Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from Pseudomonas sp. strain E-3

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 μmol h^–1 (mg protein)^–1 and a K _m of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7–8 and 30° C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as α-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1,10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed. Received: 21 May 1997 / Accepted: 5 September 1997     

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO₂-eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.     

Production ofcis,cis-muconic acid from benzoic acidvia microbial transformation

For the production ofcis,cis-muconic acidvia biocatalytic conversion reactions from a toxic cosubstrate, benzoic acid, a fed-batch process using computer-controlled DO-stat feeding was developed. The mutant strain ofPseudomonas putida BM014 producedcis,cis-muconic acid from benzoic acid with high conversion yield. More than 32 g/L ofcis,cis-muconic acid was accumulated in 42h and a productivity of 1.4 g/(L·h) was achieved.     

Short communication: Benzene metabolism via the intradiol cleavage in a Rhodococcus sp.

Benzene was metabolized by Rhodococcus sp. 33 through the intradiol cleavage (ortho-) pathway producing cis-benzene glycol, catechol and cis, cis-muconic acid as the intermediates. This is the first elucidation of the pathway by which benzene is degraded by a gram-positive organism. The enzyme assays have also suggested that Rhodococcus 33 does not have a fully functional tricarboxylic acid cycle but may have an operational glyoxylate bypass.     

cis-trans Isomerization Products from S-(cis-1-Propenyl)-l-cysteine and Its Sulfoxide Irradiated in Aqueous Solutions

In connection with food-flavor deterioration accompanied by irradiation, the formation mechanism of the degradation products from S-(cis-1-propenyl)-l-cysteine (PeCS) and its sulfoxide (PeCSO) (isomers of the naturally-occurring lachrymatory precursors of onion) irradiated by γ-ray in oxygen-free aqueous solutions was investigated.

From the comparisons of mass-spectral fragmentations, gas-chromatographic retention time and IR spectra with reference compounds, the major volatile products from γ-irradiated PeCS were identified as n-propyl cis-1-propenyl sulfide, n-propyl trans-1-propenyl sulfide, cis, cis-di-1-propenyl sulfide and cis, trans-di-1-propenyl sulfide and those from γ -irradiated PeCSO were identified as di-1-propenyl sulfides (cis-cis and cis-trans). A new cis-trans isomerization mechanism of the 1-propenyl thiyl radicals was suggested and it was elucidated to give a mixture of about 33% cis- and 67% trans-1-propenyl thiyl radicals.     

Metabolism of acenaphthylene via 1,2-dihydroxynaphthalene and catechol by Stenotrophomonas sp. RMSK

Stenotrophomonas sp. RMSK capable of degrading acenaphthylene as a sole source of carbon and energy was isolated from coal sample. Metabolites produced were analyzed and characterized by TLC, HPLC and mass spectrometry. Identification of naphthalene-1,8-dicarboxylic acid, 1-naphthoic acid, 1,2-dihydroxynaphthalene, salicylate and detection of key enzymes namely 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase in the cell free extract suggest that acenaphthylene metabolized via 1,2-dihydroxynaphthalene, salicylate and catechol. The terminal metabolite, catechol was then metabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed metabolic pathway in strain RMSK is, acenaphthylene → naphthalene-1,8-dicarboxylic acid → 1-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol → cis,cis-muconic acid.     

cis/trans Isomerase of Unsaturated Fatty Acids of Pseudomonas putida P8: Evidence for a Heme Protein of the Cytochrome c Type

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.     

trans-Stilbene degradation by Arthrobacter sp. SL3 cells

trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C_α=C_β bond cleavage.     

Identification of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid in Pseudomonas sp. strain E-3

A cell-free extract of Pseudomonas sp. strain E-3 catalyzed the conversion of 9-cis-hexadecenoic acid [16:1(9c)] to 9-trans-hexadecenoic acid [16:1(9t)] in the free acid form and when 16:1(9c) was esterified to phosphatidylethanolamine (PE). The cytosolic fraction catalyzed the isomerizations of free 16:1(9c) by itself and of 16:1(9c) esterified to PE in the presence of the membrane fraction. Tracer experiments using [2,2-²H₂]16:1(9c) demonstrated that the isomerization of free 16:1(9c) occurred independently of the isomerization of 16:1(9c) esterified to PE, indicating that this bacterium has two types of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid. Received: 29 December 1995 / Accepted: 10 April 1996     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Malonate added to peptone-meat extract medium has been shown to induce maleate cis-trans isomerase in Alcaligenes faecalis IB-l4. This enzyme played an indispensable role in the enzymatic production of L-aspartic acid from maleic acid and ammonia.

