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The fractionation of Chromobacterium viscosum lipase was performed using a polypropylene glycol-Sepharose gel. The influence of mobile phase composition on the adsorption of lipase on the gel was studied and it was found that the retention of lipase depends on the salt used and increased with increasing the ionic strength. The retention was not strongly affected by changing the pH value of the mobile phase. By using 20% (w/v) ammonium sulphate in phosphate buffer a total retention of lipase on the column was obtained and by simply decreasing the ionic strength of the buffer, desorption of lipase could be achieved. The chromatographic purification of Chromobacterium viscosum lipase by hydrophobic interaction chromatography on Sepharose CL-6B modified by covalent immobilisation of 1,4-butanediol diglycidyl ether, polyethylene glycol and polypropylene glycol was also compared.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.

A lipase-catalyzed glycerolysis reaction (a transesterification between polyunsaturated fatty acid ethyl ester [PUFA] and glycerol) was investigated. Its performance was multiply intensified by (1) using a lipase having high specific activity, high activity in organic solvent, and high tolerance in organic solvent; (2) immobilization on fine CaCO3 powder (cheap and safe material, easy physical adsorption method of immobilization, reusable); (3) reaction in vacuo resulting in 100% conversion and effective avoidance of oxidative deterioration of PUFA; (4) high volumetric productivity because of no use of solvent; and (5) no need of further separation and purification of the oil product. It is emphasized that performance of biocatalytic reactions in organic media should be enhanced manifold for industrial implementation.

In vitro experiments were conducted to study the stability of lipase activities from bacterial, fungal, and animal sources under conditions that approximate the small intestine. In the first experiment, the effects of preincubation with trypsin (500, 1,000, and 2,000 U/mL), chymotrypsin (200, 400, and 800 U/mL), and trypsin plus chymotrypsin (TC; 2,000 U/mL trypsin + 800 U/inL chymotrypsin) for 30 min at 40 C, on lipase activities from sources of Pseudomonas spp. (PL1, PL2), Chromobacterium viscosum (CVL), and Aspergillus niger (ANL) were determined. None of the enzymes were inhibited by trypsin. The chymotrypsin decreased the activity of all of the lipases. The TC had no additional negative effect on the activities of PL1 and PL2; however, ANL and CVL activities were further decreased relative to the chymotrypsin only treatment. In the second study, the effects of Na taurodeoxycholate (0.1 to 16 mM) on the activities of PL1, PL2, CVL, ANL, and crude porcine lipase (CPL) at 23 and 40 C were evaluated. At 23 C, in order of potency, Na taurodeoxycholate inhibited the activities of ANL, CPL, and CVL. At this temperature, Na taurodeoxycholate did not inhibit PL1 and PL2. An increase in the temperature to 40 C increased the activity of all of the enzymes tested. At 40 C, Na taurodeoxycholate had similar effects on lipase activities; however, higher Na taurodeoxycholate levels were required to inhibit ANL activity, and only a partial inhibition of CPL occurred. At 23 C, porcine colipase restored the activity of CPL but had no effect on ANL and CVL in the presence of inhibitory levels of Na taurodeoxycholate. At 40 C, porcine colipase had no effect on Na taurodeoxycholate inhibition of lipase activity. The results of this study indicate that PL is more stable than CVL and ANL, and that colipase addition has no beneficial effects on microbial lipase activities under conditions that approximate the avian small intestine.

In vitro experiments were conducted to characterize the activity and the stability of lipase from animal (crude porcine, CPL; lyophilized porcine, LPL), fungal (Rhizopus arrhizus, RAL; Aspergillus niger, ANL), and bacterial (two Pseudomonas spp., PL1, PL2; and Chromobacterium viscosum, CVL) sources when exposed to conditions associated with the glandular stomach. Activity was measured at pH 3 to 8, 40 C and then monitored in response to temperature (40 C), time of exposure (0 and 30 min), pH (3 and 7), and pepsin level (5, 50, and 500 U/mL). All lipases except ANL and CVL had maximum activity at pH 7 to 8. The optimal pH for ANL and CVL were 5 and 6 to 8, respectively. Exposure of lipases to 40 C and pH 7 for 30 min reduced the activity of all lipases except ANL. In contrast, 40 C increased ANL activity 2.5-fold. Although activity of all lipases was reduced by exposure to pH 3, it was nearly eliminated for CPL and LPL. Pepsin concentration had only minor effects on lipase activity and then only at high concentration. The results demonstrate that bacterial lipases (PL1, PL2, and CVL) and ANL are more stable under conditions that approximate the glandular stomach and may explain why dietary porcine lipase has been ineffective in preventing fat malabsorption in previous in vivo studies.

A gene encoding phenylalanine hydroxylase has been cloned from Chromobacterium violaceum and expressed in Escherichia coli. The purified phenylalanine hydroxylase contains copper, which does not support enzymatic activity. Upon removal of copper by dithiothreitol (DTT), the enzyme contains substoichiometric amounts of calcium and zinc but little or no redox-active metal ions. The copper-depleted hydroxylase catalyzes the phenylalanine-dependent oxidation of 6, 7-dimethyltetrahydropterin (DMPH4) by O2 in a reaction in which phenylalanine is not hydroxylated and does not appear to undergo a chemical change, and hydrogen peroxide is produced. Analogs of phenylalanine also activate the oxidation of DMPH4. Both the copper-phenylalanine hydroxylase and the copper-depleted hydroxylase catalyze the hydroxylation of phenylalanine in the presence of DTT and FeSO4 in a reaction in which hydrogen peroxide is not produced. The apparent values of Km for Fe2+ and DTT are 0.28 microM and 1.1 mM, respectively, at 1.0 mM phenylalanine, 120 microM DMPH4 and pH 7. 4 and 23 degreesC. The apparent value of kcat is 14.3 s-1 under these conditions. Glutathione, mercaptoethanol, and dihydrolipoate support the hydroxylation of phenylalanine essentially as well as DTT. Incubation of copper-depleted hydroxylase with FeSO4, phenylalanine, and DTT followed by gel permeation chromatography leads to an iron-hydroxylase containing approximately 1 molecule of iron per molecule of enzyme. The iron-hydroxylase displays an optical absorption band extending from 300 to 600 nm, and it catalyzes the hydroxylation of phenylalanine at the same maximum rate as the iron-activated hydroxylase but does not require added Fe2+. We conclude that iron participates in the hydroxylation of phenylalanine. Iron is not required for the oxidation of DMPH4, although it may exert a modest acceleration effect. A hypothetical mechanism is presented wherein the reaction of iron with the putative 4a-hydroperoxy-DMPH4 leads to 4a-hydroxy-DMPH4 and a high valent iron-oxy species. The iron-oxy species is postulated to react with phenylalanine in the hydroxylation process.

IND, a redox flavoprotein from Chromobacterium violaceum has been crystallized in the presence and absence of NADH. The crystals belong to the space group P41212 or its enantiomorph P43212 with a = 73.9 and c = 153.6 A. There is one molecule per asymmetric unit and the crystals diffract beyond 2.1 A resolution.

The purification of Chromobacterium viscosum lipase was performed using a polypropylene glycol-Sepharose gel. The influence of the mobile phase composition on the chromatographic behaviour of Chromobacterium viscosum lipase was studied and it was found that the retention of lipase depends on the salt used and increased with ionic strength. Using 20% (w/v) ammonium sulphate in the eluent, a total retention of lipase on the column was obtained.

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.

Lipase-catalysed stereoselective hydrolysis of a family of saturated gamma- and delta-lactones was investigated. Increasing the chain length from delta-octalactone to delta-dodecalactone leads to higher enantiomeric excess. Best results were found using a lipase from Pseudomonas species (KW51) yielding highest enantiomeric excesses (greater than 99% ee at 50% conversion, E > 100) at short reaction times (10 h) for delta-undecalactone and delta-dodecalactone. In contrast, gamma-lactones were resolved less efficiently. Highest enantioselectivities (70%ee at 50% conversion, E = 11) were found for gamma-nonalactone. Optimum reaction conditions were found at pH 8 and 12.5 degrees C.

Background: . Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms. True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface. Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions. Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals. Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity. This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources., Results: . We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively., Conclusions: . PcL exhibits some structural features found in other lipases. The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases. The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface. The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site. This is similar to the mechanism described for other well characterized fungal and mammalian lipases.

