Characterization of thermostability of Clostridium stercorarium xylanase.
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Bacterial Proteins↗
Biomedical subjects
K Sakka
Publications and source records attributed to K Sakka.
Identification and characterization of cellulose-binding domains in xylanase A of Clostridium stercorarium.
The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519. XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus. The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V. Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel. Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100. XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose. Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them. XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive. Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.
Amino Acid Sequence↗
Purification and Characterization of Two Dihydroxyacetone Kinases from Schizosaccharomyces pombe IFO 0354.
Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schizosaccharomyces pombe IFO 0354. They were immunologically different from each other. Although both of the enzymes had some affinity for glycerol and dl-glyceraldehyde in addition to dihydroxyacetone and glyceraldehyde, V(infmax) values for dihydroxyacetone were much higher than those for glycerol and dl-glyceraldehyde. On the basis of the K(infm) values of both enzymes for dihydroxyacetone, DHAK II plays a more important role than DHAK I in dissimilation of glycerol via dihydroxyacetone.
Journal Article↗
Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.
The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055. The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative. CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily E1 endoglucanase domain, a family J endoglucanase domain, a docking domain, and another domain of unknown function (UD-2). UD-1 has no significant similarity to UD-2. CelJ hydrolyzed carboxymethylcellulose and xylan, and xylanase activity was ascribed to the family J domain. Antiserum raised against the truncated CelJ cross-reacted with proteins contained in the cellulosome of C. thermocellum F1. These results strongly suggest that CelJ is equivalent to S2, which was identified as the largest catalytic component in the cellulosome of C. thermocellum YS. A second but incomplete ORF encoding an enzyme classified in subfamily E2 endoglucanase, was located downstream of celJ.
Amino Acid Sequence↗
Enumeration of transconjugated Ruminococcus albus and its survival in the goat rumen microcosm.
A transconjugant Ruminococcus albus A3 culture was released into a goat rumen, and the extent of its survival in the rumen microcosm was measured by distinguishing this bacterium from indigenous microbes by antibiotic resistance. A3 cells remained roughly constant for 14 days in this goat rumen.
Animals↗
Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui.
The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes.
Amino Acid Sequence↗
Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product.
The nucleotides of the xynB gene of Clostridium stercorarium F-9 were sequenced. The structural gene consists of an open reading frame of 1161 bp encoding a xylanase (XynB) in family F of 387 amino acids with a molecular weight of 44,377. The molecular weight of the enzyme purified from a recombinant Escherichia coli was around 41,000, smaller than the predicted value, on SDS-polyacrylamide gel electrophoresis due to the lack of 32 amino acids at the N-terminus. Intact XynB with a molecular weight of around 43,000 was immunologically detected in the total cell proteins of a recombinant E. coli and C. stercorarium F-9. The purified XynB was active toward xylan, carboxymethylcellulose, p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-cellobioside. The pH optimum was 7.0 and it was quite stable over the pH range of 5 to 12 at 4 degrees C. This enzyme was optimally active at 80 degrees C and retained about 50% of the original activity even after incubation at 100 degrees C for 10 min.
Amino Acid Sequence↗
Process of thermal denaturation of xylanase (XynB) from Clostridium stercorarium F-9.
The thermal denaturation process of Clostridium stercorarium F-9 xylanase (XynB) was studied by monitoring remaining activity and recovered activity of the enzyme. At pH 5.5, aggregation occurred rapidly after the thermal denaturation initiated. The aggregated protein could be dissolved in 8 M urea solution, and the enzyme activity was recovered by diluting the urea. The extent of the recovered activity was gradually decreased with two phases as the reaction time of the thermal denaturation became longer. These results suggested the thermal denaturation process to be as follows: [formula: see text] where N is the native state of the enzyme; D1 is the denatured state of the enzyme that is formed rapidly after the reaction started and can be renatured by the urea treatment, and D2 and D3 are the denatured states of the enzyme that cannot be renatured even by the urea treatment. The rate constants were k1 > 9.2, k2 = 0.33, and k2 = 0.57, and k3 = 0.13 (in min-1 unit).
Calorimetry, Differential Scanning↗
Purification and characterization of xylanase A from Clostridium stercorarium F-9 and a recombinant Escherichia coli.
Xylanase A encoded by the Clostridium stercorarium F-9 xynA gene was purified to homogeneity from a recombinant clone of Escherichia coli. The N-terminal amino acid sequence and molecular weight (54,000) estimated by SDS-PAGE of the purified enzyme were consistent with those deduced from the nucleotide sequence [Biosci. Biotech. Biochem., 57, 273-277 (1993)]. A xylanase was also purified to homogeneity from a culture supernatant of C. stercorarium F-9. Its N-terminal amino acid sequence, molecular weight, and enzymatic properties were quite in agreement with those of the recombinant enzyme, indicating that the xynA gene was predominantly expressed as a xylanase gene in C. stercorarium F-9. The purified enzyme hydrolyzed xylotriose to yield xylobiose and xylose while it was less active toward xylobiose. It was optimally active at 75 degrees C and pH 7.0 Km and Vmax were estimated to be 1.9 mg/ml and 2.8 mumol of xylose equivalent/min/micrograms for oat spelt xylan, respectively.
