Expression optimisation of recombinant α-Larabinofuranosidase from Aspergillus niger ATCC 120120 in Pichia pastoris and its biochemical characterisation

A gene encoding α-L-arabinofuranosidase (AnabfA) from Aspergillus niger ATCC 120120 was successfully cloned and expressed in Pichia pastoris under the control of the AOX1 promoter. The effect of cultural conditions on recombinant AnabfA production was studied and the enzyme was expressed as a soluble protein. Recombinant AnabfA was expressed as an active enzyme at 28°C when cultured in BMMY medium (pH 6.0) and induced with 2% methanol every 24 h. Maximum activity was observed 5 days after induction. The purified recombinant AnabfA before and after treatment with PNGase F migrated by SDS-PAGE had relative molecular masses of about 83 and 66 kDa, respectively, suggesting that the AnabfA contains N-linked oligosaccharides. Characterisation of the purified recombinant AnabfA showed an optimum temperature and pH of 50°C and 4, respectively. The enzyme was stable at a pH of 3 to 6 and retained more than 80% of its activity after pre-incubation at 40°C for 30 min. The recombinant AnabfA activity was stimulated by K+, Mn2+, Na2+ and triton X-100 and was strongly inhibited by Cu2+ and Fe2+ and the enzyme activity was relatively unaffected by Ca2+, CO2+, Mg2+ and EDTA. The Km and Vmax of the purified recombinant AnabfA activity towards ρNPA were 0.93 mM and 17.86 μmol/ml/min, respectively.Key words: Aspergillus niger, α-L-arabinofuranosidase, expression, Pichia pastoris, characterisation

Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris>/i>

Cellulases are industrially important hydrolytic enzymes applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, an endoglucanase from Aspergillus niger ATCC 10574, EglA, was expressed in the methylotrophic yeast Pichia pastoris and the properties of the recombinant protein were characterized. The full length cDNA of eglA has been cloned into a pPICZαC expression vector and expressed extracellularly as a ~30 kDa recombinant protein in P. pastoris X-33. Pure EglA displayed optimum activity at 50°C and was stable between 30 and 55°C. The pH stability of this enzyme was shown to be in the range of pH 2.0 to 7.0 and optimum at pH 4.0. EglA showed the highest affinity toward β-glucan followed by carboxymethyl cellulose (CMC) with a specific activity of 63.83 and 9.47 U/mg, respectively. Very low or no detectable hydrolysis of cellobiose, laminarin, filter paper and avicel were observed. Metal ions such as Mn2+, Co2+, Zn2+, Mg2+, Ba2+, Fe2+, Ca2+ and K+ showed significant augmentation of endoglucanase activity, with manganese ions causing the highest increase in activity to about 2.7 fold when compared with the control assay, whereas Pd2+, Cu2+, SDS and EDTA showed inhibition of EglA activity.Key words: Cellulase, endoglucanase, recombinant, Aspergillus niger, Pichia pastoris

Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1-independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/ unites/RIF/transcriptdata/.status: publishe

Fluorescence and evaporative light scattering HPLC profiling of intracellular asparagine (N)-linked oligosaccharides from Saccharomyces cerevisiae using the alg8 mutant

N-glycans are biologically important oligosaccharides associated with the asparagine residue that may exist in protein-bound or unbound forms in all eukaryotes (including yeasts) and some bacteria. The- core structure of these oligosaccharides is based on the trimannosyl chitobiose structure resulting from cellular N-glycosylation. Preparative-scale amounts of these oligosaccharides are important for chemical, structural and functional studies due to their biological significance. Therefore, we explored a biochemical approach of oligosaccharide preparation using mutant-derived monoglucosylated lipid-linked oligosaccharides (LLOs) required for the assembly of N-linked glycoproteins and non-monoglucosylated free-oligosaccharides (fOSs) from misfolded N-linked glycoproteins using an N-glycosylation (alg) mutant of Saccharomyces cerevisiae. Oligosaccharide extracts of fOSs and LLOs from the alg8 S. cerevisiae mutant lacking the ALG8 gene were profiled using fluorescence- and evaporative light scattering-based HPLC. LLOs did not produce accumulated levels of the target mutant- related monoglucosylated (Glc1Man9GlcNAc2) at 100 ml scale. However, it was possible to detect truncated oligomannose (paucimannose) structures in the fOSs of the alg8 mutant

Fluorescence and evaporative light scattering HPLC profiling of intracellular asparagine (N)-linked oligosaccharides from Saccharomyces cerevisiae using the alg8 mutant

N-glycans are biologically important oligosaccharides associated with the asparagine residue that may exist in protein-bound or unbound forms in all eukaryotes (including yeasts) and some bacteria. The- core structure of these oligosaccharides is based on the trimannosyl chitobiose structure resulting from cellular N-glycosylation. Preparative-scale amounts of these oligosaccharides are important for chemical, structural and functional studies due to their biological significance. Therefore, we explored a biochemical approach of oligosaccharide preparation using mutant-derived monoglucosylated lipid-linked oligosaccharides (LLOs) required for the assembly of N-linked glycoproteins and non-monoglucosylated free-oligosaccharides (fOSs) from misfolded N-linked glycoproteins using an N-glycosylation (alg) mutant of Saccharomyces cerevisiae. Oligosaccharide extracts of fOSs and LLOs from the alg8 S. cerevisiae mutant lacking the ALG8 gene were profiled using fluorescence- and evaporative light scattering-based HPLC. LLOs did not produce accumulated levels of the target mutant- related monoglucosylated (Glc1Man9GlcNAc2) at 100 ml scale. However, it was possible to detect truncated oligomannose (paucimannose) structures in the fOSs of the alg8 mutant