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A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+-dependent bacillar alcohol dehydrogenase.
No full textThe gene adh-hT encoding a thermostable and thermophilic NAD+-dependent alcohol dehydrogenase
(ADH) from the novel and more thermophilic Bacillus stearothermophilus LLD-R strain was
cloned and its nucleotide sequence determined. The deduced protein sequence shows remarkable
amino acid substitutions when compared to the sequence of the protein isolated from strain
NCAl503 and significant similarity with the highly thermostable ADH from the thermoacidophilic
archaebacterium Sulfolobus solfataricus. The alignment of these sequences led to the identification
of three amino acid replacements probably responsible for the higher thermostability of the novel
bacillar ADH.
Adh-hT gene expression in Escherichia coli, a fast purification procedure and the characterization
of the recombinant enzyme are also describe
celS, a gene encoding an extracellular cellulase from Sulfolobus solfataricus.
No full textCellulose is one of the most abundant biopolymer on Earth and represents an important source of renewable energy. It is an unbranched polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. Enzymatic hydrolysis of cellulose requires a consortium of enzymes, including endo--glucanases, exoglucanases, and -glucosidases. Endoglucanases randomly hydrolyse internal glycosidic linkages and are widely distributed among fungi, Bacteria and Archaea.
Cellulase activitiy was detected in the supernatant of S. solfataricus MT4 cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 49 kDa and 40 kDa. At the same time we have cloned a DNA fragment from Sulfolobus solfataricus MT4 containing an ORF (CelS) of 322 aminoacids (molecular weight 36703 Da) with significant homology to the Pyrococcus furiosus (1), Thermotoga maritima (2) and T. neapolitana (3) endo-1,4--glucanases. The gene was demonstrated to be transcribed in vivo.
In order to optimise the expression of celS we tried to grow MT4 on different -glucans, namely in minimal media containing Avicel, lichenan, carboxylmethylcellulose, cellobiose, but they resulted to be unable to support growth. Therefore enzymatic and transcriptional analysis were performed on cells cultured in more unspecific minimal and rich media. The results obtained suggested a catabolite repression by glucose and in general a down regulation by complex nutrients.
The transcription start site was unambiguously identified by primer extension and revealed coincident with translational initiatio
Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation.
No full textin pres
Transcriptional regulation of the gene encoding an alcohol dehydrogenase in the archaeon Sulfolobus solfataricus involves multiple factors and control elements.
No full textVeratryl alcoholoxidase from Pleurotus ostreatus partecipates in lignin biodegradation and prevents polymerization of laccase-oxidized substates
No full textEnzyme dynamics and hydrogen tunneling in a thermophilic alcohol dehydrogenase.
No full textBiological catalysts (enzymes) speed up reactions by many orders
of magnitude using fundamental physical processes to increase
chemical reactivity. Hydrogen tunnelling has increasingly been
found to contribute to enzyme reactions at room temperature1.
Tunnelling is the phenomenon by which a particle transfers
through a reaction barrier as a result of its wave-like property1–3.
In reactions involving small molecules, the relative importance of
tunnelling increases as the temperature is reduced4.We have now
investigated whether hydrogen tunnelling occurs at elevated
temperatures in a biological system that functions physiologically
under such conditions. Using a thermophilic alcohol dehydrogenase
(ADH), we find that hydrogen tunnelling makes a significant
contribution at 65 8C; this is analogous to previous
findings with mesophilic ADH at 25 8C (ref. 5). Contrary to
predictions for tunnelling through a rigid barrier, the tunnelling
with the thermophilic ADH decreases at and below room temperature.
These findings provide experimental evidence for a role
of thermally excited enzyme fluctuations in modulating enzyme catalysed
bond cleavage
Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus species
No full textThe gene adh encoding a NAD-dependent alcohol dehydrogenase front the novel strain RC3 of Sulfolobus sp. was cloned and sequenced. Both the adh gene from Sulfolobus sp. strain RC3 and the alcohol dehydrogenase gene from Sulfolobus solfataricus (DSM 1617) were expressed at a high level in Escherichia coli, and the recombinant enzymes were purified, characterized, and compared. Only a few amino acid replacements were responsible for the different kinetic and physicochemical features investigated
A superoxide dismutase from the archaeon sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins.
No full textAn oxygen-induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose-rich media. This protein was also identified as being associated with the cell-surface, with the amount of the released and cell-bound protein fractions depending on the growth phase of the cells. The steady decrease in cell-associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity. Characterization in comparison with the cytosolic superoxide dismutase revealed identical N-terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes. In order to clarify the physiological role of the cell-associated superoxide dismutase, the prevention of cell-bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme. Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay. Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components
Decreasing the Stability and Changing the Substrate Specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single aminoacid replacements.
No full textThe gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B. stearothermophilus NCA 1503. In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala--were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disruptive. The thermal resistance was changed displaying quite additive effects
Identification and molecular characterisation of an endoglucanase gene, celS, from the extremely thermophilic archaeon Sulfolobus solfataricus.
No full textA genomic region upstream of the alcohol dehydrogenase
(
Ssadh
) gene was cloned and sequenced from a
library of
Sulfolobus solfataricus
MT4 strain. The isolated
4,040-bp DNA fragment revealed an open reading frame
(
celS
), lying in the opposite direction to
Ssadh
, which
showed significant similarity to
endo
-
β
-1,4-glucanases from
Pyrococcus furiosus
,
Thermotoga maritima
, and
Thermotoga
neapolitana
.
celS
was shown to be a functional gene in
vivo: a specific
celS
mRNA was detected by primer extension
analysis showing a unique initiation transcription site
coinciding with the ATG translation initiation codon. The
specific gene product was detected as an extracellular cellulase
after enzyme staining by carboxymethyl cellulose
(CMC) SDS-PAGE, showing a molecular weight in agreement
with that deduced from the open reading frame.
Depending on growth conditions, different levels of cellulase
activity and specific
celS
transcript were detected,
revealing an inductive effect of CMC and suggesting a
repressive role of glucose
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