Though malonate in the medium inhibited the growth of A. faecalis IB-14 to some extent, cells grown in the medium containing 10^-1 M of malonate showed the highest level of the enzyme activity. Specific activity of the enzyme of malonate-grown cells was approximately ten times as strong as that of maleate-grown cells, while, in basal mediumgrown cells, cis-trans isomerization of maleate did not occur at all. Maleate was utilized not only as carbon source of the cell growth but also as inducer for the formation of maleate cis-trans isomerase. On the other hand, malonate was not utilized as carbon source and the metabolism of it within the cell was rather restricted within narrow limits. Thus it was concluded that malonate was a gratuitous inducer for the formation of the enzyme, while maleate, which was considerably metabolized, was normal inducer.

It was demonstrated that most of radioactivity of l-C¹⁴-malonate taken up by cells was localized in particle fraction which sedimented at 105,000×g for 120 minutes, whereas radioactivity of 1-4-C¹⁴-maleate was uniformly distributed in both particulate and soluble fractions. Maleate cis-trans isomerase activity, in turn, was detected exclusively in soluble fraction in both malonate and maleate induced cells.     

Metabolism of 2-chlorobenzoic acid inPseudomonas stutzeri

3-Chloropyrocatechol is formed as a result of oxidation of 2-chlorobenzoate byPseudomonas stutzeri. 2-Chloro-cis,cis-muconic acid is the product of oxidation of 3-chloropyrocatechol. A catabolic pathway for the degradation of 2-chlorobenzoate by a newly isolated strain ofP. stutzeri is proposed.     

Biotransformation of Unsaturated Long-Chain Fatty Acids by Eubacterium lentum

Eubacterium lentum (33 strains) isomerized the 12-cis double bond of C₁₈ fatty acids with cis double bonds at C-9 and C-12 into an 11-trans double bond before reduction of the 9-cis double bond. The 14-cis double bond of homo-γ-linolenic acid was isomerized by 29 strains into a 13-trans double bond. The same strains isomerized the 14-cis double bond of arachidonic acid into a 13-trans double bond and then isomerized the 8-cis double bond into a 7-trans double bond; the 13-cis double bond of 10-cis, 13-cis-nonadecadienoic acid was isomerized into a 12-trans double bond. None of these isomerization products was further reduced. Studies with resting cells showed optimal isomerization velocity at a linoleic acid concentration of 37.5 μM; higher concentrations were inhibitory. The pH optimum for isomerization was 7.5 to 8.5. The isomerase was inhibited by the sulfhydryl reagents iodoacetamide, bromoacetate, and N-ethylmaleimide and by the chelators EDTA and 1,10-phenanthroline.     

Metabolism of Aniline by Rhodococcus erythropolis AN–13

The metabolic pathway of aniline was examined in Rhodococcus erythropolis AN-13 that was isolated from soil when aniline was provided as a sole source of carbon and nitrogen. cis, cis-Muconic acid and β-ketoadipic acid were detected by thin-layer chromatography in an incubation mixture containing aniline and resting cells of this strain. These two carboxylic acids were also formed from catechol, when the substrate was incubated with cell-free extract of aniline-grown cells, and characterized spectrally as crystalline samples. Ammonia was released from aniline by resting cells. The cell-free extract of aniline-grown cells had a strong catechol 1,2-dioxygenase activity. Catechol, once formed from aniline, was apparently converted so rapidly to cis, cis-muconic acid that it could not be isolated. These results suggest that R. erythropolis AN-13 converted aniline to catechol with the release of ammonia and then mineralized catechol ultimately to inorganic end products, H₂O and CO₂, through the β ketoadipic acid pathway.     

