Purification and properties of an extracellular endo-1,4-β-xylanase from Penicillium citrinum and characterization of the encoding gene

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

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Cloning and characterization of a polygalacturonase-encoding gene from Penicillium janthinellum

Journal of Fermentation and Bioengineering ◽

10.1016/s0922-338x(97)82065-x ◽

1997 ◽

Vol 84 (3) ◽

pp. 257-260 ◽

Cited By ~ 5

Author(s):

Yutaka Ishida ◽

Kazumasa Kakibuchi ◽

Yuko Hirao ◽

Ken Izumori

Keyword(s):

Penicillium Janthinellum ◽

Encoding Gene

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Characterization of the New Metallo-β-Lactamase VIM-13 and Its Integron-Borne Gene from a Pseudomonas aeruginosa Clinical Isolate in Spain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00465-08 ◽

2008 ◽

Vol 52 (10) ◽

pp. 3589-3596 ◽

Cited By ~ 52

Author(s):

Carlos Juan ◽

Alejandro Beceiro ◽

Olivia Gutiérrez ◽

Sebastián Albertí ◽

Margalida Garau ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Pcr Amplification ◽

Class 1 Integron ◽

Negative Results ◽

New Variant ◽

Class B ◽

Class 1 ◽

Encoding Gene ◽

Good Agreement

ABSTRACT During a survey conducted to evaluate the incidence of class B carbapenemase (metallo-β-lactamase [MBL])-producing Pseudomonas aeruginosa strains from hospitals in Majorca, Spain, five clinical isolates showed a positive Etest MBL screening test result. In one of them, strain PA-SL2, the presence of a new bla VIM derivative (bla VIM-13) was detected by PCR amplification with bla VIM-1-specific primers followed by sequencing. The bla VIM-13-producing isolate showed resistance to all β-lactams (except aztreonam), gentamicin, tobramycin, and ciprofloxacin. VIM-13 exhibited 93% and 88% amino acid sequence identities with VIM-1 and VIM-2, respectively. bla VIM-13 was cloned in parallel with bla VIM-1, and the resistance profile conferred was analyzed both in Escherichia coli and in P. aeruginosa backgrounds. Compared to VIM-1, VIM-13 conferred slightly higher levels of resistance to piperacillin and lower levels of resistance to ceftazidime and cefepime. VIM-13 and VIM-1 were purified in parallel as well, and their kinetic parameters were compared. The k cat/K m ratios for the antibiotics mentioned above were in good agreement with the MIC data. Furthermore, EDTA inhibited the activity of VIM-13 approximately 25 times less than it inhibited the activity of VIM-1. VIM-13 was harbored in a class 1 integron, along with a new variant (Ala108Thr) of the aminoglycoside-modifying enzyme encoding gene aacA4, which confers resistance to gentamicin and tobramycin. Finally, the VIM-13 integron was apparently located in the chromosome, since transformation and conjugation experiments consistently yielded negative results and the bla VIM-13 probe hybridized only with the genomic DNA.

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N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01310-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 6

Author(s):

Stanislav Huszár ◽

Vinayak Singh ◽

Alica Polčicová ◽

Peter Baráth ◽

María Belén Barrio ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Target ◽

Functional Characterization ◽

Drug Candidate ◽

Content Type ◽

Chemical Libraries ◽

Whole Cells ◽

Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

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