1,2(2,3)-Diradylglycero O-(p-nitrophenyl) n-hexylphosphonates were synthesized, with the diradylglycerol moiety being di-O-octylglycerol, 1-O-hexadecyl-2-O-pyrenedecanylglycerol, or 1-O-octyl-2-oleoyl-glycerol, and tested for their ability to inactivate lipases from Chromobacterium viscosum (CVL) and Rhizopus oryzae (ROL). The experimental data indicate the formation of stable, covalent 1:1 enzyme-inhibitor adducts with the di-O-alkylglycero phosphonates. The differences in reactivity of diastereomeric phosphonates with opposite configuration at the glycerol backbone was less expressed with both enzymes tested as compared to the influence of the stereochemistry at the phosphorus. Both lipases exhibited the same preference for the chirality at the phosphorus that was independent from the absolute configuration at the glycerol backbone. However, with CVL and ROL the inhibitors with the active site serine-directed phosphonate linked at position sn-1 of the glycerol moiety reacted significantly faster than the corresponding sn-3 analogs, reflecting the sn-1 stereopreference of the enzymes towards triacylglycerol analogs with a sn-2 O-alkyl substituent. In contrast, the phosphonates based on the 1-O-octyl-2-oleoylglycerol did not significantly inactivate CVL. Unexpectedly, these substances were hydrolyzed in the presence of lipase.

The crystal structure of a lipase from the bacterium Chromobacterium viscosum ATCC 6918 (CVL) has been determined by isomorphous replacement and refined at 1.6 angstroms resolution to an R-factor of 17.8%. The lipase has the overall topology of an alpha/beta type protein, which was also found for previously determined lipase structures. The catalytic triad of the active center consists of the residues Ser87, Asp263 and His285. These residues are not exposed to the solvent, but a narrow channel connects them with the molecular surface. This conformation is very similar to the previously reported closed conformation of Pseudomonas glumae lipase (PGL), but superposition of the two lipase structures reveals several conformational differences. r.m.s. deviations greater than 2 angstroms are found for the C alpha-atoms of the polypeptide chains from His15 to Asp28, from Leu49 to Ser54 and from Lys128 to Gln158. Compared to the PGL structure in the CVL structure, three alpha-helical fragments are shorter, one beta-strand is longer and an additional antiparallel beta-sheet is found. In contrast to PGL, CVL displays an oxyanion hole, which is stabilized by the amide nitrogen atoms of Leu17 and Gln88, and a cis-peptide bond between Gln291 and Leu292. CVL contains a Ca2+, like the PGL, which is coordinated by four oxygen atoms from the protein and two water molecules.

The stereoselectivity of microbial lipases from Chromobacterium viscosum (CVL) and Rhizopus arrhizus (RAL) towards monoacylglycerols (rac-1(3)-oleoylglycerol and 2-oleoylglycerol), diacylglycerols (1,3-dioleoylglycerol and rac-1,2(2,3)-dioleoylglycerol) and 2-O-ether analogs (rac-1(3)-oleoyl-2-O-hexadecylglycerol and rac-1(3)-octanoyl-2-O-hexadecylglycerol) was determined. The results of the hydrolysis of 2-O-ether analogs confirmed the importance of the substituent at C-2 of acylglycerols in the stereoselective recognition by microbial lipases and also showed that acylation of mono- and diradylglycerols with oleic acid overlaps the hydrolysis reaction in aqueous medium. With the short-chain, water-soluble octanoic acid no significant esterification occurred. Using rac-1,2(2,3)-dioleoylglycerol as a substrate for the hydrolysis with RAL and CVL, the appearance of 1,3-dioleoylglycerol and of 1(3)-monooleoylglycerol was demonstrated. The possibility of chemical vs. enzyme-catalyzed isomerization of 1,2-dioleoylglycerol and of 2-oleoylglycerol is discussed.

L-Tryptophan 2',3'-oxidase, an amino acid alpha,beta-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C alpha and C beta carbons of various tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an alpha-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic beta-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of alpha,beta-dehydrotryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct alpha,beta-dehydrogenation of the tryptophan side chain is proposed.

