On the issue of interfacial activation of lipase in nonaqueous media

Louwrier, Ariel; Drtina, Gary J.; Klibanov, Alexander M.

doi:10.1002/(sici)1097-0290(19960405)50:1<1::aid-bit1>3.3.co;2-o

Cited by 25 publications

(35 citation statements)

References 2 publications

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“…However no such activation was observed in a careful investigation into the existence of interfacial activation of lipase molecules in organic media. [27] For the lyophilised preparation, 70 % of the active sites were titrated after 96 h of titration ( Figure 1). It is possible that the remaining 30 % were reversibly denatured during the freezedrying step [5] or directly denatured by the solvent; this would explain why some proportion of the enzyme molecules are not available for catalysis.…”

Section: Resultsmentioning

confidence: 99%

Factors Governing the Activity of Lyophilised and Immobilised Lipase Preparations in Organic Solvents

2002

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Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze-dried preparation. At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.

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Section: Resultsmentioning

confidence: 99%

Factors Governing the Activity of Lyophilised and Immobilised Lipase Preparations in Organic Solvents

2002

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“…We therefore attempted in the present work to compare a broad range of commercially available lipase preparations under identical reaction conditions. Hydrolysis of phosphatidylcholine (PC) in a reverse micellar system with sodium bis(2-ethylhexyl) sulfosuccinate (AOT) was chosen as the reaction for evaluating the reactivities of the lipases, our reasoning being that, because enzymes added to this system would be dissolved in the reverse micelles and adsorb onto the hydrophobic interface, they would be fully activated by what is known as "interfacial activation" (23), unlike in microaqueous organic media in which transesterification occurs (24).…”

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confidence: 99%

Comparative study of commercially available lipases in hydrolysis reaction of phosphatidylcholine

Hara

Nakashima

Fukuda

1997

J Americ Oil Chem Soc

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Nineteen commercial lipase preparations were compared with respect to their ability to hydrolyze phosphatidylcholine (PC) in a reverse micellar system. In terms of the source organism, lipases from Mucor or Rhizopus species showed comparatively high reactivity, while those from Aspergillus and Humicola sp. exhibited little PC hydrolysis activity. Almost no hydrolysis reaction was observed by positionally nonspecific lipases derived from Candida sp. The results suggest that particular attention should be paid to the source organism in selecting lipases for use in the enzymatic conversion of phospholipids.Complete hydrolysis was established in 1.5-h reaction. c 4 BIU of the enzyme was used. d These lipases were prepared using recombinant DNA technology: the genetic coding for the lipase was transferred from the source organism to a host, Aspergillus oryzae. e No information was found in the brochure obtained from the supplier.

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“…It has been suggested that the lower enzyme activity in organic solvent could be due to the fact that, after lyophilization, the enzyme is in a close conformation (less active) [24] . Then, if an additive (e.g.…”

Section: Why Do Additives Affect the Activity And Enantioselectivity mentioning

confidence: 99%

Importance of Enzyme Formulation for the Activity and Enantioselectivity of Lipases in Organic Solvents

Secundo

2008

Modern Biocatalysis

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5.1 IntroductionLipases are of remarkable practical interest since they have been used in numerous biocatalytic applications, such as kinetic resolution of alcohols and carboxyl esters (both in water and in non -aqueous media) [1] , regioselective acylations of polyhydroxylated compounds, and the preparation of enantiopure amino acids and amides [2,3] . Moreover, lipases are stable in organic solvents, do not require cofactors, possess broad substrate specifi city, and exhibit, in general, a high enantioselectivity. All these features have contributed to make lipases the class of enzyme with the highest number of biocatalytic applications carried out in neat organic solvents.The use of organic solvents is especially advantageous in transforming substrates that are unstable or poorly soluble in water. Furthermore, in the absence of water, the synthesis of ester and amide bonds by hydrolases (mainly lipases and proteases) can be favored over hydrolysis and many side reactions that are water dependent can be prevented. By varying the organic solvent it is also possible to control the substrate specifi city and the regio -and enantioselectivity of a given enzyme. However, although enzymes in organic media show numerous advantages, their catalytic effi ciency is, in most cases, orders of magnitude lower than in aqueous systems. This behavior can be ascribed to different causes such as high saturating substrate concentrations, diffusional limitations, low stabilization of the transition state of the enzyme -substrate intermediate, restricted protein fl exibility, and aggregation and distortions of the enzyme molecules caused by dehydration, which become irreversible in anhydrous solvents [4] .The present chapter briefl y examines and discusses the effects of some additives, namely lyoprotectants (sugars, crown ethers, and methoxypoly(ethylene glycol)), on the activity and enantioselectivity of several lipases. A possible rationale for the increment usually observed when using lyoprotectants, on the basis of conformational data obtained by means of Fourier -transform infrared (FT -IR) spectroscopy is also suggested.

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On the issue of interfacial activation of lipase in nonaqueous media

Cited by 25 publications

References 2 publications

Factors Governing the Activity of Lyophilised and Immobilised Lipase Preparations in Organic Solvents

Factors Governing the Activity of Lyophilised and Immobilised Lipase Preparations in Organic Solvents

Comparative study of commercially available lipases in hydrolysis reaction of phosphatidylcholine

Importance of Enzyme Formulation for the Activity and Enantioselectivity of Lipases in Organic Solvents

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