Karl Hult

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.

Ochratoxin A in human blood and Balkan endemic nephropathy

The etiology of Balkan endemic nephropathy, a kidney disease encountered among the rural population living in regions along several big rivers on the Balkan Peninsula, remains unknown in spite of many hypotheses put forward and tested. One hypothesis involves mycotoxins as the causal agent. The mycotoxin ochratoxin A has been demonstrated to have a potent nephrotoxic effect in all mammalian species tested so far.The results of analysis of ochratoxin A in human blood samples by an analytical method based on the measurement of fluorescence spectra, before and after incubation with carboxypeptidase A, is described. For a 2-g-sample the detection limit of the method is 1–2 ng/g serum. High performance liquid chromatography used for the confirmation of ochratoxin A identity by means of several derivatives of the molecule is also described. Out of more than 600 samples collected in an endemic region in Yugoslavia about 7% were positive for ochratoxin A. The highest concentration found was 40 ng ochratoxin A/g serum.

Plasma ochratoxin A levels in three Swedish populations surveyed using an ion-pair HPLC technique.

A new HPLC method for the analysis of ochratoxin A in plasma samples is described. The analysis is performed at an alkaline pH using an ion-pair technique, fluorescence detection at an excitation wavelength 380 nm, and an emission wavelength 420 nm. The detection and quantification limits are 0.02 ng and 0.05 ng ochratoxin A/ml plasma, respectively. The method was used to determine the ochratoxin A content of human plasma samples, collected in three districts of Sweden. The Visby district had a significantly higher proportion of ochratoxin A positive samples and higher levels than the other two districts--Uppsala and Ostersund. The calculated daily intake of ochratoxin A in the Visby district (0.35 ng/kg body weight), exceeds the lower tolerable daily intake (TDI) value suggested by Kuiper-Goodman and Scott (1989). The calculated daily intake by the population on the mainland of Sweden (0.04 ng/kg body weight) is below the proposed TDIs.

Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B

The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions

Michael‐type additions of various thiols and α,β‐unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild‐type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks the nucleophilic serine 105 in the active‐site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael‐type additions was initially explored by quantum‐chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular‐dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The kcat values were found to be in the range of 10−3 to 4 min−1, similar to the values obtained with aldolase antibodies. The enzyme proficiency was 107. Furthermore, the Michael‐type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

Molecular Modeling of the Enantioselectivity in Lipase-Catalyzed Transesterification Reactions

Two strategies based on the use of subsets for calculating the enantioselectivity in lipase-catalyzed transesterifications using the CHARMM force field were investigated. Molecular dynamics was used in our search for low energy conformations. Molecular mechanics was used for refining these low energy conformations. A tetrahedral intermediate with a rigid central part was used for mimicking the transition state. The energy differences between the transition states of the diastereomeric enzyme-substrate complexes were calculated. The way of defining the subsets was based on two fundamentally different strategies. The first strategy used predefined parts of the enzyme and the substrate as subsets. The second approach formed energy-based subsets, varying in size with the substrates studied. The selection of residues to be included in these energy-based subsets was based on the energy of the interaction between the specific residue or water molecule and the transition state. The reaction studied was the kinetic resolution of secondary alcohols in transesterifications using the Candida antarctica lipase B as chiral biocatalyst. The secondary alcohols used in the study were 2-butanol, 3-methyl-2-butanol, and 3,3-dimethyl-2-butanol.

Improved Enantioselectivity of a Lipase by Rational Protein Engineering

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1‐Halo‐2‐octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast‐reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivtiy as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.

Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B

The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser‐His‐Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast‐reacting enantiomer of secondary alcohols places its medium‐sized substituent in the stereospecificity pocket and its large substituent towards the active‐site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild‐type lipase increased 270 times towards heptan‐4‐ol and 5500 times towards nonan‐5‐ol; this resulted in the high specificity constants 1100 and 830 s−1 M−1, respectively. The substrate selectivity changed over 400 000 times for nonan‐5‐ol over propan‐2‐ol with both Trp104Ala and the Trp104Gln mutations.

Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme‐substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics‐based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3‐methyl‐2‐butanol with various aliphatic carboxylic acids and also 2‐butanol, as well as 3,3‐dimethyl‐2‐butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

CHIRAL RECOGNITION OF ALCOHOL ENANTIOMERS IN ACYL TRANSFER REACTIONS CATALYSED BY CANDIDA ANTARCTICA LIPASE B

A description of the substrate-enzyme interactions involved in the discrimination of see-alcohol enantiomers in acyl transfer reactions catalysed by the highly enantioselective Candida antarctica lipase B is presented. Experimentally found activities and enantioselectivities from kinetic resolutions of a series of secondary alcohol substrates were used together with molecular modelling for the elucidation of the stereoselective substrate-enzyme interactions. Matching experimental and calculated results allowed conclusions regarding the orientation of the tetra-hedral intermediates in the active site. The finding, valid for substrates of a specific activity above 1 mol min-1 mg-1 protein, describes the origin of enantioselectivity as a combination of a binding site of limited size, the ”stereospecificity pocket“, and principally different productive orientations of the two enantiomers.