Cloning And Expression Of Endoglucanase And Exoglucanase From Aspergillus Niger

In the natural course of cellulose hydrolysis into glucose, it relies on the synergy of the three components to degrade cellulose into glucose.? Endoglucanase acts on long-chain molecules of cellulose of the fiber and cuts it into short fibers,?while?Exo-glucanase acts on cellulose molecules at one end and generates cellodextrin and cellobiose by cutting two units of glucose residue once.β- glucosidase takes charge of cutting cellobiose into glucose.?It relies on the synergy ofβ- 1, 4 - endoglucanase and exo-glucanase to decompose cellulose into small molecules of cellobiose which can be eventually transformed into glucose byβ- glucosidase, so the first two components play a crucial role in cellulose degradation.Design two primers according to endoglucanase gene (egA) of Aspergillus niger in Genebank, five introns were found within the gene after TA cloning which were removed by PCR with five primer designed. Design upstream and downstream primers with EcoRⅠand XbaⅠrestriction sites at both ends. Aspergillus niger endoglucanase expression plasmid pPICZαA-Aeng was built after restriction enzyme digestion and ligation. Plasmid pPICZαA-Aeng was transformed into Pichia pastoris competent cells GS115 by electroporation after linearizing by the restriction enzyme BstⅠ. Pichia pastoris recombinants with high copy number were acquired by antibiotic gradient plate screening when the resistance was up to 500μg / ml Zeocin. Two recombinants were screened out by Congo red plate and expression of endoglucanase gene appeared in flask culture induction.A gene encoding an exo-β-1,4-glucanase was isolated from Aspergillus niger and designated as CBH II with a length of 1,548 bp. The deduced amino acid sequence encoded by CBH II showed 96.69% homology with the sequence of a not-well-characterized cellulase encoded by cbhB.The molecular weight of the enzyme was about 67 kDa. To confirm the sequence of the gene encoding the stable exo-β-1,4-glucanase , the cloned gene was ligated with a vector pPICZαA and expressed in Pichia pastoris, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The optimal temperature and pH for enzyme activity was 60℃and 6.6 separately and was stable in the range of pH 4.0-9.0.