Abstract
BackgroundFor industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.ResultsTwo Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the α-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40°C at pH8.0, they retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.ConclusionThe LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.
Highlights
For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes
TFhigeusrteru1cture of the expression plasmid pLHJ047 and p LHJ048 The structure of the expression plasmid pLHJ047 and p LHJ048. 5'AOX1, alcohol oxidase 1 promoter; S, α-factor signal sequence for secretion in Pichia; FLO, FLO gene coding for FLO protein from Saccharomyces cerevisiae; lipB52, lipase gene lipB52 cloned in frame and downstream of the FLO sequence; 3'AOX1(TT), transcriptional terminator from pichia pastoris AOX1gene; HIS4, pichia wild-type gene coding for histidinol dehydrogenase and used to complement Pichia his4- strains; kanr, Kanamycin resistance gene from Tn903 which confers resistance to G418 in Pichia; 3'AOX1, Sequences from the AOX1 gene that are further 3' to the TT sequences; CoE1, E. coli origin of replication; Ampr, Ampicillin resistance gene
The recombinant plasmids linearized with Sal I were electroporated into Pichia pastoris KM71 as described in Methods
Summary
The utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes. Pseudomonas lipases display special biochemical characteristics differing from those produced by other microorganisms [1,4,5,6]. The lipase LipB52 encoded by lipB52 (GenBank accession number AY623009), isolated from Pseudomonas fluorescens B52, displayed high level enantioselectivity with (R)-tert-leucinate [7]. The utilization of surface-displayed lipase in the form of wholecell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzyme, and can be separated . The P. pastoris expression system has gained acceptance as an important host organism[12], and is common for high level expression of foreign proteins
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