Hydroxymandelic Acid Research Articles

Co-production of biofuel, bioplastics and biochemicals during extended fermentation of Halomonas bluephagenesis.

Halomonas bluephagenesis TD1.0 was engineered to produce the biofuel propane, bioplastic poly-3-hydroxybutyrate (PHB), and biochemicals mandelate and hydroxymandelate in a single, semi-continuous batch fermentation under non-sterile conditions. Multi-product separation was achieved by segregation of the headspace gas (propane), fermentation broth ([hydroxy]mandelate) and cellular biomass (PHB). Engineering was performed by incorporating the genes encoding fatty acid photodecarboxylase (CvFAP) and hydroxymandelic acid synthase (SyHMAS) into a H.bluephagenesis hmgCAB cassette knockout to channel flux towards (hydroxy)mandelate. Design of Experiment strategies were coupled with fermentation trials to simultaneously optimize each product. Propane and mandelate titres were the highest reported for H.bluephagenesis (62 g/gDCW and 71 ± 10mg/L respectively) with PHB titres (69% g/gDCW) comparable to other published studies. This proof-of-concept achievement of four easily separated products within one fermentation is a novel achievement probing the versatility of biotechnology, further elevating H.bluephagenesis as a Next Generation Industrial Biotechnology (NGIB) chassis by producing highly valued products at a reduced cost.

Directed evolution of the PobR allosteric transcription factor to generate a biosensor for 4-hydroxymandelic acid.

Hydroxy-mandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122-126 were involved in its PobR ligand specificity. These results showed the effectiveness of directed evolution in switching the ligand specificity of a biosensor and improving HMA production.

Modification of an atmospheric pressure photoionization source for online analysis of exhaled breath coupled with quadrupole time-of-flight mass spectrometry.

Breath analysis is a promising method for metabolomics studies and clinical diagnosis, as it enables the observation of metabolites in a convenient and noninvasive way. In this work, an atmospheric pressure photoionization (APPI) source was modified for online analysis of exhaled breath by coupling with quadrupole time-of-flight mass spectrometry (QTOFMS). Three parameters, namely, the capillary voltage, the sampling flow and the curtain gas flow of the APPI source, were optimized. Five healthy volunteers, three males and two females, were enrolled to test the performance of modified APPI-QTOFMS by analyzing their exhaled breath. A total of 21 compounds were tentatively identified, and four metabolites, namely, dimethyl selenoxide, δ-valerolactam, hydroxymandelic acid and palmitic amide were detected in the exhaled breath for the first time. The result shows that modified APPI-QTOFMS can be used for the online study of exhaled breath. Graphical abstract.

Rapid prototyping of microbial production strains for the biomanufacture of potential materials monomers

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production.

Structural and Functional Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase from Escherichia coli.

The hydroxylation of phenols into polyphenols, which are valuable chemicals and pharmaceutical products, is a challenging reaction. The search for green synthetic processes has led to considering microorganisms and pure hydroxylases as catalysts for phenol hydroxylation. Herein, we report the structural and functional characterization of the flavin adenine dinucleotide (FAD)-dependent 4-hydroxyphenylacetate 3-monooxygenase from Escherichia coli, named HpaB. It is shown that this enzyme enjoys a relatively broad substrate specificity, which allows the conversion of a number of non-natural phenolic compounds, such as tyrosol, hydroxymandelic acid, coumaric acid, hydroxybenzoic acid and its methyl ester, and phenol, into the corresponding catechols. The reaction can be performed by using a simple chemical assay based on formate as the electron donor and the organometallic complex [Rh(bpy)Cp*(H2 O)]2+ (Cp*: 1,2,3,4,5-pentamethylcyclopentadiene, bpy: 2,2'-bipyridyl) as the catalyst for FAD reduction. The availability of a crystal structure of HpaB in complex with FAD at 1.8 Å resolution opens up the possibility of the rational tuning of the substrate specificity and activity of this interesting class of phenol hydroxylases.

