[Heterologous expression and characterization of Yarrowia lipolytica lipase 4 and lipase 5 in Pichia pastoris].
OBJECTIVE
To clone cDNA sequences of lipase 4 (LIP4) and lipase 5 (LIPS), analyze gene structures and express them in Pichia pastoris so as to investigate their enzymatic characteristics.


METHODS
We first cloned cDNA sequences of LIP4 and LIP5 by reverse transcription PCR and analyzed their gene structures by SignalP 3.0. Then, intracellular expression vectors pPIC3. 5K-Lip4, pPIC3. 5K-Lip5 and inducible secretion vectors pPIC9K-Lip4, pPIC9K-Lip5 were constructed. All vectors were transformed into Pichia pastoris GS115 by electroporation, resulting in a series of engineered strains. After fermentation and NTA-Ni resin purification, the enzymatic properties of LIP4 and LIP5 were examined.


RESULTS
The cloned cDNA sequences revealed that there was no intron in both of Lip4 and Lip5. The two lipases both contained catalytic triads and conserved GHSLG motifs. Their optimal substrate, pH, temperature were respectively pNP-caprylate (C8), 7.0 and 40 degrees C. The activities of LIP4 and LIPS were 10.16 U/mg and 5.1 U/mg, respectively. It was found that LIP4 was more sensitive to the variations of pH and temperature than LIP5. LIP4 and LIP5 could both be stimulated by Ca2+, besides LIPS could also be activated by Mg2+. They were both strongly inhibited by Hg2+, Phenylmethanesulfonyl fluoride (PMSF) and Dithiothreitol (DTT).


CONCLUSIONS
The cloning of Lip4 and Lip5, expression in P. pastoris and characterization of their properties would offer a solid basis for their large-scale production and future application. In addition, the results also enriched the data for a systematic research on the lipase gene family of Y. lipolytica.
Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Heyun
H
Zhao
Xiao
X
Xiao
Li
L
Xu
Yun
Y
Liu
Yunjun
Y
Yan
D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008049:Lipase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005810:Multigene Family; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D025062:Yarrowia / Q000737:chemistry / Q000201:enzymology*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase