Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.
The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.
Huge-Jensen
B
B
Novo-Nordisk A/S, Novo Alle, Copenhagen, Denmark.
Andreasen
F
F
Christensen
T
T
Christensen
M
M
Thim
L
L
Boel
E
E
D000596:Amino Acids / Q000032:analysis; D001230:Aspergillus / Q000235:genetics*; D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D007525:Isoelectric Focusing; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D009090:Mucorales / Q000201:enzymology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D000596:Amino Acids; D008049:Lipase