strain was identified as Bacillus amyloliquefaciens using physiological and biochemical tests as well as 16S rDNA sequence analysis. The characterization of spore laccase activity was investigated. The result showed that the optimum pH and temperature of the enzyme was 6.6 and 70°C, respectively. A great thermostability was observed for the spore laccase at 70°C. Laccase activity was strongly inhibited by 0.1 mmol/L NaN3, dithiothreitol and cysteine.

Improving the Catalytic Activity of HyperthermophilicPyrococcus horikoshiiProlidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases.

Environmental Effects on Mechanical Properties of Modified and Unmodified Carbon Nanotube/Epoxy Resin Nanoomposites

The composites of mechanical and thermal properties of modified and unmodified Carbon nanotube /epoxy resin nanocomposites tested under various circumstances is a major investigation in this study. Identification of functional groups of CNT surface using Fourier transforms infrared spectroscopy (FTIR) was done to male sure if the means of modification adopted in this study is successfully. The tensile and flexural strengths of modified CNT-containing nanocomposites with the amount of modified CNT around 0.75 Phr tested under room temperature circumstance increase 10.96 % and 21.44 % respectively. These two strengths of nanocomposites tested under high temperature circumstance increase 14.55 % and 10.80 % respectively. Additionally, both strengths of nanocomposites tested under high temp.-high humidity circumstance increase 11.65 % and 23.53 % respectively. According to the test results using pyris diamond thermomechanical analyzer (TMA) coefficient decreases 39.81 % with increasing the content of modified CNT and meanwhile glass transition temperature increases 4.15 %. Because of above-mentioned effect, a modified CNs-containing nanocomposite possesses great thermo stability. Overall, No matter what circumstance (high temperature or high temp.-high humidity circumstances) nanocomposites expose to, and no matter whether CNT are modified or not, CNT do significantly enhance mechanical and physical properties of composite.

Molecular cloning and characterization of trehalose synthase from Thermotoga maritima DSM3109: Syntheses of trehalose disaccharide analogues and NDP-glucoses

A gene (ORF TM0392) encoding a putative trehalose synthase (TmTreT) in Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and a glutathione-sepharose affinity column chromatography. The purified enzyme existed exclusively as a monomer in a native state. The optimum pH and temperature for this enzyme were 6.0 and 65°C. The glutathione-S-transferase (GST)-fusion enzyme had greater thermostability than thrombin-treated free enzyme. TmTreT had diverse substrate specificities. The enzyme effectively created a free trehalose from several nucleoside diphosphate (NDP)-glucoses as a donor and glucose as an acceptor. Inversely, the enzyme was also capable of employing several NDPs such as UDP, ADP, GDP, and CDP with trehalose to produce corresponding NDP-glucoses. The enzyme was able to employ other monosaccharides, such as mannose and fructose, as acceptors to synthesize disaccharide analogues of trehalose. The mannose-containing analogue was not hydrolyzed by trehalase and the rat intestinal enzymes. Furthermore, the analogue showed a competitive inhibition to the intestinal disaccharidases with Ki values of approximately 0.8–1.6mM. The results suggest that the enzyme is an useful trehalose synthase that can regenerate NDP-glucoses from NDPs and produce the inhibitory trehalose analogues of indigestible disaccharides.

Improvement of Bacillus circulans β-amylase activity attained using the ancestral mutation method

Thermostabilization of enzymes is one of the greatest challenges of protein engineering. The ancestral mutation method, which introduces ancestral residues into a target enzyme, has previously been developed and used to improve the thermostabilities of thermophilic enzymes. Herein, we report a study that used the ancestral mutation method to improve the thermostability of Bacillus circulans beta-amylase, a mesophilic enzyme. A bacterial, common-ancestral beta-amylase sequence was inferred using a phylogenetic tree composed of higher plant and bacterial amylase sequences. Eighteen mutants containing ancestral residues were designed, expressed in Escherichia coli and purified. Several of these mutants were more thermostable than that of the wild-type amylase. Notably, one mutant had both greater activity and greater thermostability. The relationship between the extent to which the amino acid residues within 5 A of the mutation site were evolutionarily conserved and the extent to which thermostability was improved was examined. Apparently, it is necessary to conserve the residues surrounding an ancestral residue if thermostability is to be improved by the ancestral mutation method.

Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye

The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this enzyme in various industrial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L(-1) of a bacterial laccase with a high purity and without any extensive purification. This is the highest production yield reported in the literature for a bacterial laccase. The secreted enzyme achieved oxidation under a wide pH range depending on the substrate: 4.0 for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and 9.0 for 2,6-dimethoxyphenol. Furthermore, this bacterial laccase was found to be quite resistant under various conditions. It withstands pH from 3.0 to 9.0, shows a great thermostability at 70 degrees C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN(3), the laccase of Streptomyces coelicolor was fully active under the same conditions. To assess application potential of this laccase, we have investigated its ability to decolourise Indigo carmine. This enzyme was able to rapidly decolourise the dye in the presence of syringaldehyde as a redox mediator.

Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppAEc and rAppASc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppASc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppAEc.

