[Expression of pectin lyase 1 from Aspergillus oryzae in Escherichia coli].
Pectin lyases from Aspergillus oryzae and Aspergillus niger are usually used for the production of traditional fermented foods, but these fungi produce less pectinases under natural conditions. The cDNA coding mature Pell (without signal peptide) was amplified from Aspergillus oryzae by RT-PCR. Pell cDNA was cloned into pET-28a ( + ) expression vector, then was transformed into E. coli Turner (DE3) plac I cells to express Pell with 6-His tag. For improving the efficiency of Pell expression in E. coli, the conditions of expressing the Pell in E. coli were optimized. E. coli Turner (DE3) plac I cells with pET-28a ( + )-pell was first cultivated at 37 degrees C, 220 r/min until OD600 reached about 0.8. Then, cultivation broth was added with 0.05-0.1 mmol/L IPTG and continuously incubated at 15 degrees C, at 170 r/min for 60 h for expressing of Pell. The recombinant expressed Pell activity could reach 400 u/mL medium, which is 4000-fold of Pell produced naturally by A. oryzae and superior than known recombinant amount of pectin lyases expressed in different fungi expression systems.
Zhao
Qing-Xin
QX
Jiangsu Key Laboratory for Biodiversity and Biotechnology, Key Laboratory for Microbial Technology in the College of Life Science, Nanjing Normal University, Nanjing 210097, China.
Yuan
Sheng
S
Zhang
Yu-Ling
YL
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C027349:pectin lyase