Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

Murai, Toshiyuki; Ueda, Mitsuyoshi; Atomi, Haruyuki; Shibasaki, Yumi; Kamasawa, Naomi; Osumi, Masako; Kawaguchi, Takashi; Arai, Motoo; Tanaka, Atsuo

doi:10.1007/s002530051086

Cited by 68 publications

(39 citation statements)

References 18 publications

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“…Yeast cell surface engineering (12,13) is an alternative approach that immobilizes an enzyme on the yeast cell surface. Proteins are immobilized by using an outer shell cell wall protein, the C-terminal half of ␣-agglutinin (10,26).…”

mentioning

confidence: 99%

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Shiraga

Kawakami

Ishiguro

et al. 2005

Appl Environ Microbiol

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Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with ␣-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 ؋ 10 4 -fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 ؋ 10 4 -fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H 2 O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three-to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.Lipases (EC 3.1.1.3) catalyze the hydrolysis of acylglycerides and other fatty acid esters under aqueous conditions and the synthesis of esters in organic solvents (5, 25). Lipase-catalyzed reactions have high substrate specificity or regioselectivity, so lipase-catalyzed ester synthesis in organic solvents is potentially an industrially feasible alternative to conventional chemical methods (1, 4). However, organic solvents may alter the structure and activity of enzymes that usually function in an aqueous environment.One alternative is to immobilize the enzymes on solid supports in order to increase their function and stability in response to organic solvents or increased temperatures. Enzymes may be stabilized by chemical and physical processes (6,15,20,24). With chemical methods enzymes are immobilized by strong covalent bonding, but changes in protein structure often result. In physical stabilization processes, the interactions between enzymes and solids usually are weaker, resulting in fewer changes in the enzyme's structure.Yeast cell surface engineering (12, 13) is an alternative approach that immobilizes an enzyme on the yeast cell surface. Proteins are immobilized by using an outer shell cell wall protein, the C-terminal half of ␣-agglutinin (10, 26). The gene encoding Rhizopus oryzae lipase (ROL), which has been used to produce diesel fuels (8), was fused to a DNA sequence encoding the C-terminal half of yeast ␣-agglutinin and expressed in Saccharomyces cerevisiae. The transformants have hydrolytic...

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confidence: 99%

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Shiraga

Kawakami

Ishiguro

et al. 2005

Appl Environ Microbiol

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“…Phage display is a cell-based method in which proteins are expressed in Escherichia coli. Another display technique using living cells is cell-surface display ( Figure 3B) in which proteins are displayed on the surface of living cells, such as yeast (Georgiou et al, 1997;Murai et al, 1997) or mammalian cells (Wolkowicz et al, 2005). These cell-based display techniques have some weaknesses; the library size is limited by the number of cells and transformation efficiency (typically below 10 9 ), and some proteins that are toxic to the cell are excluded from the library.…”

Section: Mrna Display For In Vitro Selection Of Proteinsmentioning

confidence: 99%

“…Proteins are displayed on a filamentous phage by fusing them to coat proteins of the phage. (B) Cell-surface display (Georgiou et al, 1997;Murai et al, 1997;Wolkowicz et al, 2005). Proteins are displayed on the surface of living cells.…”

Section: Mrna Display For In Vitro Selection Of Proteinsmentioning

confidence: 99%

Evolutionary Engineering of Artificial Proteins with Limited Sets of Primitive Amino Acids

Tanaka¹,

Yanagawa²,

Doi³

2012

Protein Engineering

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“…Unlike secreted enzymes, such displayed enzyme is readily produced at a low cost and is "naturally" immobilized onto the cell surface. Therefore, no tedious purification or immobilization processing is required (Murai et al, 1997). Moreover, such immobilization offers enzymes a physical support that often improves thermostability and facilitates reusability (Shusta et al, 1999;Park et al, 2006;Tanino et al, 2006;Gai and Wittrup, 2007).…”

Section: Introductionmentioning

confidence: 99%

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Guo

Zhang

et al. 2010

J. Zhejiang Univ. Sci. B

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Abstract:The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

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Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

Cited by 68 publications

References 18 publications

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Evolutionary Engineering of Artificial Proteins with Limited Sets of Primitive Amino Acids

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

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