Five microbial lipases from Chromobacterium viscosum, Candida cylindracea, Pseudomonas (source Fluka), Pseudomonas (source Genzyme) and lipoprotein lipase ex Microbial (Genzyme) have been screened for lactonisation activity towards 16-hydroxyhexadecanoic acid (HHA) in a variety of different w/o microemulsion systems. With the exception of Candida cylindracea (CC), all the lipases exhibited lactonisation activity although they were inherently more active in microemulsion systems based on the anionic surfactant sodium bis(2-ethylhexyl)sulphosuccinate (AOT) than in those based on the cationic surfactant cetyltrimethylammonium bromide (CTAB). Lactone yields are typically 50-60% and are markedly better than those reported previously using microemulsions in combination with chemical catalysts. Lipase stability is superior in the CTAB microemulsion systems, while lipase stability in the low water content AOT microemulsion systems was still good with the exception of CC lipase, which is rapidly inactivated. Buffering the water pools of AOT microemulsions using diglycine buffer at pH 8.0 improved biocatalyst stability. The lactonisation activity of lipases in CTAB w/o microemulsion systems compares favourably with that obtained using the same preparations as a solid suspension in the corresponding water-saturated organic solvent. In addition, the unusual solubility properties of microemulsions allowed the use of considerably higher concentrations of substrate in the microemulsion systems as compared to water-saturated organic solvents such as n-heptane. Lactone yields obtained at equivalent concentrations in the corresponding organic solvents containing conventional condensation catalysts were consistently measured at approx. 10%.

Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing a tetrahydrobiopterin cofactor. Several key mechanistic questions have yet to be resolved, specifically the identity of the hydroxylating species and the role of the non-heme iron which is present in all of the mammalian PAHs. Recently, we have demonstrated that a bacterial PAH from Chromobacterium violaceum does not require any redox active metal for activity [Carr, R. T., & Benkovic, S. J. (1993) Biochemistry 32, 14132-14138]. To identify the function of iron in the mammalian PAH's, we have undertaken a series of experiments to compare the mechanisms of this metal-independent PAH with the iron-dependent PAH from rat liver. Using [4-2H]phenylalanine as a substrate gave a kinetic isotope effect on hydroxylation of unity for CVPAH which is in agreement with previous values reported for RLPAH. The [4-2H]phenylalanine underwent an NIH shift upon hydroxylation by CVPAH. The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both RLPAH and CVPAH using [4-2H]phenylalanine and [2,3,5,6-2H]phenylalanine as substrates. This suggests that PAH from either source probably does not directly mediate the NIH shift mechanism. No uncoupled pterin turnover was observed for CVPAH with either L-tyrosine or p-chloro-L-phenylalanine as substrate or tetrahydropterin as cofactor, each of which causes uncoupled turnover with RLPAH. CVPAH readily accepts 4-methylphenylalanine as a substrate giving 4-(hydroxymethyl)phenylalanine as the major product and 3-methyltyrosine as the only other minor product.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, the stereoselectivity of purified lipases from Candida rugosa, Chromobacterium viscosum, Pseudomonas species and Rhizopus arrhizus towards triacylglycerols in comparison to various structural analogs were investigated. Different triacylglycerol analogs with distinct polarities at position sn-2 of the glycerol backbone (1,3-diacyl-2-X-glycerol, where 2-X = 2-acyloxy, 2-alkyloxy, 2-deoxy-2-alkyl, or 2-deoxy-2-phenyl) were synthesized. Substrate hydrophobicity and steric requirement was modified by variation of the alkyl and acyl chain length. Hydrolysis of these substrates demonstrated that minor structural variations at C2 of triacylglycerol strongly affect the stereoselectivity of the lipases tested. It was noteworthy that the variation of substrate structure did not only affect the quantity of stereoselectivity expressed as percentage enantiomeric excess, but also resulted in a reversal of stereopreference in some cases. Replacement of the acylester in position 2 of glycerol by a non-ester-linked aliphatic moiety shifted the preference of Chromobacterium viscosum lipase from sn-3 to sn-1. Lipases from Chromobacterium viscosum. Pseudomonas species and Rhizopus arrhizus exhibited sn-3 preference with 2-deoxy-2-phenyl analogs, while towards substrates with a 2-deoxy-2-alkyl moiety sn-1 stereobias was recorded. Candida rugosa lipase was rather insensitive to substrate variations concerning the polarity at position 2 of the glycerol backbone. However, variation of the acyl chain length significantly influenced stereoselectivity of this lipase.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) is known to bind an equivalent of divalent copper. The "metal-free" form of the protein is fully active, and Cu(II) is now shown to be an inhibitor of CVPAH rather than an activator of the enzyme [Carr, R. T., & Benkovic, S. J. (1994) Biochemistry 32, 14132-14138]. On the basis of amino acid sequence homology, the metal binding site may be related to those of rat liver PAH and other eukaryotic pterin-dependent hydroxylases, which require Fe(II) for activity. The conserved histidines at that site in CVPAH, histidines 138 and 143, were each mutated to serines. The mutant enzymes H138S and H143S were both catalytically inactive, but still able to bind Cu(II). Binding studies further demonstrated that both mutant enzymes still bind L-phenylalanine. Electron spin echo envelope modulation (ESEEM) studies on each of the mutants showed the presence of only a single copper-coordinating histidine, rather than the two histidine ligands suggested for the wild-type protein. This result supports a model in which Cu(II) is equatorially ligated to only two histidines in the Cu(II)-inhibited protein and allows us to unambiguously assign histidines 138 and 143 as these ligands. That the enzyme is inactive when these histidines are either bound with copper or when replaced with serines suggests that these histidines perform a catalytic function. Possible catalytic roles for these histidines in the hydroxylation mechanism of pterin-dependent monooxygenases are discussed along with potential future applications of the combination of ESEEM with site-directed mutagenesis.

