Preparation of lipases for use in organic solvents

Persson, Mattias; Mladenoska, Irina; Wehtje, Ernst; Adlercreutz, Patrick

doi:10.1016/s0141-0229(02)00184-9

Cited by 129 publications

(78 citation statements)

References 34 publications

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“…Instead, higher concentrations of the additive induce a decrease of enzyme activity. This behavior, which is in agreement with the data reported by Persson et al (2002) with Humicola lanuginosa lipase, appears related to the fact that the crown ether causes conformational changes that eventually lead to enzyme inactivation. The micronization of the enzyme powder by crown ethers (already found by Santos et al (2003) with subtilisin), which would lead to a decrease of diffusional limitations, might also be a valid explanation for the increase of the activity of lipases observed in this study.…”

Section: Discussionsupporting

confidence: 92%

“…A method, commonly employed to overcome this problem and to preserve the enzymatic activity, is the use of proper additives in the lyophilization buffer. Crown ethers (especially 18-crown-6) are among the additives that have a beneficial effect on the activity and enantioselectivity (Mine et al, 2003;Persson et al, 2002;Santos et al, 2001;Tsukube et al, 2001;van Unen et al, 1998) of hydrolases in organic solvents. Concerning the way by which they improve the enzyme activity, van Unen et al (2002) have suggested two simultaneous mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Secundo

Barletta

Dumitriu

et al. 2006

Biotech & Bioengineering

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Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Secundo

Barletta

Dumitriu

et al. 2006

Biotech & Bioengineering

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“…The esterification by the surfactant-coated lipase resulted in a higher rate and yield than that in the direct dispersion or microemulsion system (Okahata and Ijiro, 1988;Okahata and Ijiro, 1992). Lipase absorbed on the surfactant were activated 1-9 to 150 fold compared to the crude lipase since the solubility was extremely low in the reaction media (Persson et al, 2002). The enantioselectively of Pseudomonas cepacia and C. cylindracea was modulated by utilizing the surfactant-coating and molecular imprinting technique.…”

Section: Modification Of Lipasementioning

confidence: 99%

Lipase catalyzed ester synthesis for food processing industries

Rajendran

Palanisamy

Viruthagiri

2009

Braz. arch. biol. technol.

141

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“…However, the practical use of enzymes in organic solvents has been limited due to the lower catalytic activity and lower stability compared with the activity and stability of enzymes in an aqueous phase. Numerous immobilization techniques, including adsorption, entrapment, emulsion, and covalent attachment, have been proposed as alternatives to enhance activity and stability (10,20,22). However, many of these processes are expensive and have various limitations for actual application to industrial production.…”

Section: Discussionmentioning

confidence: 99%

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

Lee¹,

Lee

Park

2005

Appl Environ Microbiol

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We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.

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Preparation of lipases for use in organic solvents

Cited by 129 publications

References 34 publications

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Lipase catalyzed ester synthesis for food processing industries

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

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