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

Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD