Expression of Endopolygalacturonase A of Aspergillus oryzae in Escherichia coli

Abstract:

Pectinases are mainly used in the food industry to clarify fruit juices and wine, improve oil extraction, remove the peel from the citrus fruit, increase the firmness of some fruits and degum fibres. The filamentous fungus Aspergillus oryzae, used for the production of traditional fermented foods, only could produce less pectinases under general conditions. So far only a few of PGs expressed in yeast or E.coli were reported but they did not show higher activity. The cDNA of mature PGA (without signal peptide) was synthesized with specific primers from total RNA of Aspergillus oryzae by RT-PCR. PGA cDNA was ligated into pET-28a(+) expression vector, creating plasmid pET-28a(+)-pgA. The plasmid pET-28a(+)-pgA was transformed into E.coli Turner (DE3) placⅠcells to express PGA heterogeneously. For improving the efficiency of PGA expression in E.coli, the conditions for expression of the PGA in E.coli were optimized. E.coli Turner (DE3) placⅠcells with pET-28a(+)-pgA was first cultivated at 37℃，220r/min until OD_600nm reached about 0.8. Then, cultivation broth was added with 0.5 mmol/L IPTG and incubated at 15℃, 170r/min for other 24 h for induced-expression of PGA. Our data showed that the activity of recombinant expressed PGA could reach to 70u/mL medium, which is 87.5-fold of the activity of PGA produced in culture of A. oryzae and superior than known recombinant expression amount of PGA reported by other researchers.