Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B

Abstract

To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37% and 109%, respectively, but decreased by 26% and 43%, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris, and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells.

Immobilization of Eversa Lipase on Octyl Agarose Beads and Preliminary Characterization of Stability and Activity Features

“…In the open form, the lid is shifted and exposes to the medium a large hydrophobic pocket containing the active center. This pocket may become adsorbed and stabilized to any hydrophobic surface, like drops of oils (the so-called interfacial activation) [21,22], but also to hydrophobic proteins [23,24], another open form of a lipase [25,26] or a hydrophobic support [27].…”

Immobilization on octyl‐agarose beads and some catalytic features of commercial preparations of lipase a from Candida antarctica (Novocor ADL): Comparison with immobilized lipase B from Candida antarctica

“…In the presence of hydrophobic surfaces, like drops of substrate (e.g., oil drops), or any other hydrophobic surface (a hydrophobic support, a hydrophobic protein, or even other open form of the lipase), this lipase open form becomes adsorbed and this conformation becomes stabilized …”

“…That way, the conformational equilibrium is shifted toward the closed form. [29][30][31][32][33] In the presence of hydrophobic surfaces, like drops of substrate (e.g., oil drops), 34,35 or any other hydrophobic surface (a hydrophobic support, 36 a hydrophobic protein, [37][38][39] or even other open form of the lipase [40][41][42][43], this lipase open form becomes adsorbed and this conformation becomes stabilized. 44 Among the commercially available lipases, the ones from Candida antartica stand out.…”

Cryo-FIB specimen preparation for use in a cartridge-type cryo-TEM

“…Yeast cells from the strain Pichia pastoris were cultured in BMMY media with 1–2% methanol added, following the protocol reported previously (Wang et al, 2016). Cells were cultured for 4 days at 30°C, then harvested by 1000×g centrifugation and re-suspended in water immediately before plunge-freezing.…”