L-Tryptophan 2',3'-oxidase is a novel enzyme that specifically catalyzes the alpha,beta-dehydrogenation of L-tryptophan derivatives. It was extracted from Chromobacterium violaceum and purified 108-fold to apparent homogeneity with a 34% overall recovery. The molecular weight of the native enzyme is approximately 680,000, and its isoelectric point is nearly equal to 4. SDS-polyacrylamide gel electrophoresis showed that the enzyme is composed of two components with molecular weight of approximately 74,000 and 14,000. It also contains a noncovalently bound heme prosthetic group, as judged from a typical spectrum showing maxima at 427, 530, and 560 nm. The catalyzed reaction is completed without side-product formation over a broad pH range comprised between 3 and 8. The enzyme is reoxidized at the expense of molecular oxygen by producing one molecule of H2O2. Kinetic parameters for modification of N-acetyl-L-tryptophanamide were determined (Km = 19.5 microM; kcat = 45.2 s-1). A Hill coefficient of about 1 suggests the absence of any cooperative effect. As inferred from both its kinetic and stability optimal conditions, L-tryptophan 2',3'-oxidase constitutes a promising tool for chemical modification of tryptophanyl side chain in peptides and proteins.

The fatty acid specificity of Chromobacterium viscosum lipase was studied by comparing the pseudo-first-order rate constants for the transesterification of different fatty acid methyl esters with 1-propanol in dry acetonitrile as solvent. It was found that this enzyme shows a significant preference towards long chain fatty acids and, for chains with the same length, towards saturated ones. The same enzyme was used to study the esterification of sorbitol and decanoic acid. A mixture of mono-, di-, tri- and tetraesters was obtained. The concentration of esters was strongly increased upon raising the temperature from 35 to 70 degrees C. The structures of the di-, tri- and tetraesters were determined using 13C NMR spectrometry. The diester appeared to be sorbitol 1,6-didecanoate, the triester was sorbitol 1,5,6-tridecanoate and the tetraester was the 1,2,5,6-tetradecanoate, which indicates that the C. viscosum lipase acylates sorbitol in a regioselective manner.

Limited proteolysis converts the 39200 molecular weight catalytic domain of rat tyrosine hydroxylase to a monomer with a molecular weight of 37600. The purified monomer is almost fully active, with minor changes in kinetic parameters at pH 7. Mass spectral analysis and N-terminal sequencing of the proteolytically generated species establish that 20 amino acids have been removed from the carboxyl terminus and five from the amino terminus. Based on these results, the carboxyl terminus is responsible for tetramer formation by tyrosine hydroxylase. The sequence of amino acids which is removed is consistent with a coiled coil structure in the intact tetramer.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was classified as a copper metalloenzyme by virtue of a 1/1 Cu/enzyme stoichiometry and its inhibition with various chelators [Pember, S. O., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611]. We have prepared "copper-free" CVPAH by extraction with DTT. These preparations retained full activity though the Cu/enzyme ratio averaged 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after the specific activity was determined to be within 85% of a fully copper-complexed control. Several copper chelators were examined and were not inhibitory. The "copper-free" enzyme had significant activity without a thiol or other reducing agent capable of reducing the copper center whereas copper-complexed CVPAH had minimal activity under these conditions. Copper-complexed CVPAH can be activated, however, by nonreducing copper ligands such as imidazole. From these results, we conclude that copper is not a requirement of activity. Iron, cobalt, nickel, manganese, molybdenum, and chromium were not found in the "copper-free" preparation, indicating that the active hydroxylating species may not require a redox-active metal. The KdS for binding Cu2+ and Zn2+ were measured to be 0.48 and 0.85 microM, respectively. Both copper and zinc were found to be potent inhibitors of "copper-free" CVPAH in the absence of thiols. DTT reverses inhibition due to Cu2+ but not inhibition caused by Zn2+. The product stoichiometry indicates the same tightly coupled turnover found with all other pterin-dependent hydroxylases when using natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of AOT reverse micellar systems with various alkyl glucosides and nonionic surfactants was attempted in order to improve the activity to Chromobacterium viscosum lipase. Tweens and Tritons, having the poly(oxyethylene) chain, have been shown to be solubilized at the oil-water interface of AOT micelles. Alkyl glucosides were also solubilized at the interface. These additives form mixed micelles with AOT and increase the concentration of the micelles. In contrast, Spans were found to be solubilized in the micro water pool of AOT micelles. Addition of nonionic surfactants decreased the hydrophobicity of the reverse micellar systems. Improvement of the lipase activity was achieved by the addition of nonionic surfactants, because of suppression of the hydrophobic and electrostatic interaction between AOT and lipase. The addition of nonionic surfactant having a poly(oxyethylene) chain has been shown to effectively increase the activity of the lipase in reverse micelles.

The coordination chemistry of the Cu sites of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum has been studied by X-ray absorption spectroscopy (XAS). The EXAFS of the Cu(II) form of the enzyme resembles that of other non-blue copper proteins such as plasma amine oxidases and dopamine-beta-hydroxylase and is characteristic of a mixed N/O coordination shell containing histidine ligation. Detailed simulations of the raw EXAFS data have been carried out using full curved-wave restrained refinement methodologies which allow imidazole ligands to be treated as structural units. The results suggest a Cu(II) coordination of two histidines and two additional O/N-donor groups. A reasonable fit to both data sets can be obtained by assuming that the non-imidazole first-shell donor atoms are derived from solvent (H2O or OH-). The EXAFS of the reduced enzyme shows major differences. The amplitude of the first shell in the Fourier transform is only 50% of that of the oxidized enzyme, indicative of a substantial reduction in coordination number. In addition, the first shell of the transform is split into two components. Simulations of the reduced data can be obtained by either two histidines at a long distance of 2.08 A and an O ligand at a short distance of 1.88 A or two histidines at a short distance of 1.90 A and one second-row scatterer such as S or Cl at 2.20 A. Comparison of absorption edge data on the reduced enzyme with data from Cu(I) bis- and tris(1,2-dimethylimidazole) complexes suggests a pseudo-three-coordinate structure.

