Mats Holmquist

On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase

The interfacial activation of Candida antarctica lipase A (CALA) and B (CALB) has been investigated and compared with that of Humicola lanuginosa lipase (HLL). CALB displayed no interfacial activation towards p-nitrophenyl butyrate (PNPB) when exceeding the solubility limit of the substrate. No activation was observed towards p-nitrophenyl acetate (PNPA) at the addition of sodium dodecyl sulfate (SDS) nor in the presence of a solid polystyrene surface. The catalytic action of CALB was very different from that of Humicola lanuginosa lipase, which showed a pronounced interfacial activation with the same substrates. The basis for the anomalous behaviour of CALB is proposed to be due to the absence of a lid that regulates the access to the active site. In contrast to CALB, CALA expressed interfacial activation, but the activation was not as prominent as for Humicola lanuginosa lipase (HLL). The structural basis for the activation of CALA is unknown.

LIPASES FROM RHIZOMUCOR MIEHEI AND HUMICOLA LANUGINOSA : MODIFICATION OF THE LID COVERING THE ACTIVE SITE ALTERS ENANTIOSELECTIVITY

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:ES=8.5;H. lanuginosa:ES=10.5), but theR-enantiomer of phenyl 2-methyldecanoate (ER=2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (ER=2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (ES=2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (ES=1.9) and increased the enantioselectivity with the aromatic ester (ER=4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (ES=17.4) and a decreased enantioselectivity with the phenyl ester (ER=2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.

Trp89 in the lid ofHumicola lanuginosa lipase is important for efficient hydrolysis of tributyrin

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax=6000±300 mmol·min−1·g−1 and an apparent Km=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.

PROBING A FUNCTIONAL ROLE OF GLU87 AND TRP89 IN THE LID OF HUMICOLA LANUGINOSA LIPASE THROUGH TRANSESTERIFICATION REACTIONS IN ORGANIC SOLVENT

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theVmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheKm,app for tributyrin was not affected by the Glu87Ala mutation, but theKm,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedVmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theKm,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theKm,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theVmax,app was only 6% of that obtained with wild-type enzyme.

2-Methylalkanoic acids resolved by esterification catalysed by lipase from Candida rugosa : alcohol chain length and enantioselectivity

2-Methylalkanoic Acids Resolved by Esterification Catalysed by Candida rugosa : Alcohol Chain Length and Enantioselectivity

Reaction conditions for the resolution of 2-methylalkanoic acids in esterification and hydrolysis with lipase from Candida cylindracea

SummaryWe have demonstrated resolution of 2-methylalkanoic acids using lipase from Candida cylindracea as a catalyst. The resolution of 2-methyldecanoic acid was more successful than that of 2-methylbutyric acid both by esterification and hydrolysis. This indicates that the resolution of the acid is dependent on the chain length of the acid moiety. The chain length of the alcohol moiety of the ester affected the resolution of the long-chain acid only. Using esterification, (R)-2-methyldecanoic acid was produced in an enantiomeric excess (e.e.) of 95% (E = 40). If the enantiomeric ratio is low (E = 3.6), as in the resolution of 2-methylbutyric acid, esterification combined with a high equilibrium conversion could be used to yield the remaining acid in a high e.e. In the hydrolytic reactions, the e.e and the equilibrium conversion were dependent on the pH and the presence of CaCl2. When octyl 2-methyldecanoate was hydrolysed at pH 8.0 in the presence of CaCl2, the (S)-acid was formed with an e.e. of 80% (E = 9), but when the hydrolysis was carried out at pH 7.5 without CaCl2, a very low e.e. and a low equilibrium conversion were observed. The latter conditions allowed the esterification of 2-methyldecanoic acid with 1-octanol even in aqueous medium.

Insights into the molecular basis for fatty acyl specificities of lipases from Geotrichum candidum and Candida rugosa.

Despite immense progress in our comprehension of lipase structure and function during the past decade, the basis for lipase acyl specificities has remained poorly understood. This review summarizes some recent advances in the understanding at the molecular-level of substrate acyl recognition by two members in a group of large (Mw approximately 60 kDa) microbial lipases. Two aspects of acyl specificity will be focused upon. (i) The unique preference of a fungal Geotrichum candidum lipase for long-chain cis (delta-9) unsaturated fatty acid moieties in the substrate. Mutational analysis of this lipase identified residues essential for its anomalous acyl preference. This information highlighted for the first time parts in the lipase molecule involved in substrate acyl differentiation. These results are discussed in the context of the 3D-structure of a G. candidum lipase isoenzyme and structures of the related Candida rugosa lipase in complex with inhibitors. (ii) The mechanism by which the yeast C. rugosa lipase discriminates between enantiomers of a substrate with a chiral acyl moiety. Molecular modeling in combination with substrate engineering and kinetic analyses, identified two alternative substrate binding models. This allowed for the proposal of a molecular mechanism explaining how long-chain alcohols can act as enantioselective inhibitors of this enzyme. A picture is thus beginning to emerge of the interplay between lipase structure and fatty acyl specificity.

Alcohols as enantioselective inhibitors in a lipase catalysed esterification of a chiral acyl donor

SummaryIncreased reaction rates and increased enantioselectivities were observed with decreased concentrations of n-alkanols when resolving 2-methyldecanoic acid by esterification catalysed by immobilised lipase from Candida rugosa at controlled water activities in cyclohexane. The enantioselectivity was found to be independent of the water activity in the reaction medium at the n-heptanol concentrations investigated. However, when n-decanol was used as the acyl acceptor, not only the alcohol concentration but also the water activity in the reaction medium, influenced the enantioselectivity. The results obtained showed that the low enantioselectivity seen at a high alcohol concentration could be explained by the alcohol influencing the apparent VmaxS and VmaxR differently.

The role of arginines in stabilizing the active open-lid conformation ofRhizomucor miehei lipase

Molecular dynamics simulations for the lid covering the active site ofRhizomucor miehei lipase [EC 3.1.1.3] postulated that, among other interactions, Arg86 in the lid stabilized the open-lid conformation of the protein by multiple hydrogen bonding to the protein surface. Chemical modification of arginine residues inR. miehei lipase with 1,2-cyclohexanedione or phenylglyoxal resulted in residual activities in the hydrolysis of tributyrin of 66 and 46%, respectively. Tryptic maps of native and phenylglyoxal-reactedR. miehei lipase showed that Arg86 was the residue modified most, when the lipase was inhibited to the greatest extent. Guanidine, a structural analog to an arginine side chain, inhibited both the natibe enzyme and the arginine-modified enzymes, resulting in residual activities of 26% as compared to the native enzyme. The inhibition was not an effect of enzyme denaturation. The native enzyme was also inhibited by 1-ethylguanidine, benzamidine and urea, but to a lesser degree than by guanidine. Lipases fromHumicola lanuginosa and porcine pancreas in 100 mM guanidine showed residual activities of 88 and 70%, respectively. The lipases fromCandida antarctica, C. rugosa, Pseudomonas cepacia andP. fluoresences were not inhibited by guanidine. The inhibition ofR. miehei lipase by structural analogs of the arginine side chain and after chemical modification of arginine residues suggest a role of an arginine residue in stabilizing the active open-lid conformation of the enzyme.

Reversed enantiopreference of Candida rugosa lipase supports different modes of binding enantiomers of a chiral acyl donor

Molecular modelling identifies two different productive modes of binding the enantiomers of a 2-methyldecanoic acid ester to the active site of Candida rugosa lipase (CRL). The fast reacting S-enan ...