Cloning and Expression of Pseudomonas fluorescens 26-2 Lipase Gene in Pichia pastoris and Characterizing for Transesterification

Abstract

Pseudomonas lipases are important biocatalysts widely used in a variety of industrial fields. An extracellular lipase gene lipA with 1,854-bp open reading frame was cloned from Pseudomonas fluorescens 26-2. The multialignment assay of the putative amino acid and the secondary structure prediction revealed this enzyme could be classified into the lipolytic subfamily I.3 and secreted via adenosine-triphosphate-binding cassette pathway. The lipA gene was integrated into Pichia pastoris GS115, and the methanol-inducible recombinants with Mut(S) and Mut(+) phenotypes were acquired. The characteristics and the transesterification capacity shown by this enzyme suggested it is a useful biocatalyst for biodiesel preparation.

Molecular Cloning and Heterologous Expression of a True Lipase in <b><i>Pichia pastoris</i></b> Isolated via a Metagenomic Approach

“…The enzymatic bioconversion of olive oil into biodiesel was carried out as described by Yang et al [2009]. In brief, transesterification was conducted in a 50-ml shaking flask for 54 h at 37 ° C with rotary shaking at 200 rpm.…”

“…High-level expression of enzymes is regarded as a prerequisite for commercial biotechnological production. To date, several microbial lipase genes have been cloned and expressed in P. pastoris [Luo et al, 2006;Shi et al, 2010;Yang et al, 2009].…”

lip2, a novel lipase gene cloned from Aspergillus niger exhibits enzymatic characteristics distinct from its previously identified family member

“…A unique property of lipases that distinguishes them from other enzymes is their enhanced activity at an oil-water interface (interfacial activation). Apart from their hydrolysis capacity in aqueous medium, lipases in non-aqueous medium are efficient catalysts in the kinetic resolution of chiral compounds, synthesis of esters and peptides, and preparation biofuel by transesterification (Alatorre-Santamaría et al 2009;Yang et al 2009). The lipases from Aspergillus niger have positional selectivity toward the sn-1,3 positions of the glycerol ester with substrate preference for medium-chain length fatty acids, an optimal pH between 4 and 7.…”

Cloning of Lipase Gene From Thermomyces langinosus into Pichia pastoris with its Original Signal Peptide

“…Recombinant enzymes in eukaryotic systems are a good alternative to expression of enzymes extracellularly (Yang et al 2009). Recombinant lipase enzyme can be expressed and secreted in the methylotrophic yeast P. pastoris.…”

“…The pAOX1-regulated systems are the most common (Cregg et al 2003). T. lanuginosus lipase (Zheng et al 2011), Rhizopus chinensis lipase (Yuet al 2009), Pseudomonas fluorescens (Yang et al 2009) were expressed under the alcohol oxidase promoter. Many researchers used AOX1 promoter to control expression of heterologous protein, because the promoter was able to control protein expression in large number with methanol as sole carbon source (Cereghino and Cregg 2000).…”