Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica.
To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.
Tamalampudi
Sriappareddy
S
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan.
Talukder
Md Mahabubur Rahman
MM
Hama
Shinji
S
Tanino
Takanori
T
Suzuki
Yuya
Y
Kondo
Akihiko
A
Fukuda
Hideki
H
D001236:Aspergillus oryzae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D002384:Catalysis; D018914:Cells, Immobilized; D004951:Esterification; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica