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Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-  -L-Fucosidase
(AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95).Takane Katayama, 2004.A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the -(1 2)
linkage of 2'-fucosyllactose, and a gene encoding 1,2--L-fucosidase
(AfcA) was isolated . The afcA gene was found to comprise 1,959
amino acid residues with a predicted molecular mass of 205 kDa
and containing a signal peptide and a membrane anchor at the N and C
termini, respectively . A domain responsible for fucosidase activity
(the Fuc domain; amino acid residues 577 to 1474) was localized by
deletion analysis and then purified as a hexahistidine-tagged
protein . The recombinant Fuc domain specifically hydrolyzed the
terminal
-(12)-fucosidic
linkages of various oligosaccharides and a sugar chain of a
glycoprotein . The stereochemical course of the hydrolysis of
2'-fucosyllactose was determined to be inversion by using 1H
nuclear magnetic resonance . The primary structure of the Fuc domain
exhibited no similarity to those of any glycoside hydrolases (GHs)
but showed high similarity to those of several hypothetical proteins
in a database . Thus, it was revealed that the AfcA protein
constitutes a novel inverting GH family (GH family 95) .
Postexponential Regulation of sin Operon Expression in Bacillus subtilis. Sasha H. Shafikhani, 2002.The expression of many gene products required during the early stages of Bacillus subtilis sporulation is regulated by sinIR operon proteins . Transcription of sinIR from the P1 promoter is induced at the end of exponential growth . In vivo transcription studies suggest that P1 induction is repressed by the transition-state regulatory protein Hpr and is induced by the phosphorylated form of Spo0A . In vitro DNase I footprinting studies confirmed that Hpr, AbrB, and Spo0A are trans-acting transcriptional factors that bind to the P1 promoter region of sinIR . We have also determined that the P1 promoter is transcribed in vitro by the major vegetative sigma factor,  A, form of RNA polymerase .
Xis Protein Binding to the Left Arm Stimulates Excision of Conjugative Transposon Tn916. Kevin M. Connolly, 2002.Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase (Tn916Int) and excisionase (Tn916Xis) proteins . In this study, we have investigated the role of the Tn916Xis protein in stimulating excisive recombination . The functional relevance of Tn916Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay . Our results indicate that in Escherichia coli the stimulatory effect of the Tn916Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon . These sites lie in between the core and arm sites recognized by Tn916Int, suggesting that the Tn916Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage  , serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis . However, our finding that excision in E . coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage
recombination .
Kraft Pulp Biobleaching and Mediated Oxidation of a Nonphenolic Substrate by Laccase from Streptomyces cyaneus CECT 3335. M. Enriqueta Arias, 2003.A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity . The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed . The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70°C, respectively . The activity was strongly enhanced in the presence of Cu2+, Mn2+, and Mg2+ and was completely inhibited by EDTA and sodium azide . The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine . The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm . The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry . We demonstrated that application of the laccase from S . cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes .
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