[Ethanol production from materials containing cellulose: the potential of approaches developed in Russia].

Development of domestic studies of cellulose-degrading microorganisms and enzymes is reviewed, with emphasis on the prospects of producing ethanol from cellulose materials using cellulolytic enzymes. Domestic research groups leading in the field are presented. A section of the review analyzes problems and prospects of setting up ecologically safe production of motor biofuels from renewable raw materials of plant origin (an approach developed in Russia).

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.

Cellulase activity of a haloalkaliphilic anaerobic bacterium, strain Z-7026.

The cellulolytic activity of an alkaliphilic obligate anaerobic bacterium, Z-7026, which was isolated from the microbial community of soda-lake sediments and belongs to the cluster III of Clostridia with low G+C content, was studied. The bacterium was capable of growing in media with cellulose or cellobiose as the sole energy sources. Its maximal growth rate on cellobiose (0.042-0.046 h(-1)) was observed at an initial pH value of 8.5-9.0, whereas the maximal rate of cellulase synthesis, assayed by using a novel fluorimetric approach, was found to be 0.1 h(-1) at pH 8-8.5. Secreted proteins revealed high affinity for cellulose and were represented by two major forms of molecular masses of 75 and 84 kDa, whereas the general protein composition of the precipitated and cellulose-bound preparations was similar to cellulosome subunits of Clostridium thermocellum. The optimum pH of the partially purified enzyme preparation towards both amorphous and crystalline cellulose was in the range 6-9, with more than 70% and less than 50% of maximal activity being retained at pH 9.2 and 5.0, respectively.

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).

[Effect of antioxidants on regeneration of protoplasts in the mycelial fungus Trichoderma reesei 6/16]. / Vliianie antioksidantov na regeneratsiiu protoplastov mitselial'nogo griba Trichoderma reesei 6/16.

The relative content of antioxidants in the mycelium of Trichoderma reesei 6/16 obtained by propagation of fungal protoplasts was shown to decrease (as compared to the initial culture taken for preparation of protoplasts) and restored only in the second generation of regenerated mycelium. In this respect, the effects of various antioxidants (beta-carotene, ascorbic acid, alpha-tocopherol, and ionol) on the frequency of regeneration of T. reesei 6/16 protoplasts were studied. beta-Carotene increased the viability of fungal protoplasts to the greatest extent. The effect of ascorbic acid depended on the presence of Fe ions. Ionol did not cause any measurable protective effect.

[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.

The structure and mechanism of action of cellulolytic enzymes.

The modern structural classification of polysaccharases comprising cellulase-hemicellulase enzyme systems is discussed. Their catalytic domains are currently grouped into 15 of more than 80 known glycosyl hydrolase families, whereas substrate binding domains fall into 13 families. The structures of catalytic and substrate binding domains, as well as linker sequences, are briefly considered. A hypothetical mechanism of concerted action of catalytic and substrate binding domains of cellobiohydrolases on the surface of highly ordered cellulose is suggested.

[Cellulases from microorganisms]. / Tselliulazy mikroorganizmov.

Compositions of cellulase-hemicellulase systems of aerobic fungi (hyphomycetes, ascomycetes, and basidiomycetes), aerobic bacteria, actinomycetes, as well as anaerobic fungi and bacteria, are considered in the context of modern structural classification of glycosyl hydrolases. A new nomenclature of cellulases and relative enzymes based on their structural classification is reviewed. Some opportunities of cellulase improvement by means of protein engineering are discussed.

[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.

[Milk ultrafiltrate as a promising source of angiogenin]. / Molochnyi ul'trafil'trat--perspektivnyi istochnik angiogenina.

The use of membrane technologies in the production of soft cheese (children's food) is associated with the appearance of up to 80% of angiogenin in the ultrafiltrate. An electrophoretically homogeneous preparation of angiogenin (MW approximately 17 kDa) was obtained from milk ultrafiltrate by two-stage ion-exchange chromatography. The yield of the angiogenin was approximately 60%, which corresponds to a 586-fold purification of the raw material. The obtained preparation retained stability in the course of lyophilization and could be stored at 4 degrees C for a long time without decomposition.

[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.

[Enzyme regeneration during hydrolysis of steam-pretreated willow and requirement for cellulase complex composition]. / Regeneratsiia fermenta pri gidrolize predobrabotannoi parovmy vzyvom drevesiny ivy i trebovaniia k sostavu tselliulaznogo kompleksa.

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willow Salix caprea by mixed cellulase of Trichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and permanent mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5:1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5-10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identified T. reesei cellobiohydrolase II as the predominant component, whereas clear domination of T. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to hydrolysis residual solids.

[Enzymatic hydrolysis of willow treated with a steam burst without preliminary water extraction with a high concentration of substrate]. / Fermentativnyi gidroliz obrabotannoi parovym vzryvom drevesiny ivy bez predvaritel'noi vodnoi ékstraktsii pri vysokoi kontsentratsii substrata.

A laboratory reactor equipped with a screw press was used for hydrolysis of steam-SO2 exploded willow Salix caprea by a composition of Trichoderma reesei and Aspergillus foetidus enzyme preparations at high substrate concentrations. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude initial washing of steam-pretreated plant raw material by large volumes of water, which is necessary to eliminate the inhibitory effect of explosion by-products on enzymatic hydrolysis. The first procedure included a one-hour-long enzymatic prehydrolysis of the substrate, then separation of sugar syrup containing 40-60 g/l of glucose, 20-25 g/l of xylose, and up to 10% of disaccharides, as well as up to 35% of the initial enzymatic activity, then addition of a diluted acetate buffer (pH 4.5), and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by added enzymes for 24 hours. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons of inhibition of enzymatic hydrolysis are discussed.

[Polyclonal antiserum against noncatalytic part of cellobiohydrolase I from Trichoderma reesei]. / Poliklonal'naia antisyvorotka k nekataliticheskoi chasti tsellobiogidrolazy I iz trichoderma reesei.

A specific antiserum to the noncatalytic part of cellobiohydrolase I from Trichoderma reesei was obtained by exhaustion of rabbit antiserum to the native enzyme with its catalytic domain prepared by papain treatment of cellobiohydrolase I tightly adsorbed onto microcrystalline cellulose.

Enhancement of the affinity of cellobiohydrolase I and its catalytic domain to cellulose in the presence of the reaction product--cellobiose.

The catalytic domain of cellobiohydrolase I from Trichoderma reesei has been obtained by papain treatment of the native enzyme adsorbed onto the surface of microcrystalline cellulose. Both the intact and the truncated enzyme are almost equally active toward soluble fluorogenic derivatives of cellobi-, -tri-, -tetra-, and -pentaose, the fastest and the slowest fluorophore liberation being observed for MUF-cellopenta- and -tetraose, respectively. Titration of the active centers of the intact enzyme and its catalytic domain with MUF-cellotetraose showed their molecular masses to be 49 and 39 kD, respectively, the dissociation constants of the enzyme-soluble ligand complexes being almost equal (65 and 70 nM at 20 degrees C, respectively). In contrast, the intact enzyme and its catalytic core have been shown to significantly (50-60 times) differ in their affinity to insoluble microcrystalline cellulose at low enzyme loading (up to 10 mg per g of the substrate). At 20 degrees C the dissociation constants for the two forms of the enzyme are estimated to be 10 and 500 nM, respectively. Surprisingly, under these conditions the reaction product and inhibitor, cellobiose (Ki = 10 microM), at the concentration 10 mM, increased 3-4-fold the affinity of both the intact cellobiohydrolase and its catalytic domain to cellulose.