[Expression of endopolygalacturonase A of Aspergillus oryzae in Escherichia coli].
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) plac I cells 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) plac I cells with pET-28a( + )-pgA was first cultivated at 37 degrees, 220r/min until OD600nm reached about 0.8. Then, cultivation broth was added with 0.5 mmol/L IPTG and incubated at 15 degrees C, 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.
Zhang
Yu-Ling
YL
Jiangsu Key Laboratory for Biodiversity and Biotechnology , Key Laboratory for Microbial Technology in the College of Life Sciences, Nanjing Normal University, Nanjing 210097, China.
Zhao
Qing-Xin
QX
Zhu
Hong
H
Sun
Jing
J
Han
Feng-Min
FM
Yuan
Sheng
S
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D055162:Biocatalysis; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011096:Polygalacturonase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D011096:Polygalacturonase