Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

Pencreach, Gaëlle; Baratti, J.

doi:10.1016/0141-0229(95)00120-4

Cited by 152 publications

(73 citation statements)

References 29 publications

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“…A atividade hidrolítica da enzima livre e imobilizada foi determinada usando p-nitrofenilpalmitato (p-NPP, massa molecular = 377,5 g/Mol) como substrato, de acordo com metodologia descrita por Pencreac'h e Baratti 20 . O substrato foi incubado em uma cubeta no espectrofotômetro (Varian, Modelo Cary 50) com circulação de água na temperatura de 37 ºC.…”

Section: Atividade Hidrolíticaunclassified

Comparação do desempenho da lipase de candida rugosa imobilizada em suporte híbrido de polissiloxano-polivinilálcool empregando diferentes metodologias

Paula¹,

Moreira²,

Braga³

et al. 2008

Quím. Nova

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a. Sara Mesquita, 2270, 60511-110 Fortaleza -CE, Brasil Recebido em 16/10/06; aceito em 25/5/07; publicado na web em 9/11/07 COMPARATIVE PERFORMANCE OF Candida rugosa LIPASE IMMOBILIZED ON POLYSILOXANE POLYVINYL ALCOHOL HYBRID SUPPORT USING DIFFERENT METHODOLOGIES. The efficiency for immobilizing microbial Candida rugosa lipase on a hybrid matrix of polysiloxane polyvinyl alcohol, by adsorption, covalent coupling and encapsulation was compared. The activities of immobilized derivatives were evaluated using p-nitrophenylpalmitate (hydrolysis) and butyric acid and butanol (esterification) as substrates. Operational stability and storage tests were also performed. Among the procedures tested, the proposed matrix was efficient for immobilizing C. rugosa lipase by adsorption and covalent coupling techniques and unsuitable for encapsulation purposes. The results reveal that better catalytic properties in both aqueous and organic media were demonstrated by the covalent coupling POS-PVA immobilized lipase, including also satisfactory half-life and good storage stability.Keywords: lipase; hybrid support; adsorption. INTRODUÇÃOEnzimas na sua forma nativa (enzimas livres) têm sido usadas por séculos na indústria de alimentos e mais recentemente nas indústrias farmacêuticas e químicas 1 . Sua estrutura e modo de ação vêm sendo gradativamente elucidados por métodos experimentais bem estabelecidos, como técnicas clássicas de Raio-X e avançadas de Ressonância Nuclear Magnética (RNM). Modernas metodologias de engenharia genética têm possibilitado a produção de enzimas em larga escala ou a modificação de sua estrutura primária, alterando, portanto, algumas de suas características físico-químicas e biológicas 2 . De igual significância são as recentes técnicas de evolução direcionada, as quais têm permitido, via modificação do DNA, a preparação de enzimas especialmente dirigidas para uma determinada finalidade, isto é, enzimas que atuam em valores extremos de pH e temperatura, bem como em presença de meios não convencionais (solventes orgânicos, fase gasosa e CO 2 supercrítico) 3 .O valor de venda de enzimas para uso em indústrias de alimentos, detergentes, especialidades químicas perfizeram um total de aproximadamente US$ 700 milhões em 1992 e aumentaram para US$ 2 bilhões em 2004 4 . De acordo com as projeções, o crescimento no mercado de enzimas é de aproximadamente 4-5% ao ano, acompanhada pela redução de preço, devido ao grande núme-ro de empresas que vêm comercializando enzimas com preços mais competitivos. Como resultado, o mercado previsto para 2009 é da ordem de 2,4 bilhões 4 .De interesse particular é o aumento do uso de enzimas imobilizadas, termo empregado por Katchalski-Katzir 5 na primeira Conferência de Engenharia Enzimática realizada em Henniker, NH, EUA em 1971, para designar enzima fisicamente confinada ou localizada numa certa região do espaço, com retenção de sua atividade catalítica, podendo ser usada repetida e continuamente 6 . Nesta técnica, a enzima fica retida no interior (poros) ou na superfície de u...

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Section: Atividade Hidrolíticaunclassified

Comparação do desempenho da lipase de candida rugosa imobilizada em suporte híbrido de polissiloxano-polivinilálcool empregando diferentes metodologias

Paula¹,

Moreira²,

Braga³

et al. 2008

Quím. Nova

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“…The K m and v max values were 0.7 mg/mL and 0.97 Â 10 À 3 U/min, respectively. For a P. cepacia lipase, Pencreac'h and Baratti (1996) reported K m and v max values of 12 mM and 30 mmol/min, respectively, when the substrate was pNPP. For a lipase of Rho.…”

Section: Purification and Kinetic Characterization Of Lipasesmentioning

confidence: 99%

Production, purification, characterization, and applications of lipases

Sharma

Chisti

Banerjee

2001

Biotechnology Advances

1,269

712

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Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing. D

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“…In heptane, the pNPP of the supernatant was extracted in 0.1 M Tris-HCl (pH 8.0) before measurement. A reaction rate was calculated using a molar extinction coefficient of 12.75 ϫ 10 6 cm 2 /mol (14). One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 mol of pNPP per min.…”

Section: Strains and Media Escherichia Colimentioning

confidence: 99%

“…Hydrolytic activity in aqueous and organic media was measured with p-nitrophenyl palmitate (pNPP) as the substrate (14). In aqueous solvents, the commercial lipases or ROL-displaying yeast cells (0.5, 1.0, or 2.0 mg) were added to 2 ml 0.1 M Tris-HCl (pH 8.0) containing 1.5 mM pNPP, 0.4% (wt/vol) Triton-X, and 0.1% (wt/vol) arabic gum.…”

Section: Strains and Media Escherichia Colimentioning

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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Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

Cited by 152 publications

References 29 publications

Comparação do desempenho da lipase de candida rugosa imobilizada em suporte híbrido de polissiloxano-polivinilálcool empregando diferentes metodologias

Comparação do desempenho da lipase de candida rugosa imobilizada em suporte híbrido de polissiloxano-polivinilálcool empregando diferentes metodologias

Production, purification, characterization, and applications of lipases

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

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