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K Hult

Publications and source records attributed to K Hult.

At least 37 records · Page 2Linked to original sources
PubMed1995

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 of Humicola 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 the Vmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The Km,app for tributyrin was not affected by the Glu87Ala mutation, but the Km,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 decreased Vmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the Km,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 the Km,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 the Vmax,app was only 6% of that obtained with wild-type enzyme.

Binding Sites↗
PubMed1994

Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa.

The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme. Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.

Binding Sites↗
PubMed1994

Trp89 in the lid of Humicola 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) from Humicola 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Detergents↗
PubMed1994

Enhanced stereoselectivity in pig liver esterase catalysed diester hydrolysis. The role of a competitive nucleophile.

The enantioselectivity of pig liver esterase catalysed hydrolysis of cis-N-benzyl-2,5-bis(methoxy-carbonyl)pyrrolidine (1) has previously been shown to be very dependent on the reaction conditions. Hydrolysis performed in media buffered with tris(hydroxymethyl)aminomethane (Tris) afforded a monoester with much higher optical purity than hydrolysis in media without Tris. Detailed product studies in a Tris-buffered medium have been performed using NMR-techniques and a 13C-labelled ester. The NMR-studies revealed the presence of (2S,5R)-N-benzyl-2-methoxycarbonyl-5-[[[2-hydroxy-1,1- bis(hydroxymethyl)ethyl]amino]carbonyl]pyrrolidine (4) as an intermediate, which together with the isolated product (2S,5R)-N-benzyl-2-carboxy-5-[[[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino]carbonyl]pyrrolidine (3) suggested Tris as a competitive nucleophile to water. The increased enantioselectivity seen in the produced (2R,5S)-N-benzyl-2-methoxy-carbonyl-5-carboxypyrrolidine (2) was explained by the preference of Tris to react faster with one of the diastereomeric acyl enzymes over the other.

Animals↗
PubMed1994

Molecular dynamics simulations of an enzyme surrounded by vacuum, water, or a hydrophobic solvent.

We report on molecular dynamics simulations of a medium-sized protein, a lipase from Rhizomucor miehei, in vacuum, in water, and in a nonpolar solvent, methyl hexanoate. Depending on force field and solvent, the molecular dynamics structures obtained as averages over 150 ps had root-mean-square deviations in the range of 1.9 to 3.6 A from the crystal structure. The largest differences between the structures were in hydrogen bonding and exposed surface areas of the protein. The surface area increased in both solvents and became smaller in vacuum. The change of surface exposure varied greatly between different residues and occurred in accordance with the hydrophobicity of the residue and the nature of the solvent. The fluctuations of the atoms were largest in the external loops and agreed well with crystallographic temperature factors. Root-mean-square fluctuations were significantly smaller in the nonpolar solvents than they were in water, which is in accordance with the notion that proteins become more rigid in nonpolar solvents. In methyl hexanoate a partial opening of the lid covering the active site occurred, letting a methyl hexanoate molecule approach the active site.

Computer Simulation↗
PubMed1994

Ochratoxin A in human serum samples collected in southern Italy from healthy individuals and individuals suffering from different kidney disorders.

Ochratoxin A was determined in human serum samples, collected in the south of Italy in November 1992, using ion-pair liquid chromatography and fluorescence detection. The samples were collected from healthy people (65 subjects) as well as from people with different kidney disorders. Five different kinds of kidney disorders were represented: transplanted subjects (13), chronic glomerulonephritis (8), renal calculus or cyst (6), chronic renal failure (13), and subjects treated by dialysis (28). The mean and median concentrations of ochratoxin A in the healthy group was 0.53 and 0.44 ng/ml serum, respectively. The highest mean concentration was found in the group of patients treated by dialysis, 1.4 ng/ml serum. A higher incidence of samples containing > 0.44 ng ochratoxin A/ml serum was found in the dialysis group, compared to the other groups. Comparing the mean concentrations by Student's t-test, a significant difference was found between the mean concentrations of the healthy group and of the group of patients treated by dialysis (P < 0.01). No other significant differences were found when comparing the groups two at a time.

