Properties of neutral cellobiose dehydrogenase from the ascomycete Chaetomium sp. INBI 2-26(-) and comparison with basidiomycetous cellobiose dehydrogenases.

The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.

Cellobiose dehydrogenase formation by filamentous fungus Chaetomium sp. INBI 2-26(-).

Laccase-negative filamentous fungus INBI 2-26(-) isolated from non-sporulating laccase-forming fungal association INBI 2-26 by means of protoplast technique was identified as Chaetomium sp. based on partial sequence of its rRNA genes. In the presence of natural cellulose sources, the strain secreted neutral cellobiose dehydrogenase (CDH) activity both in pure culture and in co-culture with laccase-positive filamentous fungus INBI 2-26(+) isolated from the same association. INBI 2-26(-) also secreted CDH during submerged cultivation in minimal medium with glucose as the sole carbon source. Maximal CDH activity of 1IU/ml at pH 6 with 2,6-dichlorophenolindophenol (DCPIP) as an acceptor was obtained on 12th day of submerged cultivation with filter paper as major cellulose source. Cellulase system of Chaetomium sp. INBI 2-26(-) capable of adsorption onto H(3)PO(4)-swollen filter paper consisted of four major proteins (Mr 200, 95, 65 and 55K) based on SDS-polyacrylamide gel electrophoresis and was capable of DCPIP reduction without exogenous cellobiose.

[Degradation of the herbicide atrazine by the soil mycelial fungus INBI 2-26(-)--a producer of cellobiose dehydrogenase]. / Razlozhenie gerbitsida atrazina pochvennym mitselial'nym gribom INBI 2-26(-)--produtsentom tsellobiozodegidrogenazy.

Nonsporulating mycelial fungi producing cellobiose dehydrogenase (CDH) and isolated from soils of South Vietnam with high residual content of dioxins are capable of growing on a solid medium in the presence of high atrazine concentrations (to 500 mg/l). At 20 and 50 mg/l atrazine, the area of fungal colonies was 1.5-1.2-fold larger, respectively, compared with control colonies of the same age, whereas development of the colonies at 500 mg/l atrazine was delayed by 5 days, compared with controls grown in the absence of atrazine. Surface cultivation of the fungus on a minimal medium with glucose as a sole source of carbon and energy decreased the initial concentration of atrazine (20 mg/l) 50 times in 40 days; in addition, no pronounced sorption of atrazine by mycelium was detected. This was paralleled by accumulation in the culture medium of extracellular CDH; atrazine increased the synthesis of this enzyme two- to threefold. Accumulation of beta-glucosidase (a mycelium-associated enzyme) and cellulases preceded the formation of CDH.

[Decomposition of natural aromatic structures and xenobiotics by fungi]. / Razlozhenie prirodnykh aromaticheskikh struktur i ksenobiotikov gribami.

The review deals with transformation of natural and synthetic aromatic compounds by fungi (causative agents of white rot, brown rot, or soft rot, as well as soil filamentous fungi). Major enzyme types involved in the transformation of lignin and aromatic xenobiotics are discussed, with emphasis on activity regulation under the conditions of secondary metabolism and oxidative stress. Coupling of systems degrading polysaccharides/lignin and non-phenolic lignin structures (without the involvement of lignin peroxidase) is analyzed, together with non-enzymatic mechanisms (involving lipoperoxide free radicals, cation-radicals, quinoid mediators, or transition metal ions). Metabolic pathways resulting in the formation of aromatic and haloaromatic compounds in fungi are described. Consideration is given to the mechanisms of fungal adaptation to aromatic xenobiotics.

[Degradation of lignin-carbohydrate substrate by soil fungi--producers of laccase and cellobiose dehydrogenase]. / Razlozhenie lignouglevodnogo substrata pochvennymi gribami--produtsentami lakkazy i tsellobiozodegidrogenazy.

The growth of nonsporulating mycelial fungi INBI 2-26(+), producer of laccase; INBI 2-26(-), producer of cellobiose dehydrogenase; and their mixed culture on lignin-carbohydrate substrates under conditions of submerged fermentation were studied. The degrees of degradation of lignin, cellulose, and hemicellulose of cut straw over 23 days amounted to 29.8, 51.4, and 72% for the laccase producer; 15.8, 33.9, and 59.1% for the cellobiose dehydrogenase producer; and 15.8, 39.4, and 64.5% for the mixed culture, respectively. The laccase activity in the medium when strain 2-26(+) was cultivated individually reached its maximum on day 28; the activity of cellobiose dehydrogenase of strain 2-26(-), on days 14 to 28. A method for determining cellobiose dehydrogenase activity in the presence of laccase was developed. In the mixed culture, both enzymes were formed; however, the level of laccase synthesis was 1.5-fold lower compared to that of strain 2-26(+), while synthesis of cellobiose dehydrogenase was similar to that of the corresponding producer. Cellobiose dehydrogenase failed to boost the action of laccase while degrading the lignin of straw.