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima.

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.

Partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation

Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. It was concluded that hydrophobic sites become available and that catalytically active immobilized preparations may be finally obtained. In the present study, alkyl-substituted Sepharose 4B containing octyl, dodecyl or palmityl residues (Sepharose-lipid) were used for adsorptive immobilization of heat-denatured forms of this protein. The enzyme adsorbed on octyl-Sepharose showed the highest k cat/ K m(app) and the greatest thermostability as compared to the other two preparations. Moreover, the pH stability and resistance to the effect of KSCN, as a chaotropic salt, was higher for this preparation of the immobilized enzyme. Thermal stability and aggregation studies suggested that the enzyme is protected against irreversible thermoinactivation by interaction with the alkyl residues upon adsorption. Proline prevented both aggregation of the free enzyme at elevated temperatures and its interaction with the hydrophobic support. The higher degree of substitution in octyl-Sepharose appears to provide it with a greater capacity for multipoint interaction. It is suggested that the type of studies presented may provide useful information in connection with formation of intermediate states, in addition to its importance related to protein immobilization by adsorption.

Isolation of the patC gene encoding the cystathionine beta-lyase of Lactobacillus delbrueckii subsp. bulgaricus and molecular analysis of inter-strain variability in enzyme biosynthesis.

The patC gene encoding the cystathionine beta-lyase (CBL) of Lactobacillus delbrueckii subsp. bulgaricus NCDO 1489 was cloned and expressed in Escherichia coli. Overexpression of CBL complemented the methionine auxotrophy of an E. coli metC mutant, demonstrating in vivo that this enzyme functions as a CBL. However, PatC is distinguishable from the MetC CBLs by a low identity in amino acid sequence, a sensitivity to iodoacetic acid, greater thermostability and a lower substrate affinity. Homologues of patC were detected in the 13 Lb. delbrueckii strains studied, but only seven of them showed CBL activity. In constrast to CBL(+) strains, all CBL-deficient strains analysed were auxotrophic for methionine. This supports the hypothesis that CBLs from lactobacilli are probably involved in methionine biosynthesis. Moreover, the results of this study suggest that post-transcriptional mechanisms account for the differences in CBL activities observed between strains of Lb. delbrueckii.

Thermostable dextranases: screening, detection and preliminary characterization

Screening methods have been developed for detection of micro‐organisms producing thermostable dextranases. They utilize the incorporation of Blue Dextran into agar or liquid culture media for isolation of active dextranase producers growing at temperatures above 55°C. A variety of high temperature environments in sugar factories and naturally occurring thermal water samples were excellent sources of dextranase producers. A number of aerobic and anaerobic thermophilic bacteria, isolated from these sources, were found to produce thermostable dextranases. Dextranases with the greatest thermostability were found in cultures of anaerobic bacteria grown above 65°C. Temperature optima were determined for several crude enzyme preparations, four of which exhibited temperature optima in the range 65–85°C.

Adaptation of fish to different environmental temperature by qualitative and quantitative changes in gene expression

In fish that coccupy a specific thermal habitat the contractile apparatus has been adapted during evolution for that specific temperature range. In Antarctic fish the myofibrillar ATPase activity is relatively high at temperatures around 0°C, but this is at the expense of enzyme thermostability as this enzyme from antarctic fishes is heat inactivated at comparatively low temperatures (Johnston and Goldspink, 1975c; Johnston and Walesby, 1977). Such thermal characteristics may be a general feature of proteins from Antarctic fish (Somero, 1991) and so may contribute to their extreme cold stenothermy. Some curythermal species of fish have the ability to rebuild their contractile systems by expressing a different set of genes at low temperature to that expressed at warm environmental temperatures. We have cloned and are in the process of characterising warm and cold as well as embryonic myosin heavy chain isoform genes of carp. These genes encode different types of myosin crossbridges (the force generators for muscular contraction) and therefore determine the contractile characteristics of the muscle. The physiological result of the switches in gene expression are that the muscles of fish acclimated to low environmental temperatures develop more force and more power at these temperatures than muscles from fish acclimated to warm environmental temperatures. Carp acclimated to warm temperatures have myosin with a greater thermostability but at a lower myofibrillar ATPase.

Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M r of 250 000. It consists of four, apparently identical subunits each of M r 60 000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.

Purification and properties of pyruvate kinase fromThermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native Mr of 250 000. It consists of four, apparently identical subunits each of Mr 60 000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.

Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome‐inactivating protein α‐sarcin from Aspergillus giganteus

An immunotoxin was synthesized by the attachment of alpha-sarcin, the ribosome-inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2 antibody Fib75. The alpha-sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the alpha-sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with ricin A chain. Fib75-alpha-sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The alpha- and beta-phase half-lives were 0.8 h and 6 h, respectively, similar to the serum half-lives of analogous Fib75 immunotoxins made with ribosome-inactivating proteins derived from plants. alpha-Sarcin was completely stable in physiological saline buffer at 37 degrees C, whereas the ribosome-inactivating activity of ricin A chain was gradually lost under identical conditions. alpha-Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.