Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Abstract

Two alternative cell-surface display systems were developed in Pichia pastoris using the alpha-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins alpha-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with alpha-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with alpha-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using alpha-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with alpha-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

“…P. pastoris GS115 and expression plasmid pPIC9K were purchased from Invitrogen Corporation (Carlsbad, CA). The vector pKNS-CALB, which contained the mature CALB cDNA, has been described previously (20). P. pastoris GS115 cells were grown in yeast peptone dextrose medium (1% yeast extract, 2% tryptone, 2% dextrose) and on minimal dextrose (MD) agar plates (1.34% yeast nitrogen base, 2% dextrose, 2% agar) supplemented with 0.4 mg/liter biotin.…”

“…The disruption of GAS1 in P. pastoris production strains for obtaining human trypsinogen and human serum albumin did not result in an enhancement of product secretion, whereas Rhizopus oryzae lipase secretion could be improved 2-fold (17). In addition, GPI-modified cell wall proteins from S. cerevisiae, such as ␣-agglutinin, Tip1p, Flo1p, and Sed1p, have been used as anchor proteins for P. pastoris cell surface display of heterologous proteins (18)(19)(20). However, very few endogenous GPImodified cell wall proteins of P. pastoris have been confirmed and used in P. pastoris cell surface display.…”

Poly(styrene–divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

“…A small amount of water is essential for the enzymatic activity during ester synthesis reaction and excess water leads to a lower reaction rate as reported earlier. The necessary amount of water for the optimal esterification activity of lipases in organic media depends on the origin of lipase, the surface properties of supports and the polarity of organic solvents [44,45]. In this study, the effect of water content on the esterification activity of immobilized lipase for synthesis of butyl butyrate from butyric acid and butyl alcohol was carried out in n-heptane medium by addition of different amount of water.…”

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

“…The secretory route of cell wall proteins has been exploited to link heterologous proteins to the cell wall of yeasts (Su et al 2010;Müller 2011;Tanaka et al 2012) including C. utilis (Kunigo et al 2013). Surface (cell wall) display requires an N-terminal secretion signal sequence and a specific Cterminal signal sequence for addition of a GPI-anchor; the GPI anchor is cleaved at the plasma membrane and the protein is linked via its GPI remnant to ß-1,6-glucan in the cell wall (Eisenhaber et al 2004;Frieman and Cormack 2004).…”