[Fungal decomposition of oat straw during liquid and solid state fermentation]. / Razlozhenie ovsianoi solomy gribami pri zhidkofaznom i tverdofaznom kul'tivirovanii.

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.

[Isolation and characterization of a cellobiose dehydrogenase formed by a asporogenic mycelial fungus INBI 2-26(-)]. / Vydelenie i kharakteristika tsellobiozodegidrogenazy, obrazuemoi nesporuliruiushchim mitselial'nym gribom INBI 2-26(-).

A nonsporulating fungus isolated from dioxine-containing tropical soils forms cellobiose dehydrogenase, when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M(r) of 95 kDa; its pH optimum was in the range 5.5-7.0; more than 50% activity was retained at pH 4.0-8.0 (citrate-phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K(M) values at pH 6.0 equaled 4.5 +/- 1.5 and 56 microM) in the presence of dichlorophenolindophenol (K(M) app = 15 +/- 3 microM at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl-beta-D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited affinity to amorphous cellulose. At 55 degrees C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the single-electron acceptor cytochrome c3+ (K(M) app = 15 microM at pH 6.0).

[Consumption of the triazine herbicide atrazine by laccase and laccase-free variants of the soil fungus Mycelia sterilia INBI-2-26]. / Potreblenie triazinovogo gerbitsida atrazina lakkaznym i bezlakkaznym variatami pochvennogo griba Mycelia sterilia INBI 2-26.

Asporogenic fungus Mycelia sterilia INBI 2-26 isolated from tropical soils with high residual dioxin content (as a result of Agent Orange defoliant treatment during the Vietnamese-American war) and capable of atrazine decomposition was treated to obtain protoplasts. This technique resulted in isolation of laccase-positive and laccase-negative clones. Atrazine consumption by liquid surface cultures of Mycelia sterilia INBI 2-26 was monitored by using enzyme immune assay and reversed phase HPLC. Atrazine (20 micrograms/l) stimulated fungal growth. Laccase-positive clone consumed up to 80% of atrazine within four weeks. However, no correlation of atrazine consumption and laccase activity in the culture medium was observed. Moreover, the laccase-negative clone was also capable of consuming at least 60-70% of atrazine within three weeks. Surprisingly, in the corresponding control set (cultivation of laccase-negative clone without atrazine) an unidentified metabolite having a retention time and UV-spectrum similar to those of atrazine was also found. It was concluded that the presence of laccase was not a crucial factor in atrazine consumption by this fungus.

[Immunoenzyme analysis of decomposition of herbicides by soil and wood-rot fungi]. / Immunofermentnyi analiz razlozheniia gerbitsida pochvennymi i drevorazrushaiushchimi gribami.

The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicillium sp. 13 and noncellulolytic ones Humicola lanuginosa spp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using enzyme immunoassay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. Hyphomycete Mycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80-90% in 40 days. Mycelia sterilia INBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.

[Isolation and characteristics of micromycetes--producers of neutral phenol oxidase from trophic soil with a high level of dioxins]. / Vydelenie i kharakteristika mikromitseta--produtsenta neitral'nykh fenoloksidaz iz tropicheskikh pochv s vysokim soderzhaniem dioksinov.

Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with elevated phenol oxidase activity. As a result, fast-growing non-sporulating strain producing neutral phenol oxidases was isolated and identified as Mycelia sterilia INBI 2-26. The strain formed extracellular phenol oxidases during surface growth on liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of cultural liquid has revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by ultrafiltration, ion exchange chromatography and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40 degrees C they retained at least 50% of activity after incubation for 50 h. At 50 degrees C PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3-6 h. The pH optimums for PO1 and PO2 towards catechol were equal to 6 and 6.5, and the Km values were 1.5 +/- 0.35 and 1.25 +/- 0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 with Km values 1.6 +/- 0.18 and 0.045 +/- 0.01 mM, respectively, but displayed no activity towards tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.

