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The Thermostability of Two Kinds of Recombinant ∆6-Fatty Acid Desaturase with Different N-Terminal Sequence Lengths in Low Temperature

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Abstract

Two recombinant Rhizopus stolonifer ∆6-fatty acid desaturase enzymes with different-length N-termini were cloned and expressed in Saccharomyces cerevisiae strain INVScl: LRsD6D begins with the sequence of the N-terminal of the R. stolonifer ∆6-fatty acid desaturase native, encoding a deduced polypeptide of 459 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A-M-K-F), whereas SRsD6D begins with the amino acid sequence of the predicted ORF, encoding a deduced polypeptide of 430 amino acids (M-K-F) and LRsD6D is longer than SRsD6D by 29 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A). Bioinformatic analysis characterized the two recombinant ∆6-fatty acid desaturase enzymes with different-length N-termini, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5-like domain in the N-terminus. When the coding sequence was expressed in S. cerevisiae strain INVScl, the coding produced ∆6-fatty acid desaturase activity exhibited by RsD6D, leading to a novel peak corresponding to γ-linolenic acid methyl ester standards, which was detected with the same retention time. The residual activity of LRsD6D was 74 % at 15 °C for 4 h and that of SRsD6D was 43 %. Purified recombinant LRsD6D was more stable than SRsD6D, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant LRsD6D.

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Acknowledgments

This research was supported by the Key Project of Chinese Ministry of Education, China National “948” Program, China National “863” Program, etc.

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Authors and Affiliations

  1. Department of Microbiology, Chongqing University of Medical Sciences, Chongqing, 400016, People’s Republic of China

    He Lu

  2. Department of Immunology, Chongqing University of Medical Sciences, Chongqing, 400016, People’s Republic of China

    Yu Zhu

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  1. He Lu
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  2. Yu Zhu
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Correspondence to He Lu.

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Lu, H., Zhu, Y. The Thermostability of Two Kinds of Recombinant ∆6-Fatty Acid Desaturase with Different N-Terminal Sequence Lengths in Low Temperature. Appl Biochem Biotechnol 171, 165–172 (2013). https://doi.org/10.1007/s12010-013-0363-3

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  • DOI: https://doi.org/10.1007/s12010-013-0363-3

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