G. A. Velikodvorskaya

Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery

Clostridial acetone–butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis.

A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose

The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley beta-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl beta-D-cellotetraoside, blockage of the hydrolysis of NaBH(4)-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.

The binding pattern of two carbohydrate-binding modules of laminarinase Lam16A from Thermotoga neapolitana: differences in β-glucan binding within family CBM4

Carbohydrate-binding modules (CBMs) are often part of the complex hydrolytic extracellular enzymes from bacteria and may modulate their catalytic activity. The thermostable catalytic domain of laminarinase Lam16A from Thermotoga neapolitana (glycosyl hydrolase family 16) is flanked by two CBMs, 148 and 161 aa long. They share a sequence identity of 30%, are homologous to family CBM4 and are thus called CBM4-1 and CBM4-2 respectively. Recombinant Lam16A proteins deleted for one or both binding modules and the isolated module CBM4-1 were characterized. Proteins containing the N-terminal module CBM4-1 bound to the soluble polysaccharides laminarin (1,3-beta-glucan) and barley 1,3/1,4-beta-glucan, and proteins containing the C-terminal module CBM4-2 bound additionally to curdlan (1,3-beta-glucan) and pustulan (1,6-beta-glucan), and to insoluble yeast cell wall beta-glucan. The activity of the catalytic domain on soluble 1,3-beta-glucans was stimulated by the presence of CBM4-1, whereas the presence of CBM4-2 enhanced the Lam16A activity towards gelatinized and insoluble or mixed-linkage 1,3-beta-glucan. Thermostability of the catalytic domain was not affected by the truncations. Members of family CBM4 can be divided into four subfamilies, members of which show different polysaccharide-binding specificities corresponding to the catalytic specificities of the associated hydrolytic domains.

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

The multi-modular non-cellulosomal endo-1,3(4)-beta-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca2+. The highest affinity to CBMX was shown for xylan (binding constant K=3.1x10(4) M(-1)), yeast cell wall glucan (K=1.4x10(5) M(-1)) and chitin (K=3.3.10(5) M(-1) in the presence of Ca2+). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5

Abstract The upstream region of the cellobiohydrolase gene cbhA of Clostridium thermocellum F7 was sequenced. It was found that this region contains the previously sequenced gene celK encoding an enzyme closely related to CbhA (cellulosomal subunit S3). The presence of a putative transcription terminator in the 524-bp intergenic region indicates that celK and cbhA are not cotranscribed as an operon. Sequence comparison between the two cellobiohydrolases revealed high sequence conservation in the catalytic domain and in the N-terminal cellulose-binding domain (CBD) homologous to CBD family IV, which binds specifically to amorphous cellulose and soluble cellooligosaccharides. In contrast to CbhA, CelK lacks a family III CBD capable of binding to crystalline cellulose. By partial amino acid sequence determination CelK was shown to be identical to cellulosomal subunit S5. CelK and CbhA were found to be members of subfamily E1 of cellulase family E (glycosylhydrolase family 9). Sequence comparison of catalytic domains of family E1 cellulases with C. thermocellum CelD, a family E1 endoglucanase of known three-dimensional structure, revealed a significant variation in the lengths of substrate-binding loops connecting the helices of the (α/α)6 barrel fold. The extended loops of CelK and CbhA might form an active-site tunnel, as found in the catalytic domains of fungal cellobiohydrolases.

Purification and cellulosomal localization ofClostridium thermocellum mixed linkage β-glucanase LicB (1,3–1,4-β-D-glucanase)

SummaryClostridium thermocellum recombinant β-(1,3–1,4)-glucanase LicB was expressed inEscherichia coli K-12 and purified to electrophoretic homogeneity. Specific activity of the final preparation was 12,000 U/mg. Antibodies against recombinant LicB protein reacted with a specific band of purifiedC. thermocellum F7 cellulosomes, suggesting localization in the extracellular multienzyme complex. This indicates that the cellulosome takes part in the hydrolysis of other polysaccharides besides cellulose.

Synergism between Clostridium thermocellum cellulases cloned in Escherichia coli

We have obtained a synergistic effect during degradation of Avicel and filter paper byClostridium thermocellum cellulases (two endoglu-canases and one cellobiohydrolase) cloned inEscherichia coli. The highest degree of synergism was found at early stages of reaction, during the first 20 h: 2.5 and 2.9 on Avicel and filter paper, respectively. During combined action of all three cellulases the main product is cellobiose.

Extracellular glycosyl hydrolase activity of the Clostridium strains producing acetone, butanol, and ethanol

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% wheat flour medium amounted to 12.7–15 g/l with butanol constituting 51.0–55.6%. Activities of these strains towards xylan, β-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acetobutylicum 6, C. acetobutylicum 7, and C. acetobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824 in lab-scale butch cultures. It was demonstrated that starch in the medium could be partially substituted with plant biomass.

Thermoanaerobacter ethanolicus Gene Cluster Containing the α- and β-Galactosidase Genes melA and lacA and Properties of Recombinant LacA

The nucleotide sequence of a 4936-bp genomic DNA fragment from the thermophilic bacterium Thermoanaerobacter ethanolicus has been determined. The fragment contains three open reading frames (ORFs). The product of the incomplete first ORF is highly homologous to α-galactosidases (melibiases). The second ORF corresponds to the lacA gene for a thermostable β-galactosidase. The product of the third ORF is similar to α-D-mannosidases. A putative ρ-independent terminator is located immediately downstream of the lacA stop codon, suggesting a cotranscription of the α- and β-galactosidase genes. The deduced molecular weight of LacA is 86 kDa. LacA belongs to glycosylhydrolase family 2 (GH2). Native recombinant LacA is a dimer and shows the highest activity at pH 5.7–6.0 and 75–80°C. LacA is most active with lactose (480 units per mg protein, Km = 30 mM); the activities with pNP-Gal and oNP-Gal are 330 and 420 units per mg protein, respectively. Immobilized on aldehyde silochrome, LacA is even more thermostable and retains its high activity.

Purification and properties ofClostridium thermocellum endoglucanase 5 produced inEscherichia coli

Endoglucanase 5 (EG5) has been isolated from the strain ofE. coli TGI harboring recombinant plasmid pCU108, which contains thecel5 gene ofC. thermocellum. The enzyme has been produced with 98-fold purification and a final yield of 27% by using subsequent twofold high performance ion-exchange chromatography on Mono Q and high performance chromatofocusing on Mono P. The protein has a mol mass of 35 kDa and includes 3 multiple forms with pI 4.4–4.8 as evidenced by analytical gel isoelectrofocusing. EG5 cleaves CMC (Km = 0.097 g/L, Vmax = 8.2 mg/min·mg of protein), amorphous cellulose, xylan, lichenan as a substrate with an optimum temperature of 80‡C and pH 6.0 and Avicel (Km = 18.2 g/L, Vmax = 0.035 mg/min·mg of protein) with an optimum temperature of 60‡C and pH 6.O. Cellobiose in concentrations up to 200 Μg/mL do not inhibit the hydrolysis of CMC by EG5, but 10–30 Μg/mL of glucose significantly decrease the activity of this enzyme. The stimulating role of calcium chloride and concentration of protein in the system has been demonstrated for Avicel hydrolysis by EG5.