Modulation of the enantioselectivity of lipases via controlled immobilization and medium engineering: hydrolytic resolution of mandelic acid esters
Introduction
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are perhaps the most popular enzymes in biocatalysis [1], [2], [3], [4], [5], [6], [7], [8], [9] because they couple a wide specificity to a high regio and enantioselectivity and specificity, therefore, may be used in many different reactions.
However, lipase catalysis implies dramatic conformational changes of the enzyme molecule. Lipases may be in two different structural forms. One of them, where the active center of the lipase is secluded from the reaction medium by a polypeptide chain called “lid”, is considered an inactive (closed) form. The other one, which presented the lid displaced and the active center exposed to the reaction medium, is considered as the lipase in an active (open) form. The lipase molecule is in equilibrium between the open-active and the closed-inactive structures of the immobilized lipases. This equilibrium shifts towards the open form in the presence of hydrophobic interfaces by the adsorption of the open form [10], [11], [12], [13], [14], [15], [16].
It is very likely that if this equilibrium or the exact shape of the enzyme is altered in any way, the catalytic properties of the enzyme may be dramatically altered. This could be achieved via immobilization techniques involving different areas of the enzyme, giving different rigidity to the enzyme structure or even generating a certain special microenvironment surrounding the enzyme. This could reduce the freedom of the lid of the enzyme to move it, altering the shape of the final open form of the lipase (Scheme 1). In fact, enzyme derivatives from lipases exhibiting different catalytic properties may be found in the literature [17], [18], [19], [20]. Also, the change of the reaction conditions could present an intense effect on the lipases features, perhaps because the change in the global interactions of the open–closed forms of lipase could alter the exact shape of lipase open structure. The interaction between conditions and immobilization protocols could offer also variety of results.
This modulation of the enzyme properties trying to alter the exact form of the active center of lipases via physicochemical tools could be denominated “conformational engineering” and it has been used successfully to modulate the behavior of different enzymes which suffer drastic conformational changes during catalysis penicillin G acylase [21], [22] and lipases [17], [18], [19], [20].
In this paper we have intended to use different immobilization protocols to prepare enzyme derivatives of lipase from Candida rugosa (CRL) involving different areas of the protein, promoting different degrees of enzyme rigidity or altering the microenvironment, together to a study on the effect of the reaction conditions on the different enzyme preparations.
This glycoprotein presents different isoforms and isoenzymes [23], [24] with some differences in their catalytic properties. For this reason, special care has been taken to immobilize all lipase. Nonlipase esterases have been discarded by the previous purification using interfacial adsorption on octyl-agarose [25].
Three different derivatives have been compared (see Scheme 2): interfacially adsorbed lipase (using octyl-Sepharose 4BCL) [25], ionically adsorbed lipase (using polyethylenimine (PEI)-coated Sepabeads) [26], and a covalently immobilized derivative (using glutaraldehyde-Sepabeads) [27], [28], [29]. These three derivatives should be quite different in terms of orientation, environment, and rigidity.
Mandelic acid esters have been used as model substrates to compare the acyl donor and the nucleophile sides of the enzyme. From one side, methyl mandelate placed the chiral group in the acyl donor site, while 2-phenyl-2-butyroylacetic acid placed this group in the nucleophile side.
Optically pure isomers of R- and S-mandelic acid and their esters are very useful in organic synthesis. (R,S)-2-Phenyl-2-butyroylacetic acid used as displacer with the Cyclobond-II chiral stationary phase eliminates much of the trial and error effort traditionally involved in the development of a displacement chromatographic separation on cyclodextrin silica stationary phases [30]. R-Mandelic acid is used for synthesis of the interesting antibiotics (cephamandole and cephonicid) [31] and optically pure acids may be used in the resolution of racemates by selective precipitation [32].
Section snippets
Materials
The lipase from C. rugosa (Tipo VII) (specific activity 875 U/mg solid), Triton X-100, p-nitrophenyl propionate (pNPP), butyric acid ethyl ester, and (R,S)-mandelic acid methyl ester were from Sigma. Octyl-agarose 4BCL was purchased from Pharmacia Biotech (Uppsala, Sweden). Sepabeads FP-EA (amine) and Sepabeads FP-EP (epoxide) were kindly donated by Resindion srl. Glutaraldehyde-Sepabeads and PEI-Sepabeads were prepared as previously described [26], [27], [28], [29]. (R,S
Immobilization of Candida rugosa lipase on different supports
The immobilization courses of the lipase from C. rugosa on the different supports followed by pNPP hydrolysis are shown in Fig. 2. Using a hydrophobic support like octyl-agarose, the immobilization was extremely rapid (Fig. 2a), with no soluble lipase activity detectable after 60 min of incubation and promoting a certain degree of activity hyperactivation (by twofold factor).
When the glutaraldehyde-Sepabeads support was used to prepare the covalent derivative, the immobilization rate was much
Discussion
The results showed in this paper suggest that the properties of a lipase may be strongly modulated via the so called “conformational engineering”: directed immobilization (altering rigidity and environment) and design of the experimental conditions.
Thus, for a fixed condition, the same lipase immobilized on different supports having different rigidity and microenvironment exhibited very different catalytic properties: different activity (even by a 10-fold factor) and different E value. Thus,
Acknowledgements
The authors gratefully recognize the support from the Spanish CICYT with the project BIO2000-0747-C05-02. Authors thanks CAM for a postdoctoral fellowship for Dr. Fernández-Lorente and a Ph.D. fellowship for J.M. Palomo. We thank to Resindion srl for the gift of Sepabeads resins and we gratefully recognize the help from Daminati (Resindion).
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