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| Expression, Purification of Mortierella alpina Δ9 Desaturase and Characterization of Its Cytochrome b5 Domain |
| WANG Ming-xuan, CHEN Hai-qin, GU Zhen-nan, CHEN Wei, CHEN Yong-quan |
| School of Food Science and Technology, State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China |
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Abstract The integral Δ9-I desaturase gene from Mortierella alpina ATCC 32222 was expressed and purified from Pichia pastoris. The codon-optimized gene for theΔ9-I desaturase was appended to a cassette containing the human rhinovirus 3C protease cleavage site, the IgG-specific ZZ-tag, and an RGS-His10-tag. The highest expressing clone was determined by Western blotting and SDS-PAGE analysis. A membrane fraction was isolated by gradient centrifugation and detergents were used for efficient extraction of desaturase. Affinity purification of desaturases was conducted using the C-terminally fused IgG-binding ZZ-tag. The enzymatic activity was determined using cell lysate of Saccharomyces cerevisiae as substrate. The cytochrome b5 domain was characterized by wavelength scan and Na2S2O4 reduction experiment. Results showed that the Mortierella alpina Δ9-I desaturase was expressed successfully and transformant with high expression level was selected. The membrane fraction was collected at a centrifugation of 20 000g for 1h and only Fos-Choline-16 effectively extracted the desaturases from the membrane, when compared to the efficiency of SDS. The purified desaturase was intact and the cytochrome b5 domain was present. In the yeast lysate, the conversion rate of C16:0 and C18:0 substrate were (16.88±9.32)% and (20.61±7.55)%, respectively. Wavelength scan of the purified protein revealed an absorbance feature at 411nm and it shifted to 422nm in the presence of Na2S2O4. This result indicated that the cytochrome b5 domain can be reduced in vitro. Thus, the first successful purification of an integral desaturase that contained the fusion cytochrome b5 domain lays the foundation for the mechanism study of heme integral desaturase.
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Received: 06 September 2016
Published: 25 March 2017
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