RESUMO
Under the conditions of submerged cultivation in a medium containing microcrystalline cellulose, the Cerrena unicolor VKM F-3196 basidiomycete is capable of producing xylanase and cellulase. Electrophoretically homogeneous cellulase and xylanase were obtained using ion exchange and hydrophobic chromatography. The molecular weight of both cellulase and xylanase was -44 kDa. It was shown that xylanase catalyzed the hydrolysis of xylan with the production of xylose, xylobiose, and xylotetrose and it exhibited properties of endoxylanases. Cellulase hydrolyzed carboxymethylcellulose, xylan, and microcrystalline cellulose with the formation of cellotriose and cellotetraose. For both enzymes, the pH optimum was -4.0. The enzymes exhibited moderate thermostability: xylanase retained 35% of the initial activity for an hour at 60 degrees C; cellulase, 10% under the same conditions. Xylanase, cellulose, and a mixture of these enzymes saccharified plant material (wheat, rye, wheat middling, and oat), indicating the possible use of these enzymes in biotechnology.
Assuntos
Agaricales/enzimologia , Celulase/química , Endo-1,4-beta-Xilanases/química , Agaricales/química , Avena/química , Carboximetilcelulose Sódica/química , Celulase/isolamento & purificação , Celulose/química , Meios de Cultura , Dissacarídeos/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Secale/química , Especificidade por Substrato , Triticum/química , Xilanos/química , Xilose/químicaRESUMO
An original method of immobilization of nongrowing microorganism cells on xerogel of silicon dioxide containing insoluble hydroxyl compounds of cobalt(III) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3-5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62-65 degrees C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was -60 and -25 days, respectively.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Engenharia Metabólica/métodos , Dióxido de Silício/química , Aldose-Cetose Isomerases/genética , Arthrobacter/enzimologia , Arthrobacter/genética , Proteínas de Bactérias/genética , Biocatálise , Reatores Biológicos , Células Imobilizadas , Cobalto/química , Escherichia coli/genética , Frutose/metabolismo , Glucose/metabolismo , Glutaral/química , Temperatura Alta , Hidrogéis , Cinética , Plasmídeos/genéticaRESUMO
The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject to catabolite repression, was inhibited by xylulose, an intermediate product ofxylose utilization and the final product of its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated with its influence on the catalytic activity of the enzyme.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Arthrobacter/enzimologia , Proteínas de Bactérias/biossíntese , Arthrobacter/efeitos dos fármacos , Arthrobacter/crescimento & desenvolvimento , Proteínas de Bactérias/farmacologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Xilose/metabolismo , Xilulose/metabolismo , Xilulose/farmacologiaRESUMO
Arthrobacter nicotinanae cells, producers of glucose isomerase, were immobilized in xerogel of silicium dioxide, and properties of the resulted heterogeneous biocatalysts were investigated in the process of isomerization of monosaccharide (glucose and fructose). The glucose isomerase activity of the resulted biocatalysts was shown to be 10 U/g, on average, taking into account the loss of the activity upon the immobilization, which amounted to 50% of the cell activity in suspension. The rate of the fructose isomerization increased linearly in the range of 55-80 degrees C with the temperature coefficient 1.3. The biocatalysts were stable in this range; they were rapidly inactivated, however, at increasing temperature. The half-inactivation time was six to seven h and five min or less at 80 degrees C and 85 degrees C, respectively. The half-inactivation time of heterogeneous biocatalysts was 50-90 h in the periodic process of isomerization of 2 M monosaccharides at 60 degrees C in the presence of the immobilized Arthrobacter nicotinanae cells.
Assuntos
Aldose-Cetose Isomerases/química , Arthrobacter/enzimologia , Proteínas de Bactérias/química , Frutose/química , Glucose/química , Dióxido de Silício/química , Aldose-Cetose Isomerases/biossíntese , Proteínas de Bactérias/biossíntese , Células Imobilizadas/enzimologia , Temperatura AltaRESUMO
The effect of a specific substrate as well as other carbon sources on the biosynthesis of xylose isomerase in the actinobacterium Arthrobacter ureafaciens BIM B-6 has been studied. It was established that xylose and its structural analogue xylite induced the production of the enzyme by bacterial cells. The inducing effect peaked at a concentration of specific substrates of 0.025% (as carbon) and then remained unchanged irrespective of the substrate amount. It has been shown that the synthesis of xylose isomerase by A. ureafaciens is controlled by catabolite repression occurring at the transcription level and mediated by cyclic 3',5'-AMP.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Arthrobacter/metabolismo , Proteínas de Bactérias/biossíntese , Xilose/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Arthrobacter/crescimento & desenvolvimento , Meios de Cultura , Cetoses/metabolismo , Especificidade por Substrato , Xilose/análogos & derivadosRESUMO
Kinetics of monosaccharide isomerization has been studied in suspensions of intact, non-growing Arthrobacter nicotianae cells. Under the conditions of the study, glucose and fructose were isomerized at the same maximum rate of 700 micromol/min per 1 g dried cells, which increased with temperature (the dependence was linear at 60-80 degrees C). The proposed means of adsorption immobilization of A. nicotianae cells involve inorganic carriers differing in macrostructure, chemical nature, and surface characteristics. Biocatalysts obtained by adsorbing the cells of A. nicotianae on carbon-containing foam ceramics in the coarse of submerged cultivation were relatively stable and retained original activity (catalysis of monosaccharide isomerization) throughout 14 h of use at 70 degrees C. Maximum glucose isomerase activity (2 micromol/min per 1 g) was observed with biocatalysts prepared by adsorption of non-growing A. nicotianae cells to the macroporous carbon-mineral carrier Sapropel and subsequent drying of the cell suspension together with the carrier.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Arthrobacter/enzimologia , Frutose/metabolismo , Glucose/metabolismo , Adsorção , Óxido de Alumínio , Silicatos de Alumínio , Benzopiranos , Carbono , Catálise , Cerâmica , Substâncias Húmicas , Porosidade , SuspensõesRESUMO
The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Bactérias/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Bactérias/crescimento & desenvolvimento , Especificidade da Espécie , Especificidade por Substrato/fisiologiaRESUMO
A plate method was developed to screen for xylose isomerase-producing microorganisms based on the use of 2,3,5-triphenyltetrazolium as an indicator of D-xylulose, the D-xylose isomerization product. The use of this method allows microorganisms to be differentiated by the character of the enzyme synthesis (inducible or constitutive).
Assuntos
Aldose-Cetose Isomerases/análise , Arthrobacter/isolamento & purificação , Aldose-Cetose Isomerases/biossíntese , Arthrobacter/metabolismo , Contagem de Colônia Microbiana/métodos , Corantes , Sais de TetrazólioRESUMO
The study of the xylose/glucose isomerase-containing Arthrobacter sp. B-5 cells immobilized in cobalt hydroxide gel showed that immobilization increases the substrate affinity of the enzyme, its thermo- and pH-optima of action and stability, and makes unnecessary the addition of stabilizing cobalt ions to the isomerization medium.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Arthrobacter/enzimologia , Aldose-Cetose Isomerases/química , Cobalto , Géis , Temperatura Alta , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Fatores de TempoRESUMO
The composition of pectin hydrolase complexes produced by various Aspergillus alliaceus strains was studied under the conditions of induction, catabolite repression, or constitutive synthesis. The strains were found similar in terms of the polygalacturonase spectrum and different with regard to the levels of endo- and exoenzyme activities. The analysis of the zymograms of inducible polygalacturonases revealed that all tested cultures contained at least 24 molecular forms of polygalacturonase. Taking into account only the three molecular forms typical of all analyzed strains of A. alliaceus with pI values of 5.7, 5.9, and 6.3, one can use the spectrum of constitutive, catabolite repression-resistant polygalacturonases as an additional taxonomic species criterion.
Assuntos
Aspergillus/metabolismo , Poligalacturonase/química , Poligalacturonase/metabolismo , Focalização Isoelétrica , Isoenzimas/química , Isoenzimas/metabolismoRESUMO
The substrate specificity of isomerases produced by six strains of Arthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A. ureafaciens B-6 strains showed low activity toward D-ribose, Arthrobacter sp. B-5 was slightly active toward L-arabinose, and A. ureafaciens B-6 and Arthrobacter sp. B-2239, toward L-rhamnose. In Arthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources). The synthesis of xylose/glucose isomerase induced by D-xylose in Arthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol in A. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in the Arthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Arthrobacter/enzimologia , Proteínas de Bactérias/metabolismo , Ativação Enzimática , Especificidade por SubstratoRESUMO
Of three molecular forms of polygalacturonases synthesized by Aspergillus alliaceus on glucose media, two were exopolygalacturonases (exoPG1 and exoPG2) and one was an endopolygalacturonase (endoPG). Low-methoxylated beet pectin was the preferred substrate for the endoPG and exoPG2 whereas pectic acid was the optimal substrate for exoPG1. The highest activities of endoPG, exoPG1 and exoPG2 were at pH 5.5, 3.5, and 6.0 and at 35, 45 to 50 and 35°C, respectively. Disks of potato-tuber tissue were macerated by endoPG, but not by exoPG1 or exoPG2.
RESUMO
Pectinlyase complexes of Penicillium adametzii, P. citrinum and P. janthinellum occur as multiple molecular forms distinguished by their biosynthetic control. AMP is involved in derepression of pectinlyase formation.
RESUMO
The enzymatic activity of micromycetes of Aspergillus and Penicillium genera cultivated on substrate containing substances media and without them has been studied. The promising producers of pectin-transeliminases, proteases and model organisms for study of constitutive synthesis of exoenzymes have been selected.