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca<Superscript>2+</Superscript>-induced cysteine relative mobility/mitochondrial permeability transition pore

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca²⁺-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys⁵⁶ relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca²⁺-induced ANT conformational change and mitochondrial swelling. ANT-Pro⁶¹ isomerization increased ANT-Cys⁵⁶ relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca²⁺ induced ANT “c” conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca²⁺-induced ANT “c” conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca²⁺ induces the ANT “c” conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

Summary The conformationalcis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that thecis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing thecis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on thecis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L-or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of thecis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bound undergoing thecis/trans isomerisation moves the equilibrium to a significant amount of thecis form.     

Geometrical Isomers of Monohydroperoxides Formed by Autoxidation of Methyl Linoleate

Isomeric monohydroperoxides produced from autoxidized methyl linoleate were separated into two geometrical isomers (cis-trans and trans-trans) by silver nitrate TLC. Purified monohydroperoxides were converted into hydroxy octadecadienoates. Trimethylsilyl (TMS) derivatives of these compounds (four components) were separated into three peaks in the gas chromatogram; the mixture of 9-hydroxy-cis,trans-isomer and 13-hydroxy-cis,trans-isomer, 9-hydroxy-trans,trans-isomer and 13-hydroxy-trans,trans-isomer. The trans-trans isomers became more dominant than the cis-trans isomers in the later stage of autoxidation and with the rise of temperature. At the degradation of monohydroperoxides, the decrease of trans- trans isomers was apparently slower than that of cis-trans isomers. It is proposed that cis,trans isomerization of monohydroperoxides takes place at the process of autoxidation of methyl linoleate.     

固定化胞外邻苯二酚1，2－双加氧酶的研究

首次将胞外邻苯二酚1,2－双加氧酶固定化,并用于制备顺,顺—己二烯二酸.该固定化酶表观活力高,使用范围扩大,耐酸性及耐碱性都有显著提高,并且使用稳定性好,得到的产物浓度及纯度均较高,酶与产物容易分离,整个工艺简单、独特、新颖,有利于工业化应用.     

<Emphasis Type="Italic">Trans</Emphasis> Fatty Acids in Membranes: The Free Radical Path

The double bond geometry of most of the naturally occurring unsaturated fatty acid residues is cis. Due to the relevance of fatty acids as structural components of cell membranes and as biologically active molecules, the change of the cis geometry means a change of the associated functions and activities. The finding that the cis to trans isomerization is effective in phospholipids by the intervention of radical species led to the discovery that there can indeed occur an endogenous formation of trans fatty acids, whose significance in biological systems started to be addressed with in vitro and in vivo studies. Studies of liposome models simulating the formation of isomerizing species and evaluating their ability to interact with the hydrophobic part of the membrane bilayer has contributed to the gain in knowledge of the fundamental features of the lipid isomerization in membranes. Further work is in progress for the identification of the real culprits of the in vivo lipid isomerization, and recent results are shown on oleic acid micelles, where ^•NO₂ radicals are not able to induce double bond isomerization in comparison with amphiphilic thiol, such as 2-mercaptoethanol. H₂S and sulfur-containing amino acid residues are two of the possible species involved in this process at a biological level. An update of the scenario of the geometrical isomerization in membranes by free radicals is provided, together with applications and perspectives in life sciences.     

C-nuclear magnetic resonance studies of 85% C-enriched amino acids and small peptides: pH effects on the chemical shifts, coupling constants, kinetics of cis-trans isomerisation and conformation aspects

The ¹³C chemical shifts of several 85% ¹³C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH going from 7 to 1 all the δC are shifted more or less upfield with the exception of the carbonyl group of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal.The percentage of cis and trans conformers of glycyl-l-proline and glycyl-l-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-l-proline and only from 77 to 80% for glycyl-l-prolylglycine.The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-l-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the α-protons of the glycine residue, by a lower pK₁ and by a larger ΔδH_α of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-l-proline molecule.The ¹³C-¹³C coupling constants were also studied as a function of pH. The results show that J_Co-Cα of a C-terminal residue, varying from 5 to 6 Hz and reflecting the pK of the carboxylate group, is a linear function of δ_Co and δ_Cα as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the C_α−C_o bond, however, is practically the same in both cases.Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and δ_Co, δ_Cα on the one hand and the J_Co-Cα on the other were established.     

Biosynthesis of cis,cis-Muconic Acid and Its Aromatic Precursors,Catechol and Protocatechuic Acid,from Renewable Feedstocks by Saccharomyces cerevisiae

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.