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Birgisson H Fridjonsson O Bahrani-Mougeot FK Hreggvidsson GO Kristjansson JK Mattiasson B 《Biotechnology letters》2004,26(17):1347-1351
An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C. AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51. 相似文献
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Multiple isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-
transferase (ppGaNTase) have been cloned and expressed from a variety of
organisms. In general, these isoforms display different patterns of
tissue-specific expression, but exhibit overlapping substrate
specificities, in vitro . A peptide substrate, derived from the sequence of
the V3 loop of the HIV gp120 protein (HIV peptide), has previously been
shown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennett
et al. , 1996). To determine if this isoform- specificity is maintained in
vivo , we have examined the glycosylation of this substrate when it is
expressed as a reporter peptide (rHIV) in a cell background (COS7 cells)
which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIV
was greatly increased by coexpression of a recombinant ppGaNTase-T3.
Overexpression of ppGaNTase- T1 yielded only partial glycosylation of the
reporter. We have also determined that the introduction of a proline
residue at the +3 position flanking the potential glycosylation site
eliminated ppGaNTase- T3 selectivity toward rHIV observed both in vivo and
in vitro .
相似文献
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Bahrani-Mougeot FK Buckles EL Lockatell CV Hebel JR Johnson DE Tang CM Donnenberg MS 《Molecular microbiology》2002,45(4):1079-1093
Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants. 相似文献
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Hobel CF Hreggvidsson GO Marteinsson VT Bahrani-Mougeot F Einarsson JM Kristjánsson JK 《Extremophiles : life under extreme conditions》2005,9(1):53-64
A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far. 相似文献
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