Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul;100(13):5883-95.
doi: 10.1007/s00253-016-7431-x. Epub 2016 Mar 12.

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

Pan Wang  1 , Jie He  2 , Yufei Sun  1   3 , Matthew Reynolds  2 , Li Zhang  1 , Shuangyan Han  1 , Shuli Liang  1 , Haixin Sui  2   4 , Ying Lin  5
Affiliations

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

Pan Wang et al. Appl Microbiol Biotechnol. 2016 Jul.

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.

Keywords: Candida antarctica lipase B; Co-display; Hydrophobicity; Hydrophobin; Pichia pastoris.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Figures

Fig.1
Fig.1
Schematic diagram of plasmid construction and outline of the vector expression cassette used in this study. a The vectors pZS61 and pZH61contained a SC3-GCW61 fusion and a HFBI-GCW61fusion, respectively. b The vector pKfC51 contained a CALB-GCW51 fusion. S,alpha signal sequence; TT, transcription termination.
Fig.2
Fig.2
Analysis of the surface hydrophobicity of the three strains. Recombinant cells were induced to display CALB in BMMY medium containing 1% (v/v) methanol for 120 h. a The interfacial phenomena of the recombinant cells. b Hydrophobicity of the cells as measured by the modified microbial adhesion to hydrocarbon method. Data was obtained from three independent experiments.
Fig. 3
Fig. 3
Analysis of cell wall composition of three strains. a Levels of β-1,3-glucan as a percentage of the amount present in strain GS115. b Sensitivity of the three strains to Congo red in YPD medium.
Fig.4
Fig.4
Fluorescence microscopy, flow cytometry and halo formation analysis of the expression of CALB on the surface of recombinant P. pastoris strains. The x-axis of flow cytometry represents the fluorescence intensity, and the y-axis represents the cell count. Strain GS115 was set as the background, and the gray-filled peaks show increased fluorescence intensity on the cell surface.
Fig. 5
Fig. 5
Analysis of the surface hydrophobicity of the three strains co-displaying CALB and hydrophobins. Recombinant cells were induced to display CALB in BMMY medium containing 1% (v/v) methanol for 120 h. The hydrophobicity of the cells was measured using the modified microbial adhesion to hydrocarbon method. Data was obtained from three independent experiments.
Fig.6
Fig.6
Analysis of the hydrolytic and synthetic activity of cells co-displaying CALB and hydrophobins on the cell surface. Recombinant cells were induced to display CALB in BMMY medium containing 1% (v/v) methanol for 120 h. The hydrolytic activity was measured using pNPB as the substrate, and the synthetic activity was evaluated by the synthesis of ethyl hexanoate. Data was obtained from three independent experiments.
Fig.7
Fig.7
Analysis of the degree of glycerol adsorption on the surface of recombinant cell with gas chromatography. The recombinant cells used in the transesterification reaction as whole cell catalysts. After the reactions finished, cell were rinsed with tert-butanol to remove the glycerol adsorbed on the cell surface, and then the amount of glycerol was detected. a GS115/CALB-GCW51, b GS115/SC3-61/CALB-51, and c GS115-51/HFBI-61/CALB-51.
Fig.8
Fig.8
Analysis of recombinant yeast cell wall composition. a Levels of β-1,3-glucan as a percentage of that of strain GS115/CALB-GCW51. b The sensitivity of the three strains to Congo red in YPD medium.
Fig.9
Fig.9
Electron microscopy of the three recombinant yeast strains. The cells were rapidly frozen after methanol induction for 96 h. Following freeze-substitution, infiltration and polymerization, the sample blocks were serially sectioned to a thickness of about 100 nm. The structural preservation is very good as demonstrated in the whole-cell micrographs in a GS115/CALB-GCW51, b GS115/SC3-61/CALB-51, and c GS115-51/HFBI-61/CALB-51. The cell wall regions, marked by the dash-line squares in the left figure column, are further magnified in d GS115/CALB-GCW51, e GS115/SC3-61/CALB-51 and f GS115-51/HFBI-61/CALB-51, respectively. The difference of the inner layers and the outer mannan fibrils are visible among the three strains.
Fig.10
Fig.10
Analysis of the hydrolytic activity of cells co-displaying CALB with different substrates. The three substrates were p-nitrophenyl butyrate (pNPB), p-nitrophenyl caproate (pNPC) and p-nitrophenol octanoate (pNPO), respectively. The methods were the same as analysis of CALB hydrolytic activity with pNPB. Data was obtained from three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bastida A, Sabuquillo P, Armisen P, Fernandez-Lafuente R, Huguet J, Guisan JM. A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng. 1998;58(5):486–493. - PubMed
    1. Brzozowski A, Derewenda U, Derewenda Z, Dodson G, Lawson D, Turkenburg J, Bjorkling F, Huge-Jensen B, Patkar S, Thim L. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature. 1991;351(6326):491–494. - PubMed
    1. Chen B, Pernodet N, Rafailovich MH, Bakhtina A, Gross RA. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading. Langmuir. 2008;24(23):13457–13464. - PubMed
    1. Espino-Rammer L, Ribitsch D, Przylucka A, Marold A, Greimel KJ, Acero EH, Guebitz GM, Kubicek CP, Druzhinina IS. Two novel class II hydrophobins from Trichoderma spp. stimulate enzymatic hydrolysis of poly (ethylene terephthalate) when expressed as fusion proteins. Appl Environ Microb. 2013;79(14):4230–4238. - PMC - PubMed
    1. Hama S, Yoshida A, Nakashima K, Noda H, Fukuda H, Kondo A. Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media. Appl Microbiol Biot. 2010;87(2):537–543. - PubMed

LinkOut - more resources