Amino Acid Sequence↗
Cellulases, xylanases, and their genes from bacteria.
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Amino Acid Sequence↗
Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A: identification of catalytic and cellulose binding domains.
The nucleotides of the xynA gene of Clostridium stercorarium were sequenced. The structural gene consists of an open reading frame of 1533 bp encoding 511 amino acids with an M(r) of 56,519. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of the enzyme produced by a recombinant Escherichia coli. Xylanase A consists of a catalytic domain belonging to family G at the N-terminus and two direct repeats of about 90 amino acids with a short spacing at the C-terminus. Deletion analysis showed that the repeated sequences were responsible for binding the enzyme to Avicel and were not essential for catalytic activity. The catalytic domain of this enzyme is highly homologous to xylanase A of Clostridium acetobutylicum (identity: 69%) and xylanase B of Bacillus pumilus (identity: 64%).
Amino Acid Sequence↗
Nucleotide sequence of the Clostridium stercorarium xylA gene encoding a bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities, and properties of the translated product.
The nucleotides of the beta-xylosidase (xylA) gene from Clostridium stercorarium were sequenced. A single open reading frame of 473 codons specifying the subunit (MW 53,340) of xylosidase was identified. The N-terminal amino acid sequence and molecular weight estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme were quite in agreement with those deduced from the nucleotide sequence. Analysis of the enzyme by gel filtration on an HPLC column gave a molecular weight of 220,000, suggesting that the native enzyme is a tetramer composed of 4 identical subunits. The pH optimum was 7.0 and quite stable over the pH range of 5 to 10 at 4 degrees C. The optimum temperature was 65 degrees C. Vm was estimated to be 5.9 nmol/min/micrograms for p-nitrophenyl-beta-D-xylopyranoside and 16.7 nmol/min/micrograms for p-nitrophenyl-alpha-L-arabinofuranoside, while Km was estimated to be 2.5 mM for p-nitrophenyl-beta-D-xylopyranoside and 17.6 mM for p-nitrophenyl-alpha-L-arabinofuranoside.
Amino Acid Sequence↗
Nucleotide sequence of celC307 encoding endoglucanase C307 of Clostridium sp. strain F1.
The celC gene, encoding endoglucanase C, of Clostridium thermocellum was recently sequenced while a promoter region was not identified. In this study, the nucleotide sequence of celC307 of Clostridium sp. strain F1 was identified and compared with that of the C. thermocellum gene. The open reading frame was composed of 1029 nucleotides and the deduced amino acid sequence corresponded to a protein of a molecular weight of 40,905. We identified promoter sequences (TGGACA and TATAAT) at a position about 150 nucleotides upstream of the initiation codon. Six substitutions were found in the coding region, 3 leading to amino acid replacements. Five substitutions and 1 deletion of a nucleotide were found in the region upstream of the initiation codon, 1 present at the promoter sequence. Overproduction of endoglucanase C307 (EGC307) in Escherichia coli strongly inhibited the cell growth of the host strain. Around 50% of EGC307 produced in E. coli was detected in the periplasmic fraction. The N-terminal amino acid sequence suggested that this protein was exported into the periplasm without processing of a signal peptide.
Amino Acid Sequence↗
Isolation and characterization of a new globomycin-resistant dnaE mutant of Escherichia coli.
We isolated a globomycin-resistant, temperature-sensitive mutant of Escherichia coli K-12 strain AB1157. The mutation mapped in dnaE, the structural gene for the alpha-subunit of DNA polymerase III. The in vivo processing of lipid-modified prolipoprotein was more resistant to globomycin in the mutant strain 307 than in its parent. The prolipoprotein signal peptidase activity was also increased twofold in the mutant, and there was a threefold increase in the activity of isoleucyl-tRNA synthetase. The results suggest that a mutation in dnaE may affect the expression of the ileS-lsp operon in E. coli. In addition, strain 307 showed a reduced level of streptomycin resistance compared with its parental strain AB1157 (rpsL31). Strain 307 was killed by streptomycin at a concentration of 200 micrograms/ml, which did not affect the rate of bulk protein synthesis in this mutant. A second mutation which was involved in the reduced streptomycin resistance in strain 307 was identified and found to be closely linked to or within the rpsD (ramA, ribosomal ambiguity) gene. Both dnaE and rpsD were required for the reduced streptomycin resistance in strain 307.
Anti-Bacterial Agents↗
[Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301].
A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301. In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.
Acetobacteraceae↗