Search

Abstract: Aims: Isolate and characterize a laccase‐encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long‐distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563‐pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α‐factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6‐dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I‐encoding gene could be successfully sequenced having cis‐acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α‐factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis‐acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively. [ABSTRACT FROM AUTHOR]

Aims: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential., Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. K m and highest k cat /K m values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees., Conclusions: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes., Significance and Impact of the Study: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively., (© 2018 The Society for Applied Microbiology.)

Aims: Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences., Methods and Results: Fermenter-scale production led to productivity (P(r)) and yield (Y(P/C)) values higher at 48 h (P(r) = 0.542 g l(-1) h(-1); Y(P/C) = 0.74) than at 72 h (P(r) = 0.336 g l(-1) h(-1); Y(P/C) = 0.50). Both EPSs were neutral glucose-homopolysaccharides with a beta-(1,3)-glycosidic backbone and single beta-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm(-1) confirmed beta-glycosidic linkages, while gentiobiose released by beta-(1,3)-glucanases confirmed single beta-1,6-glycosidic branching for both EPSs., Conclusions: The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour., Significance and Impact of the Study: Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.

The measurement of the colony radial growth rate (Kr) on solid medium of colonies of Sclerotium rolfsii Proimi F-6656 for the evaluation of scleroglucan production medium and other different media, incubation temperature and tolerance to diverse concentrations of sucrose and NaCl were studied. The optimum growth temperature observed was 30 degrees C. The Kr value reached on the Production Medium used (0.66 mm.h-1) showed no differences compared with those of the other media tested, indicating that all the requirements for growth were provided. Poor growth was only observed on Soil Extract Agar. The fungus tolerated concentrations of sucrose from 0.15 to 1.17 M, on both Czapek and production medium. Growth was limited by the highest concentrations of sucrose tested (0.88 and 1.17 M), as indicated by a slower increase in colony size. Addition of 0.86 M NaCl to the production medium and YM agar did not inhibit growth completely, but decreased the radial growth rate considerably (80 and 70% respectively).