Analysis of Variance↗
PubMed1993

Transfer of ochratoxin A from lactating rats to their offspring: a short-term study.

A dose-dependent transfer of ochratoxin A into the milk of lactating rats was found after a single oral dose of ochratoxin A, given in the dose levels of 10, 50, and 250-micrograms ochratoxin A/kg body weight by gastric intubation. The milk/blood concentration ratio of ochratoxin A at 24 and 72 h was 0.4 and 0.7, respectively. A linear relationship was found between the concentration of ochratoxin A in the dam's milk and in the blood of the pups at 72 h, as well as in the dam's milk and in the kidneys of the pups. The pup blood/milk concentration ratio of ochratoxin A was approximately 6. At 72 h the sucklings had higher levels of ochratoxin A than their dams in both blood and kidneys. The results show that the concentration of ochratoxin A in milk can be used as an indicator of the continuously administered dose to the suckling.

Animals↗
PubMed1993

Lipases from Rhizomucor miehei and Humicola lanuginosa: modification of the lid covering the active site alters enantioselectivity.

The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: ES = 8.5; H. lanuginosa: ES = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (ER = 2.9). Chemical arginine specific modification of the R. 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 the H. 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.

Arginine↗
PubMed1993

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

Molecular dynamics simulations for the lid covering the active site of Rhizomucor 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 in R. 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-reacted R. 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 native 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 from Humicola lanuginosa and porcine pancreas in 100 mM guanidine showed residual activities of 88 and 70%, respectively. The lipases from Candida antarctica, C. rugosa, Pseudomonas cepacia and P. fluorescens were not inhibited by guanidine. The inhibition of R. 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.

Amino Acid Sequence↗
PubMed1993

Theoretical studies of Rhizomucor miehei lipase activation.

Computational methods have been used to study the extensive conformational change of Rhizomucor miehei lipase upon activation. The present study considers the possible activation route, the energies involved and molecular interactions during the conformational change of the lipase in a hydrophobic environment. The conformational change was studied by conventional molecular dynamics methods and with a combined molecular dynamics and mechanics protocol, in which the conformational change was simulated by restraining C alpha pseudotorsional angles in small steps between the two crystallographically observed positions of the lid. In the closed conformer of the enzyme the active site is completely buried under a short helical loop, 'the lid'. The activation of the lipase consists of a movement of the lid, which results in an open conformer with an exposed active site. From the results of the simulations in the present work we suggest that the lipase in a hydrophobic environment is stabilized in the open form by electrostatic interactions.

Computer Simulation↗
PubMed1992

Surfactant interference on lipase catalysed reactions in microemulsions.

The hydrolysis of palm oil with lipase as a catalyst was carried out in two different microemulsion systems. One system was based on a nonionic surfactant, pentaethylene glycol monododecyl ether (C12EO5) and the other system was based on an anionic surfactant, sodium bis(2-ethylhexyl)sulphosuccinate (AOT). The yield of free fatty acid produced by the reaction was found to be much lower in the C12EO5 system compared to the AOT system. Radiochromatography showed that the low yield was due to enzymatic esterification on the nonionic surfactant. Kinetic measurements showed that the reaction rate is about ten times faster in the AOT based microemulsion than in the nonionic system. Differences in the microemulsion structure and interfacial tensions in the two systems were found to be of no significant importance for explaining this difference. Kinetic data of mixed surfactant microemulsions indicated that the observed difference in the reaction rates of the AOT and the C12EO5 microemulsion systems was a consequence of the C12EO5 surfactant competing with the substrate for the active site of the enzyme. The reason why C12EO5 surfactant inhibited the reaction, and AOT surfactant did not, was found to be related to differences in the structures of the hydrophobic part of the surfactant.

Dioctyl Sulfosuccinic Acid↗
PubMed1992

Ochratoxin A in blood and its pharmacokinetic properties.