Bacterial bifunctional chorismate mutase-prephenate dehydratase PheA increases flux into the yeast phenylalanine pathway and improves mandelic acid production

Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (hmaS). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing S. cerevisiae strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional E. coli enzyme PheA (PheAfbr) in an aro7 deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheAfbr turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120 mg/L. Moreover, our results indicate that utilizing PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.

Effect of 6-hydroxydopamine on the metabolism of endogenous octopamines and catecholamines.

The daily excretion in rat urine of o-(OHMA) (0.2), m-(MHMA) (0.9), p-(PHMA) hydroxymandelic acid (78), vanillylmandelic acid (VMA) (3.5) and homovanillic acid (30 micrograms day-1), terminal metabolites of the isomeric octopamines and catecholamines, has been determined by gas chromatography-mass spectrometry. Chemical sympathectomy with 6-hydroxydopamine (i.p.) caused the daily output of each acid (except OHMA) to drop by 50% and that of PHMA was restored to normal the most rapidly after cessation of treatment. These results indicate that m- and p- (but not o-) octopamine coexist with catecholamines in sympathetic nerve terminals, are subject to the same compensatory biosynthetic mechanism following chemical sympathectomy and that p-octopamine has the highest turnover rate of these biogenic amines in the rat.

Synthesis of polyhydroxylated aromatic mandelic acid amides and their antioxidative potential

Six hydroxymandelic acid amides of phenolic amines were synthesized by condensation of 3,4-dihydroxymandelic or 4-hydroxy-3-methoxymandelic acid N-succinimidyl esters and several phenolic benzylamino and phenethylamino hydrochlorides in moderate to good yield. The radical scavenging activities determined by 2,2-diphenyl-1-picrylhydrazyl assay and superoxide trapping assay were superior compared to standards l-ascorbic acid, α-tocopherol, and butylated hydroxytoluene (BHT). The antioxidative activities tested by accelerated autoxidation of bulk lipids were 2–3.5 times more potent compared to standards α-tocopherol and BHT. 3,4-Dihydroxymandelic acid dopamide (4b) and 4-hydroxy-3-methoxymandelic acid dopamide (4a) showed the best overall performance as antioxidants.

Synthesis of DL‐2‐(p‐hydroxyphenyl)glycine by the ammonolysis of p‐hydroxymandelic acid obtained by the condensation of phenol with glyoxylic acid

AbstractSynthesis of DL‐2‐(p‐hydroxyphenyl)glycine by the ammonolysis of p‐hydroxymandelic acid obtained by the condensation of phenol and glyoxylic acid was investigated. Different procedures for the isolation of p‐hydroxymandelic acid from the product mixture after the condensation step were considered. The ammonolysis of the mandelic acid could be suitably carried out at 115°C using a 10:1 mole ratio of ammonia to p‐hydroxymandelic acid in the presence of ammonium chloride. A yield of glycine as high as 92% could be obtained when an overall conversion of the mandelic acid was about 80 %. The reaction was found to be first order with respect to the mandelic acid up to a reaction period of 6 h.

Characterisation of an octopamine-sensitive adenylate cyclase in haemocyte membrane fragments of the American cockroach Periplaneta americana L.

An octopamine-sensitive adenylate cyclase has been studied in a fragmented haemocyte-membrane preparation of the American cockroach, Periplaneta americana. The enzyme complex is similar to that of adenylate cyclases from other eukaryotic cells in having a requirement for magnesium ions and GTP and in displaying elevated prodution of cyclic AMP in the presence of sodium fluoride and forskolin. The preparation responds to GTP in a dose-dependent manner and a positive dose-dependent response to calmodulin is also reported. Octopamine and synephrine are the most potent stimulators of cyclic AMP production in the haemocyte-membrane preparation and additivity studies indicate that synephrine and another activator, tyramine, are interacting with the enzyme complex through the octopamine-receptor. The adrenergic agonists, naphazoline and clonidine increased cyclic AMP production and the formamidines, demethylchlordimeform and BTS 27271 also were effective in this regard. Hydroxymandelic acid caused a slight but statistically significant decrease in cyclic AMP production. The most potent antagonists of the octopamine-mediated response were mianserin > promethazine > phentolamine > gramine > cis- flupenthixol > dibenamine. Lineweaver-Burk plots indicate that the mianserin, promethazine and gramine-effects, as with earlier reports for phentolamine, result from competitive inhibition