The presence of core lipids in lipoproteins expressed and secreted by transfected HepG2 cells was demonstrated by measuring the densities of these lipoproteins before and after treatment with a bacterial lipase specific for neutral lipids. HepG2 cells were reproducibly transfected with pRSV/B48, containing a truncated human apolipoprotein B-100 (apoB-100) cDNA (nucleotides 1 to 6860, where nucleotide 129 is the start of translation). Northern blots of cellular message probed with apoB-48 showed abundant transcription of an apoB-48-sized message as well as endogenous apoB-100 message. When grown in the presence of [35S]methionine, pRSV/B48-transfected cells secreted lipoproteins containing an apoB-48-like apolipoprotein. This lipoprotein banded at a density of 1.11 g/ml in isopycnic NaBr gradients. Electron microscopy of the apoB-48-containing lipoproteins demonstrated spherical particles with an average diameter of 124A. A sedimentation rate of 8.4S was measured by sucrose gradient sedimentation. When the apoB-48-containing particles were treated with a bacterial lipase (from Chromobacterium viscosum), shown to hydrolyze triglycerides and cholesteryl esters but not phospholipids, their density increased to 1.18 g/ml, consistent with removal of core lipids. When the secreted lipoprotein was modeled as a spherical particle containing a single molecule of apoB-48, a triglyceride-filled core, and a surface monolayer of phospholipid and protein, the hydrodynamic properties were consistent with the observed sedimentation coefficient, buoyant densities before and after lipase treatment, and the diameter as seen with the electron microscope. These data indicate that transfected HepG2 cells assembled and secreted lipoproteins possessing the same physical structure as naturally occurring lipoproteins.

The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.

The complete amino acid sequence (296 amino acids) of Chromobacterium violaceum phenylalanine hydroxylase (PAH) was determined by nucleotide analysis of a DNA clone isolated using both a synthetic oligonucleotide probe based on the NH2-terminal amino acid sequence and an antibody against this enzyme. The ApaL I fragment (approximately 1.9 kilobase pairs) containing the entire PAH gene was subcloned in pBluescript II and induced by isopropyl-beta-D-thiogalactopyranoside. In order to eliminate fusion proteins the XbaI/ClaI fragment which contained the PAH gene from the Bluescript construct was subcloned into pMAC 5-8 containing the TAC promoter. The recombinant protein reacts with antibody raised to authentic C. violaceum PAH and its NH2-terminal 20-amino acid sequence and COOH-terminal amino acid residue were identical with the wild-type protein. Key physical and chemical characteristics of the recombinant protein, i.e. its copper content and Michaelis-Menten parameters, were the same as wild-type. Comparison of amino acid sequences revealed a highly conserved region between C. violaceum PAH and three different mammalian aromatic amino acid hydroxylases. This conserved area may well be a catalytically important domain of these pterin- and metal-requiring aromatic amino acid hydroxylases. The over-expression of C. violaceum PAH in Escherichia coli will facilitate the analysis of the enzyme mechanism by various spectroscopic methods.

Lipase from three different sources has been immobilised in microemulsion-based gels (MBGs) with retention of catalytic activity. Such lipase-containing MBGs prove to be novel solid-phase catalysts for use in apolar organic solvents such as n-heptane. Using these systems, preparative-scale synthesis of a wide variety of esters under mild conditions was possible with products easily isolated and obtained in high yield. Stereoselective esterification of octan-2-ol was observed for all three lipases with Chromobacterium viscosum (CV) lipase yielding product with an enantiomeric excess of 92%. Repeated usage of a CV lipase-containing MBG resulted in a visually unchanged gel whose activity was 75% of the initial value after 30 days. The sectioned MBGs were well suited for use in column flow reactors and were also found to be effective esterification catalysts at temperatures as low as -20 degrees C.

1. The size of two bacterial lipases was studied by SDS/PAGE, sedimentation velocity and sedimentation equilibrium to test for possible self-association behaviour. 2. Mr values of selected lipases were obtained from SDS/PAGE and sedimentation-velocity measurements, together with an absolute determination by sedimentation equilibrium 3. The Mr values obtained in a variety of aqueous solvents indicate that lipases do not self-associate in solution, suggesting the absence of surface hydrophobic patches.

Fatty acids prevented adsorption of purified Chromobacterium lipase [triacylglycerol acylhydrolase, EC 3.1.1.3] onto palmitoyl cellulose (Pal-C) and also increased the activity of the purified lipase. These effects increased with increase in the concentration and chainlength (up to 16 carbon atoms) of the fatty acids, and long-chain unsaturated fatty acids, such as oleic acid, linoleic acid and erucic acid, were most effective. When the lipase was adsorbed (immobilized) on Pal-C, its activity was elevated to 20 times that of the free lipase in detergent-free reaction mixture (olive oil-buffer system). Thus lipase was adsorbed to Pal-C through a hydrophobic site distinct from its catalytic site and the binding of fatty acids to the hydrophobic site seems to result in stimulation of the lipase activity.