Since there are pathomorphological similarities between porcine mycotoxic nephropathy caused by ochratoxin A and Balkan endemic nephropathy (BEN), it has been suggested that the same aetiological agent has a role in BEN. Based on the results from several field and experimental studies carried out on pigs, an appropriate analytical method of monitoring possible human exposure to ochratoxin A was developed. The toxicokinetic properties of the toxin were species specific, although in all the animal species studied (with the exception of fish), as well as in humans, two binding proteins were found in the plasma. The monkey had the longest elimination half-life of the toxin, 510 hr, in contrast to the fish whose elimination half-life was only 0.68 hr. The fish kidney displayed a specific pattern of distribution. In the laying quail the most prominent observation was the accumulation of labelled ochratoxin A in egg yolk. Generally, [14C]ochratoxin A was eliminated rapidly from the quail body, but had a long retention time in the circulating blood in the mouse. Although the elimination of ochratoxin A from the body depending on its binding to plasma constituents, the existence of enterohepatic circulation might have been partially responsible for its prolonged retention and elimination from the body of mammals. The toxicokinetic profile of ochratoxin A did not contradict the mycotoxic hypothesis in the aetiology of BEN.

Animals↗
PubMed1992

Synthesis of 14C-ochratoxin A and 14C-ochratoxin B and a comparative study of their distribution in rats using whole body autoradiography.

Methods for preparation of labelled ochratoxin A and B are described. The method for preparation of labelled ochratoxin B involves the synthesis of the azide of ochratoxin beta via the mixed anhydride and subsequent conjugation to labelled phenylalanine to yield 14C-ochratoxin B. The labelled ochratoxins were injected into male Wistar rats and after different survival times they were sacrificed and subjected to whole body autoradiography. The distribution pattern of ochratoxin A in the rat did not differ from that earlier registered for mouse. The previously known, high susceptibility of rats (and not mice) to ochratoxin A-induced cancer could thus not be explained by an accumulation of the toxin in specific cells or organs. The distribution patterns of ochratoxin A and B were almost congruent--the only apparent difference being a much longer retention of the labelled ochratoxin A in the blood compared to ochratoxin B, which was much faster excreted. When analyzing tissue extracts for labelled metabolites only the extracts from the rats injected with ochratoxin B were found to contain easily detectable concentrations, while no metabolites of ochratoxin A were seen.

Animals↗
PubMed1991

Penicillium verrucosum in feed of ochratoxin A positive swine herds.

Ochratoxin A contamination of cereal feed grain was monitored during October 1989-September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2-45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (greater than or equal to ng/ml blood) and ochratoxin A negative herds (less than 2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p less than 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p less than 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5%) of the feed samples collected from the ochratoxin A negative herds.

Animal Feed↗
PubMed1991

Human exposure to ochratoxin A.

Over a nine-year period during screening campaigns in villages where Balkan nephropathy is endemic, human blood samples were collected and analysed for ochratoxin A. The incidence of positive samples was 0.5-2.5%. Dried beans were found to be more frequently contaminated with the toxin than other food commodities. In view of a specific accumulation of ochratoxin A observed in eggs, more attention should be paid to contamination of this food with ochratoxin A.

Balkan Nephropathy↗
PubMed1991

Possible sources of ochratoxin A in human blood in Poland.

Samples of plant origin and human and porcine blood samples were screened over a long period for the presence of ochratoxin A. Of 1353 cereal samples, 11.7% contained the mycotoxin; of 1372 samples of feed, 1.5%; of 368 bread samples, 17.2%; of 215 flour samples, 22.3%; of 894 porcine serum samples, 37.4%; and of 1065 human serum samples, 7.2%. Seasonal variations in the natural occurrence of ochratoxin A were observed, with an increased percentage of positive samples in the spring. Individual daily intake of the mycotoxin, estimated on the basis of residues in human serum, was found to be 0.4 ng/g of food consumed.

Animal Feed↗