Surface display of active lipases Lip7 and Lip8 from Yarrowia lipolytica on Saccharomyces cerevisiae.
Lipase has been used widely in industry. In this study, we have constructed two recombinant Saccharomyces cerevisiae strains displaying two active lipases on the cell surface by cell surface engineering. The genes encoding Yarrowia lipolytica lipases Lip7 and Lip8 were fused with the gene encoding small binding subunit Aga2 of a-agglutinin. Localization of the Lip7 and Lip8 on the cell surface was confirmed by immunofluorescence microscopy. Besides, the putative signal sequences of Lip7 and Lip8 were removed to compare their effect on the activities of surface-displayed lipases. The results showed that the activities towards p-nitrophenyl caprylate of surface-displayed Lip7 and Lip8 were 283 U/g (dry cell) and 121 U/g (dry cell), much higher than that using Flo1 as anchor protein in Pichia pastoris, and the putative signal sequences have significant effect on the activities of the displayed lipases; when deleted, the lipases' activities were declined to 65 U/g (dry cell) and 80 U/g (dry cell), respectively. The displayed lipases exhibit a preference for middle chain fatty acids and a high thermal stability. Additionally, from the study, to surface-display a target protein, it is recommendable that the structure feature of the protein should be assayed through bioinformatics methods and then the cell wall proteins with the anchor domain far away from the activity center should be chosen as anchor proteins.
Liu
Wen-Shan
WS
Key Laboratory of Molecular Biophysics, the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Pan
Xiao-Xing
XX
Jia
Bin
B
Zhao
He-Yun
HY
Xu
Li
L
Liu
Yun
Y
Yan
Yun-Jun
YJ
D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013499:Surface Properties; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase