Chimeric lactase capable of spontaneous and strong immobilization on cellulose and development of a continuous-flow system for lactose hydrolysis at high temperatures.

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.

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.

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity.

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg(-1) protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg(-1) of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.

Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5.

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 (alpha/alpha)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.

Cloning and expression of genes coding for carbohydrate degrading enzymes of Anaerocellum thermophilum in E.coli.

A gene library of Anaerocellum thermophilum Z-1320 was constructed in Escherichia coli using plasmid pUC18. Clones were selected by screening for hydrolysis of CMC, MU-Cel, lichenan and xylan. Recombinant clones expressing two beta-1,4-glucanases and two different xylanases were obtained. Both beta-1,4-glucanases were capable of degrading Avicel. Clones with xylanase activity also cleft PNPXyl, MU-Glu and MU-Cel. The genes of one beta-1,4-glucanase and one xylanase are probably located in the same region of A.thermophilum chromosome.

Cloning and expression in Escherichia coli of Thermotoga neapolitana genes coding for enzymes of carbohydrate substrate degradation.

A genomic library of thermophilic anaerobic eubacterium Thermatoga neapolitana was constructed in E. coli using the pTZ19R plasmid vector. Some groups of recombinant clones with different cellulase activities were revealed: clones carrying genes for an 1,4-beta-glucanases, 1,3-beta-glucanases, beta-xylanases, beta-glucosidases and beta-xylosidases. One clone possessing avicelase activity was obtained. Some clones were selected with amylolytic activities toward amylose, amylopectin and pullulan.

Purification and properties of Clostridium thermocellum endoglucanase 5 produced in Escherichia coli.

Endoglucanase 5 (EG5) has been isolated from the strain of E. coli TG1 harboring recombinant plasmid pCU108, which contains the cel5 gene of C. 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 degrees 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 degrees C and pH 6.0. Cellobiose in concentrations up to 200 micrograms/mL do not inhibit the hydrolysis of CMC by EG5, but 10-30 micrograms/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.

Synergism between Clostridium thermocellum cellulases cloned in Escherichia coli.

We have obtained a synergistic effect during degradation of Avicel and filter paper by Clostridium thermocellum cellulases (two endoglucanases and one cellobiohydrolase) cloned in Escherichia 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.

Cloning and expression of Clostridium thermocellum genes coding for thermostable exoglucanases (cellobiohydrolases) in Escherichia coli cells.

By special screening approach two independent Cl. thermocellum genes directing the synthesis of thermostable glucanases with an exo-mode of action have been isolated from pUC19-based gene bank in E. coli TG1. The genes are located on 3.4 and 11.3 kb DNA fragments showing no homology. E. coli-derived exoglucanases, presumably, cellobiohydrolases, are able to cleave lichenan, carboxymethyl cellulose, xylan and p-nitrophenyl derivatives of cellobioside and lactoside. Cellobiose is the main degradation product of carboxymethyl cellulose, treated with the identified exoglucanases. With p-nitrophenil-beta-D-cellobioside as substrate the enzymes had a pH optimum around 6.5 and a temperature optimum at 65 degrees C. The identified and expressed enzymes differ from all other Cl. thermocellum proteins known to date.

Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli.

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.

Mini-Mu transposition of bacterial genes on the transmissible plasmid.

Using the pRM30 plasmid, an Aps deletion derivative of broad host range plasmid RP4 with integrated new miniMu 5 (11 kb), we followed the transfer of Escherichia coli chromosomal genes to the recipient strain. The miniMu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated miniMu 5 rather than onto the "recipient" plasmid pNH602. The plasmid DNA in recipient cells was detected by electrophoresis. One of the acquired hybrid plasmids pTB2 was analyzed genetically and by restriction endodeoxyribonuclease digestion. A structure consisting of miniMu-chromosomal segment-miniMu as a product of Mu-mediated transposition was detected.