Abstract
A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca2+ and inhibited by Hg2+ and Ag+. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).
Highlights
Lipases (EC 3.1.1.3) are a class of hydrolases that catalyze a variety of reactions, such as the hydrolysis of fatty acid ester, trans-esterification, and ester synthesis at the interface between the insoluble substrate and water [1]
In this article we describe the high-level expression of the ProROL in P. pastoris in a 50-L bioreactor, with the aim of allowing the production of high amounts of the relevant enzyme
ProROL was functionally expressed and secreted in P. pastoris. This is the first report of heterologous high-level expression and characterization of ProROL in
Summary
Lipases (EC 3.1.1.3) are a class of hydrolases that catalyze a variety of reactions, such as the hydrolysis of fatty acid ester, trans-esterification, and ester synthesis at the interface between the insoluble substrate and water [1]. Microbial lipases are currently receiving much attention because of their biotechnological potential, such as broad substrate specificity, high yield and low cost production and so on. They have been widely used in industrial applications, such as biodiesel production, organic synthesis, food, pharmaceutical, and detergents [2,3]. In E. coli, ProROL could be efficiently produced in high yield at high specific activity (166 U/mL) [10] This expression system has the bottleneck that a cell disruption process is necessary, when compared with other expression systems in which the enzyme is secreted in the culture broth. The purification and characterization of the recombinant ProROL (rProROL) was investigated
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