Phenylalanine hydroxylase was purified approximately 3000-fold to apparent homogeneity with a 13% yield and crystalized from L-phenylalanine-induced cells of Chromobacterium violaceum. The enzyme was shown to be composed of a single polypeptide chain with an estimated molecular weight of approximately 32,000. Some of the physical properties of the enzyme include: a Stokes radius of 26.0 A, a sedimentation coefficient of 2.71 S, a diffusion coefficient of 8.20 X 10(-7) CM2/S, a frictional ratio of 1.23, and an isoelectric point of pH 4.5. No detectable iron was found in the purified enzyme. Apparent Km values for L-phenylalanine and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 140 and 54 muM, respectively.

The particulate fraction of disrupted Chromobacterium violaceum grown under cyanide-evolving conditions was unable to oxidize ascorbate plus N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD), but oxidized NADH and succinate by a linear respiratory pathway which was very resistant to inhibition by cyanide. When the bacteria were grown under conditions where little cyanide evolution occurred, particulate fractions developed the ability to oxidize ascorbate-TMPD by a pathway highly sensitive to cyanide inhibition; respiratory activity with NADH and succinate proceeded via both the cyanide-sensitive and-resistant pathways. Studies with respiratory inhibitors, and the cytochrome compositions of the fractions derived from cultures grown under both conditions, are presented. A soluble, carbon monoxide-binding cytochrome c was found, and this appears similar to those found recently in Beneckea natiegens, methylotrophic bacteria and the marine pseudomonad B16.

In our studies on newly synthesized compounds for their potential analgesic effect, we decided for purposes of convenience and economy to investigate non-mammalian sources for the presence of enkephalin degrading enzymes. An aminopeptidase that catalyzes the hydrolysis of the tyrosylglycyl bond of leucine- and methionine enkephalin was purified from the mesophiles Pseudomonas sp ATCC 11299a (Ps) and Chromobacterium violaceum ATCC 12540 (Cv). Each preparation also hydrolyzed to varying extents neutral dipeptides, tripeptides, tetrapeptides and amino acid beta-naphthylamides. The Ps enzyme has a pH optimum of 6.8, Km of 80 microM and a Vmax of 6.7 nmoles/min/mg of protein. The Cv enzyme has a pH optimum of 6.8-7.2, Km of 111 microM and a Vmax of 42 nmoles/min/mg of protein. Both are sulfhydryl enzymes since they are activated by dithiothreitol (DTT) and inactivated by p-chloro- and p-hydroxymercuribenzoate. They are not glycoproteins since they pass unretained through a Con A-Sepharose column. The activity lost by dialysis against EDTA can be restored, wholly or in part, by Co+2, Mg+2, Mn+2 and Ni+2; ions exerting an inhibitory effect were A1+3, Cd+2, Cu+2, Hg+2 and Zn+2. From a range of organic compounds, the greatest inhibition was elicited by the microbial peptides amastatin and bestatin. Several dipeptide analogues of bestatin, synthesized from DL-threo-2-amino-3-hydroxy-3-phenylpropanoic acid (AHPP) as the N-terminal residue in order to define the stereospecific requirements of the alpha, beta-functional groups for maximal activity, were not as active as the parent compound.

The review surveys current concepts concerning properties of lipases from gram-negative bacteria. It discusses the effects of cultivation conditions (composition, temperature and pH of the medium) of bacteria-producers on the biosynthesis of lipases.

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

A strain of Chromobacterium violaceum isolated from a fatally infected patient was found to produce a beta-lactamase. When the organism was grown in drug-free medium, beta-lactamase activity was barely detectable, but when it was grown in the presence of penicillin G, a much larger amount of activity was produced. The beta lactamase was active primarily against cephalosporins; it was sensitive to inhibition by cloxacillin but resistant to p-chloromercuribenzoate. Thus this enzyme closely resembled the common type of beta-lactamase found in Pseudomonas aeruginosa. The organism was relatively susceptible to ticarcillin, carbenicillin, and cefoxitin, which resected hydrolysis by its beta-lactamase, but was quite resistant to 11 other beta-lactam antibiotics. Production of the beta-lactamase appeared to be mediated by chromosomal genes.