Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel

Biotechnology for Biofuels
Jinjin HuangYing Li

Abstract

We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased tra...Continue Reading

Citations

Sep 5, 2015·International Journal of Molecular Sciences·Grazia M Borrelli, Daniela Trono
May 28, 2015·Journal of Industrial Microbiology & Biotechnology·Dong HeZhenhong Yuan
Jan 4, 2015·Biotechnology Letters·Tanja NarancicKevin E O' Connor
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Mar 4, 2014·PloS One·Martin Axelsson, Francesco Gentili
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Datasets Mentioned

BETA
A02536.1

Methods Mentioned

BETA
protein folding
glycosylation
electrophoresis

Software Mentioned

NetNGlyc
DNAMAN

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