Recombinant Candida rugosa LIP2 expression in Pichia pastoris under the control of the AOX1 promoter

Ferrer, Pau; Alarcón, Manuel; Ramon, Ramon; Benaiges, M. Dolors; Valero, Francisco

doi:10.1016/j.bej.2009.05.018

Cited by 28 publications

(17 citation statements)

References 42 publications

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“…This tendency to form multimolecular aggregates has also been reported in the lipase from C. parapsilosis [58] and in the sterol esterases from Streptomyces species [6]. In addition, recombinant C. rugosa LIP2, expressed in P. pastoris , resulted in an aggregated, inactivated form of the protein, and only after diaultrafiltration lipolytic activity was recovered [10]. Monomolecular forms from C. rugosa , Humicola lanuginosa (synonym T. lanuginosus ) , and Mucor miehei lipases were found only at low enzyme concentrations and in the presence of detergents.…”

Section: Discussionmentioning

confidence: 53%

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Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

2012

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BackgroundThe ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture.ResultsRecently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steady-state kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme.ConclusionP. pastoris resulted to be an optimum biofactory for the heterologous production of recombinant sterol esterase from O. piceae, yielding higher activity levels than those obtained with the saprophytic fungus. The enzyme showed improved kinetic parameters because of its modified N-terminus, which allowed changes in its aggregation behaviour, suggesting that its hydrophobicity has been modified.

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Section: Discussionmentioning

confidence: 53%

“…An additional reason to choose this system to express the O. piceae sterol esterase was that other fungal lipases have been successfully expressed in this yeast, such as the different isoenzymes (Lip1-Lip5) from C. rugosa [8,10,40-44] and G. candidum [45,46], as well as the sterol esterase from M. albomyces [15]. …”

Section: Discussionmentioning

confidence: 99%

Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

2012

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“…The supernatant was concentrated by ultrafiltration with a Centrasette V R Pall Filtron (Pall, New York) system equipped with an Omega membrane of 10 kDa cut-off, and dialyzed against 10 mM Tris-HCl buffer, pH 7.5, and thereafter lyophilized. 36 …”

Section: Methodsmentioning

confidence: 97%

Immobilization and stability of a Rhizopus oryzae lipase expressed in Pichia pastoris: Comparison between native and recombinant variants

Guillén

Benaiges

Valero

2011

Biotechnology Progress

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The stability of a soluble extract containing a recombinant lipase from Rhizopus oryzae (Cursive) lipase (rROL) produced by Pichia pastoris (Cursive), as well as that for the commercial extract containing the lipase produced by the native organism (nROL), was investigated. The results showed higher residual activity values of the commercial protein compared with the recombinant one. Moreover, two different kinds of support, the polypropylene powder EP100 and Eupergit®C, were tested to immobilize the enzymes. The residual activity of the immobilizated derivatives was also tested to determine whether their stability was enhanced. The results showed a slight improvement in rROL using both supports but a decrease in nROL using Eupergit®C. The study of the residual activity of soluble and immobilized enzymes was performed by means of a central composite rotatable experiment design. In addition, EP100 adsorption isotherms were determined.

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“…The culture broth was centrifuged and microfiltered to remove the biomass. The supernatant was concentrated by ultrafiltration with a Centrasette Pall Filtron system equipped with an Omega membrane of 10 kDa cut‐off, and subsequently dialyzed against 10 m M Tris‐HCl buffer (pH = 7.5) and thereafter lyophilized 41…”

Section: Methodsmentioning

confidence: 99%

Immobilized Heterologous Rhizopus Oryzae Lipase as an Efficient Catalyst in the Acetylation of Cortexolone

et al. 2012

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The enzymatic preparation of a monoacetyl derivative of the corticosteroid cortexolone, through a transesterification reaction, is described. The heterologous Rhizopus oryzae lipase, immobilized on three different supports, proved to be an efficient catalyst in the acylation reaction using a complex substrate such as cortexolone. Immobilization of the enzyme on Lewatit 1600 resin at pH = 7 and 25 °C was the best condition for catalysis of the acetylation reaction. The influence of various reaction parameters, such as the nature of the acetylating agent, the solvent, the temperature, and the ratios of acetylating agent to substrate, and enzyme to substrate, was evaluated. Using the response surface methodology and a central composite rotatable design, the specific yield of acetylated cortexolone was optimized by means of the study of the effect of the enzyme (E)/substrate (S) and the acylating agent (A)/substrate ratios. The ratios of 5 (E/S) and 31.6 (A/S) were predicted as the optimal values to reach the maximum specific yield of the product (P): 1.59 mmol P/mmol A·g E. The mild reaction conditions and low environmental impact make the biocatalytic procedure a convenient way to prepare the reported derivative of this biologically active steroid.

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Recombinant Candida rugosa LIP2 expression in Pichia pastoris under the control of the AOX1 promoter

Cited by 28 publications

References 42 publications

Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

Immobilization and stability of a Rhizopus oryzae lipase expressed in Pichia pastoris: Comparison between native and recombinant variants

Immobilized Heterologous Rhizopus Oryzae Lipase as an Efficient Catalyst in the Acetylation of Cortexolone

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