Expression of Rhizopus oryzae lipase gene in Saccharomyces cerevisiae

Ueda, Mitsuyoshi; Takahashi, Shouji; Washida, Masanobu; Shiraga, Seizaburo; Tanaka, Atsuo

doi:10.1016/s1381-1177(02)00018-8

Cited by 34 publications

(19 citation statements)

References 75 publications

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“…This yeast is generally recognized as a safe micro-organism (GRAS), lacking endotoxins and lytic viruses, being able to perform many post-translational modifications, including glycosylation, acylation and folding of proteins (Alberghina et al, 1991). S. cerevisiae has been used as a host micro-organism to produce different heterologous proteins such as β-galactosidase (Cheng et al, 1997), glucoamylase (Hardjito et al, 1993), α-2,6(N)-sialyltransferase (Borsig et al, 1995), antithrombotic hirudin (Kim et al, 2003), sporamin (Chen et al, 2003), Closterium sex pheromone (Sekimoto, 2002), cutinase (Calado et al, 2002), Rhizopus oryzae lipase (Ueda et al, 2002), capsid proteins of human polyomaviruses BK and JC (Hale et al, 2002), human interleukin-18 (Lim et al, 2002), α 1 -antitrypsin (Tamer and Chisti, 2001), human XPA and XRCC1 DNA repair proteins (Pushnova et al, 2001), Aspergillus ficuum endoinulinase (Park et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Fed-batch bioreactor process with recombinant Saccharomyces cerevisiae growing on cheese whey

Rech

Ayub

2006

Braz. J. Chem. Eng.

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-Saccharomyces cerevisiae strain W303 was transformed with two yeast integrative plasmids containing Kluyveromyces lactis LAC4 and LAC12 genes that codify β-galactosidase and lactose permease respectively. The BLR030 recombinant strain was selected due to its growth and β-galactosidase production capacity. Different culture media based on deproteinized cheese whey (DCW) were tested and the best composition (containing DCW, supplemented with yeast extract 1 %, and peptone 3 % (w/v)) was chosen for bioreactor experiments. Batch, and fed-batch cultures with linear ascending feeding for 25 (FB25), 35 (FB35), and 50 (FB50) hours, were performed. FB35 and FB50 produced the highest β-galactosidase specific activities (around 1,800 U/g cells), and also the best productivities (180 U/L.h). Results show the potential use of fed-batch cultures of recombinant S. cerevisiae on industrial applications using supplemented whey as substrate.

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Section: Introductionmentioning

confidence: 99%

Fed-batch bioreactor process with recombinant Saccharomyces cerevisiae growing on cheese whey

Rech

Ayub

2006

Braz. J. Chem. Eng.

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“…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).…”

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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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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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“…Experiments exposing Lip7p and Lip8p to the surface of S. cerevisiae revealed that the putative signal sequences of the enzymes were necessary for maximum enzyme activities . It was speculated that the signal peptides assist in correct folding of the lipases and sustaining protein stability as has been shown before for pro-sequences of lipases from R. oryzae (Ueda, 2002).…”

Section: Yeast Lipolytic Enzymes Different From Tgl3p Tgl4p and Tgl5pmentioning

confidence: 83%

Triacylglycerol lipases of the yeast

Grillitsch

Daum

2011

Front. Biol.

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All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.

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Expression of Rhizopus oryzae lipase gene in Saccharomyces cerevisiae

Cited by 34 publications

References 75 publications

Fed-batch bioreactor process with recombinant Saccharomyces cerevisiae growing on cheese whey

Fed-batch bioreactor process with recombinant Saccharomyces cerevisiae growing on cheese whey

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

Triacylglycerol lipases of the yeast

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