[Genetic mapping and characteristics of the actinophage phi C31 deletion mutants of Streptomyces coelicolor A3(2) incapable of lysogenization]. / Geneticheskoe kartirovanie i kharakteristika deletsionnykh mutantov aktinofaga fi C31 Streptomyces coelicolor A3(2), ne sposobnykh k lizogenizatsii.

Actinophage phi C31 deletion c mutants with impaired ability to make repressor were genetically studied. Genetic crosses indicate that the c28 deletion mutant is situated with the c-region of the phi C31 genetic map. Based on the results of a qualitive test for recombination between several c mutants, a scheme of their order relative to deletion mutants was presented. The approximate distances between eight c mutants have been represented in units of the physical DNA map estimation. Genetic studies of actinophage lyg deletion mutants which cannot lysogenize sensitive cultures were carried out. Mutants failed to lysogenize upon mixed infection with lyg+ phages. The absence of the effect of lyg+ gene in trans suggests that lyg deletions cause a structural defect in an integration site of the phage. Preliminary data on alignment of lyg positions on physical and genetic maps of phi C31 phage have been obtained. According to evidence from genetic crosses, lyg mutation has been located in the right half of the phi C31 genome.

[Physical mapping of Streptomyces coelicolor A3(2) actinophage DNA. II. Transposon-like structure in DNA molecules of phiC43 phage DNA, localization of region responsible for establishment of the lysogenic state]. / Fizicheskoe kartirovanie DNK aktinofagov Streptomyces coelicolor A3(2). II. Transpozonopodobnaia struktura v molekuliakh DNK faga phiC43, lokalizatsiia oblasti, otvetstvennoi za ustanovlenie lizogennogo sostoianiia.

Structural properties of DNA molecules of phages phi C43, phi C43del and mutant phage phi C311yg33 were studied. Actinophages phi C43del and phi C311yg33 have been isolated and shown to have a phenotype characteristic of phages defective in integration, i. e. turbid plaques and inability to establish the lysogenic state. By means of heteroduplexing, deletions were mapped in the genomes of these phages. DNA molecules of phi C43del and phi C11yg33 are devoid of the common fragment, which suggests that the mutant phenotypic character is associated with structural alterations in DNA molecules. The presence of transposon-like structure in phi C43 DNA molecules has been inferred from the analysis of phi C43/phi C43del heteroduplexes and phi C43 homoduplexes. Also, a deletion in phi C43 genome has been detected covering the same region where deletions in phi C43del and phi C11yg33 DNAs were located.

[Physical mapping of Streptomyces coelicolor A3(2) actinophage DNA. I. Location of the c-region of actinophage phiC31]. / Fizicheskoe kartirovanie DNK aktinofagov Streptomyces coelicolog A3(2). I. Lokalizatsiia c-oblasti aktinofaga phiC31.

Mutants of temperate actinophage phi C31 Streptomyces coelicolor A3(2) having an increased resistance to chelating agents (sodium EDTA and sodium citrate) were isolated. Most of these mutants were not able to lysogenize sensitive cultures (c-mutants). DNA molecules of four c-mutants resistant to chelating agents were shown to be deleted by electron microscopy of DNA heteroduplexes. The four deletions were located in the central region of phi C31 DNA molecule. The deleted segment of 1000 base pairs common for molecules of all c-mutants is located in a region 47,2--49,9% and indicates the position of c-region on the physical map of phi C31 actinophage. The size of the region containing delections of all analyzed c-mutants is 1700 base pairs. The c-region on the heteroduplex map was oriented in relation to the known genetic map of phi C31.

[Determination of Streptomyces coelicolor A3(2) resistance to erythromycin]. / Determinatsiia ustoichivosti Streptomyces coelicolor A3(2) k éritromitsinu.

Resistance to erythromycin is genetically unstable in strains of Streptomyces coelicolor A3(2). The frequent loss of resistance as well as reversion of sensitive variants to the original unstable resistance phenotype excluded the possibility that plasmid elimination is involved. The spontaneous frequency of occurrence of sensitive clones was 0.14 to 1.5%, the rate of reversion ranging from 1.10(-6) to 1.10(-8). Resistance to erythromycin has been mapped on the chromosomes of two S. coelicolor A3(2) derivatives in different sites: between markers adeC (v 10) and ArgA1 in the strain A617, between pheA1 and SCP1 in the strain S18. It is suggested that genetic instability of erythromycin resistance determinants having chromosomal location is due to transposition of genetic material.