The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

Background  

Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

A combinatorial biocatalysis approach to an array of cholic acid derivatives

A 39-member library of bile acid derivatives was prepared starting from 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid methyl ester using a combinatorial biocatalytic approach. A regioselective oxidation step, catalyzed by hydroxysteroid dehydrogenases, followed by an acylation step with a series of different acyl donors catalyzed by Candida antarctica lipase B, led to the modification of the bile acid scaffold. Each member of the library was obtained in high purity and good yield.     

Spectroscopic investigation of lipase from Pseudomonas cepacia solubilized in 1,4‐dioxane by non‐covalent complexation with methoxypoly(ethylene glycol)

Lipase from Pseudomonas cepacia was made soluble in 1,4‐dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme–PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far‐ and near‐UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near‐UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase–PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme–substrate interactions. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 624–629, 1999.     

Molecular mechanism of deactivation of C. antarctica lipase B by methanol

The catalytic activity of Candida antarctica lipase B upon alcoholysis of a constant concentration of 15.2% vinyl acetate (vol/vol) and varying concentrations of methanol (0.7–60%) in toluene was determined experimentally by measuring the initial reaction velocity. The molecular mechanism of the deactivation of the enzyme by methanol was investigated by fitting the experimental data to a kinetic model and by molecular dynamics simulations of C. antarctica lipase B in toluene–methanol–water mixtures.     

Different Structural Behaviors Evidenced in Thaumatin-Like Proteins: A Spectroscopic Study

Three proteins belonging to the thaumatin-like proteins family were compared in this study from a structural point of view: zeamatin, a new recently isolated PR-5 from Cassia didymobotrya and the commercial sweet-thaumatin. The former two proteins possess antifungal activities while commercial thaumatin is well known to be a natural sweetener. Intrinsic fluorescence studies have evidenced that the three proteins behave differently in unfolding experiments showing different structural rigidity. All the three proteins are more stable at slight acidic buffers, but sweet-thaumatin has a major tendency to destructurate itself. Similar observations were made from circular dichroism studies where a structural dependence relationship from the pH and the solvent used confirmed a hierarchic scale of stability for the three proteins. These structural differences should be considered to be significant for a functional role.     

Structural and functional characterization of the porcine proline-rich antifungal peptide SP-B isolated from salivary gland granules.

A 1905-Da cationic proline-rich peptide, named SP-B, was recently isolated by our group as the main component of salivary gland granules, and its primary sequence fully characterized by means of automated Edman sequencing and LC-MS/MS tools. In the present study SP-B is shown to possess antifungal activity when challenged with strains of Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, while only negligible antibacterial activity was detected. Furthermore, SP-B was found to be non-cytotoxic when tested on fibroblast cell lines. To obtain information regarding its structure affinity, capillary electrophoresis (CE), circular dichroism (CD) and attenuated total reflection (ATR)-FT/IR experiments were performed. CE revealed a pH dependence of the hydrodynamic radial dimensions both in aqueous and 2,2,2-trifluoroethanol solutions. CD and ATR-FT/IR measurements confirmed the structure-pH relationship, revealing a secondary structure composed of mixed proportions of polyproline-II, unordered and turn motifs, the last being more evident in the zwitterionic form of the peptide. From these findings SP-B peptide could be classified as a new member of the proline-rich antimicrobial peptide family.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.