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Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
21072 43205 2011 5 PDF Available
Title
Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris
Abstract

Hydroxynitrile lyase gene (hnl) from Eriobotrya japonica was successfully amplified using the method of SEFA PCR (Self-Formed Adaptor PCR). The complete sequence was 5.5 kbp in length, including 3100 bp of the upstream promoter region, 1659 bp of the coding sequence, three introns and 315 bp of the downstream transcription terminator. The phylogenetic analysis illustrated that the obtained hnl exhibited 66–70% identity to the reported isozymes from almond, black cherry and Japanese apricot. The EjHNL had 552 amino acids including a 25 amino acid-long signal peptide. The conserved characteristic structures of HNLs, such as FAD-binding motif, N-glycosylation sites and active sites were observed. The coding sequence of the hnl was inserted into pPIC9K vector for heterologous expression in Pichia pastoris. The HNL activity of the culture supernatant reached 15 U/ml after 96 h of induction by methanol. The specific activity of the recombinant HNL was about 197 U/mg. The enantiomeric excess value of the product R-mandelonitrile attained 98.6% and the value of Km of the recombinant HNL was determined to be 0.47 mM based on the kinetic data. The optimum temperature and pH of the recombinant HNL were 40°C and 6.0 respectively. The experimental data indicated that the obtained recombinant HNL showed similar catalytic characteristics with the natural EjHNL. The expression of the recombinant HNL in P. pastoris could present another available biocatalyst for the synthesis of R-selective cyanohydrins.

Keywords
EjHNL, hydroxynitrile lyase from Eriobotrya japonica; PaHNL, hydroxynitrile lyase from Prunus amygdalus (almond); PmHNL, hydroxynitrile lyase from Prunus mume (Japanese apricot); PsHNL, hydroxynitrile lyase from Prunus serotina (black cherry).Hydroxynitri
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Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 112, Issue 4, October 2011, Pages 321–325
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
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Any Questions? feel free to contact us