[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

Purification, biochemical characterization, and structure of recombinant endo-1,4-ß-xylanase XylE.

The gene xylE encoding endo-1,4-ß-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding ß-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-ß-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

[Multigene families of endo-(1-4)-beta-xylanases of Penicillium canescens].

Four novel genes of the enzymes of the endoxylanase (EC 3.2.1.8) families found in the mycelial fungus Penicillium canescens have been cloned. The xylB, xylC, and xylD genes encode endoxylanases of glycosyl hydrolase family 11; the xylEgene, those of family 10. In the promoter region of the xylB, xylC, and xylD genes, the binding sequences for the protein activator of xylanolytic gene transcription have been found; the promoter region of the xylB gene contains the binding sequences for the catabolite repression protein. Since the TATAA sequence, which is an element of the minimal eukaryotic promoter, has not been found in the promoter region of the xylC gene, in contrast to those of the xylB and xylD genes, it may be assumed that this gene is silent. Comparative phylogenetic analysis has shown that the cloned genes are highly homologous to some endoxylanase genes of mycelial fungi of the genera Penicillium and Aspergillus. However, within the species P. canescens, they exhibit a low homology both within and between families, and they diverge into different branches of the phylogenetic tree, which suggest divergence of the genes of this group at an early stage of evolution.

[The mutational analysis of carbon catabolite repression in filamentous fungus Penicillium canescens].

Penicillium canescens strain F178 is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. The transcription of genes bgaS and xylA, coding for these proteins, is subject to carbon catabolite repression. The system for selective isolation of regulatory mutants in P. canescens is developed. Two strains from the mutant collection are studied in details. It is shown that both mutations can be complement by creA gene of P. canescens, encoding global regulator of carbon catabolite repression in filamentous fungi. creA(-) alleles contain frameshift mutations in C-domain of CreA. Gene xylA is derepressed in mutants at transcription level in the presence of D-glucose. A transcription of creA gene in mutants is also derepressed proving effect of autoregulation for this gene.

[Transcriptional regulator of carbon catabolite repression CreA in filamentous fungus].

Penicillium canescens strain F178 is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. Tanscription of genes bgaS and xylA coding for these proteins is subject to carbon catabolite repression which proceed mainly in filamentous fungi by transcriptional repressor CreA. creA gene of P. canescens was cloned. It was demonstrated that creA transcription is also subject to carbon catabolite repression. CreA protein remains intranuclear independently of nature of carbon source and glucose concentration in culture medium. In vitro experiments confirm availability of four CreA-binding sites in bgaS promoter, four sites in xylA promoter and one such site in creA promoter.

[A heterologous production of the Trametes hirsuta laccase in the fungus Penicillium canescens].

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta laccase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency ofheterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.

Regulatory activity of heterologous gene-activator xlnR of Aspergillus niger in Penicillium canescens.

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, beta-xylosidase, alpha-L-arabinofuranosidase, alpha-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while beta-galactosidase, beta-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-beta-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous beta-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.

Enzymological properties of endo-(1-4)-beta-glucanase Eg12p of Penicillium canescens and characteristics of structural gene egl2.

Gene egl2 of secreted endo-(1-4)-beta-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding beta-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60 degrees C, respectively, exhibited specific activity of 33 IU, and had K(m) and V(max) in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 micromol/sec per mg, respectively.

[Cloning of the Penicillium canescens endo-1,4-beta-glucanase gene egl3 and the characterization of the recombinant enzyme].

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.

Isolation and characterization of extracellular alpha-galactosidases from Penicillium canescens.

Two alpha-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH- and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding alpha-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both alpha-galactosidases from P. canescens could be successfully used as feed additives. alpha-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and alpha-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder.

Isolation and characterization of extracellular pectin lyase from Penicillium canescens.

Pectin lyase A (molecular weight 38 kD by SDS-PAGE, pI 6.7) was purified to homogeneity from culture broth of the mycelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens (pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH- and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice.

[Cloning of an endo-1,4-beta-xylanase gene from Penicillium canescens and construction of multicopy strains]. / Klonirovanie gena éndo-1,4-ksilanazy Penicillium canescens i polucheni mul'tikopiinykh shtammov.

The complete gene xylA that encodes endo-1,4-beta-xylanase secreted by Penicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven-eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30-50% of the total secreted protein.