Expression and characterization of Aspergillus niger endoglucanase and Trichoderma reesei cellobiohydrolase

Abstract

Cellulases are industrially important hydrolytic enzymes especially in the bioconversion of agro-industry waste materials. Two cellulases with high activity against cellulose hydrolysis were chosen as the targeted extracellular recombinant cellulases to be produced in methylotrophic yeast in which heterologous protein expression is strongly drived by a methanol induced promoter. EglA from Aspergillus niger is an endoglucanase which is highly active towards fJ-glucan hydrolysis while CbhIl Trichoderma reesei is a cellobiohydrolase which hydrolyses specifically at cellulose chain ends to produce cellobiose. Full length cDNAs of eglA and cbhIl have been amplified by Reverse- Transcription PCR and cloned into cloning vector. Specific primers were designed to amplify the mature endoglucanase and cellobiohydrolase genes and fused in frame with the AOXl promoter, a-factor signal peptide and 6xHis tag sequences in the pPICZaC expression vector. The expression plasmids were linearized and transformed separately into Pichia pastoris yeast cells via electroporation. Integration of the targeted gene into the yeast genome was verified by PCR and multi-copy integrants were screened using high concentration ofZeocin antibiotic. EglA and CbhIl were expressed as a ~30 kDa and ~ 60 kDa extracellular recombinant proteins from the yeast. Both of the polyhistidine tag fusion proteins were purified using immobilized metal affinity chromatography and the yield of pure CbhII and EglA from P. pastoris yeast were approximately I mglL and 9 mglL respectively. Biocharacterization of the recombinant EglA showed that it has an optimum activity at pH 4.0 and 50??C and retained more than 70% of the residual activity at 60??C. High affinity of EglA towards fJ-glucan was revealed from the KM value of 88.93 mglmL and specific activity of 63.83 Ulmg respectively. EglA has shown a significant increment activity of 2.7 fold with the addition of manganese ions in the endoglucanase assay. CbhII produced an optimum activity at pH 6.0 and 50??C and stable within the range of 30 ??C-50 C with more than 50% of res idual activity remained at 55 ?? C.