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Golubev W.I.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms
Mikologiya I Fitopatologiya | Year: 2015

The strain VKM Y-2517 of Ogataea nonfermentans was revealed to secrete fungicidal mycocin active at pH of the medium within the range 3.5-6.5. The spectrum of its action is narrow and includes only several species of methylotrophic yeasts in the genera Ogataea, Candida and also some representatives related to them phylogenetically in the genera Ambrosiozyma and Nakazawaea. Strain heterogeneity of species (such as Ogataea minuta and Candida boidinii) in sensitivity to mycocin is indicative of their taxonomic heterogeneity. Source

Hilden K.,University of Helsinki | Hilden K.,University of Nottingham | Makela M.R.,University of Helsinki | Lundell T.,University of Helsinki | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2013

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics. © 2012 Springer-Verlag. Source

But S.Y.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms | Khmelenina V.N.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms | Reshetnikov A.S.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms | Trotsenko Y.A.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms
FEMS Microbiology Letters | Year: 2013

The aerobic obligate methylotroph Methylobacillus flagellatus KT was shown to synthesize sucrose in the presence of 0.5-2% NaCl in the growth medium. In the genome of this bacterium, an open reading frame (ORF) encoding a predicted 84-kD polypeptide homologous to the plant and cyanobacterial sucrose phosphate synthases (SPSs) was found. Using heterologous expression of the putative sps gene in Escherichia coli, followed by affinity chromatography, pure recombinant protein SPS-His6 was obtained. The enzyme catalyzed two reactions: conversion of fructose 6-phosphate and UDP-glucose into sucrose 6-phosphate and hydrolysis of sucrose 6-phosphate to sucrose. The bifunctional sucrose phosphate synthase/phosphatase (SPS/SPP) was a 340 kDa homotetrameric Mg2+-dependent enzyme activated by fructose 1,6-phosphate2 and ATP but inhibited by glucose 6-phosphate, fructose 1-phosphate, AMP and inorganic phosphate. The amino acid sequence of the protein had a C-terminal domain homologous to SPPs. This correlated with the absence of the spp gene in the M. flagellatus chromosome. The ORFs homologous to the M. flagellatus SPS were found in the genomes of another obligate methylotroph Methylovorus glucosetrophus as well as the lithoautotrophic bacteria Acidithiobacillus ferrooxidans, Nitrosomonas europaea and Nitrosospira multiformis whose genomes lacked the spp genes. Thus, data extending the knowledge of biochemical properties of bacterial SPSs have been obtained. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved. Source

Khomutov S.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms | Donova M.V.,Gk Skryabin Institute Of Biochemistry And Physiology Of Microorganisms
Journal of Inclusion Phenomena and Macrocyclic Chemistry | Year: 2011

Molecular-imprinting by cross-linking of ligands of β-cyclodextrin (CD) complex with steroids has been developed for the synthesis of tailor-made CD dimer. Steroids of androstane (9α-hydroxy-androst-4-en-3,17-dione, androst-4-en-3,17-dione, androsta-1,4-dien-3,17-dione (ADD)) and pregnane (hydrocortisone, 6-methyl-hydrocortisone, 20-hydroxymethylpregna-1,4-diene-3-one (HMPD)) series were used as template molecules. For imprinting procedure, crystalline β-CD complexes of exact stoichiometry (β-CD:steroid template = 2:1) were synthesized following by toluene 2,4-diisocyanate (TDI) cross-linking. The attempts to produce CD dimer for steroid without hydrophobic side chain failed, while tailor-made CD dimer has been obtained using HMPD as a template. The dimer was characterized by 1H NMR and mass-spectrometry. The complex stability constant (K S) towards HMPD template exceeded 10 7 M -1. The K S of CD dimer with ADD exceeded the corresponded value of TDI-modified CD monomer by more than an order of magnitude. The dimer was applied for quantitative extraction of ADD from aqueous solution using dialysis membranes impermeable for CD. The value of K S for ADD estimated from balanced concentrations of dialysis data corresponded to that calculated by nonlinear spectrometric method. © 2010 Springer Science+Business Media B.V. Source

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