Resolution of the enantiomers of m‐ and p‐hydroxymandelic acid by high performance liquid chromatography

Resolution of the enantiomers of <i>m</i>‐ and <i>p</i>‐hydroxymandelic acid by high performance liquid chromatography

Octopamine in leeches—II. Synthesis, release and reuptake of octopamine by the ventral nerve cord of Erpobdella octoculata

1. Octopamine was synthesized by the ventral nerve cords of E. octuculata during in vitro incubation in radiolabelled tyrosine and tyramine. Trace amounts of dopamine were also synthesized from tyrosine and tyramine. 2. In vitro uptake of 3H-octopamine revealed the presence of a sodium sensitive and a sodium insensitive component which was linear up to 10.54 μM. The sodium sensitive component showed at least one component that was fully saturated at 1.54 μM octopamine, possessed a K m of approximately 4 × 10 −7 M and a V max of 15.5 mM/10 ventral nerve cord ganglia/min. 3. Release of preloaded 3H-octopamine from ventral nerve cords was by a calcium-dependent process. A high-potassium-induced calcium-dependent release was also demonstrated. 4. Hydroxymandelic acid production was decreased in the presence of iproniazid phosphate suggesting the presence of a monoamine oxidase. 5. The synthesis, release, reuptake and metabolism of octopamine are discussed in the light of octopamine as a central transmitter in the leech.

Catecholamine stimulation of the gibberellin action that induces lettuce hypocotyl elongation

Effects of catecholamines and their derivatives on gibberellic acid (GA)-induced lettuce hypocotyl elongation was studied, because catecholamines have a chemical structure similar to the dihydroconiferyl alcohol that has been isolated from lettuce cotyledons as a GA synergist. Epinephrine, norepinephrine, dopamine and 3,4-dihydroxymandelic acid synergistically enhanced the promoting effect of GA on hypocotyl elongation. In contrast, metanephrine, normetanephrine, DOPA and 3-methoxy-4- hydroxymandelic acid did not enhance the GA effect. The action of catecholamines was inhibited by trans-cinnamic acid which competitively inhibited the action of dihydroconiferyl alcohol; this suggests that the receptor site for catecholamines is the same as that for dihydroconiferyl alcohol. The basic ethyl acetate fraction from lettuce seedlings synergistically enhanced the GA effect. TLC analyses of this basic ethyl acetate fraction revealed that the chromatographic area corresponding to authentic catecholamines could enhance the GA effect. From these results, a possible role for catecholamines in the regulation of lettuce hypocotyl elongation caused by GA was posited, and is discussed here.

A gas chromatographic-mass spectrometric assay for nine hydroxyphenolic acids in uremic serum.

A mass fragmentographic method for the determination of m-hydroxybenzoic acid, p-hydroxybenzoic acid, o-hydroxyphenylacetic acid, m-hydroxyphenylacetic acid, p-hydroxyphenylacetic acid, vanillic acid, p-hydroxymandelic acid, homovanillic acid and vanilmandelic acid in uremic serum is described. The hydroxyphenolic acids were extracted from one ml of serum ultrafiltrate. After trimethylsilylation, the extract was analyzed by a gas chromatograph-mass spectrometer. Concentrations of nine hydroxyphenolic acids are higher in uremic serum. In particular, p-hydroxybenzoic acid and p-hydroxyphenylacetic acid are markedly increased in uremic serum. The influence of hemodialysis on the hydroxyphenolic acids was studied by quantitating the acids before and after hemodialysis.

A semi automated procedure for the measurement of 3-methoxy-4 hydroxy mandelic acid

A semi automated procedure for the determination of 3-methoxy-4-hydroxy-mandelic acid (HMMA) in urine has been evaluated. This procedure offers advantages in simplicity, speed and precision over existing manual techniques which utilize spectrophotometric fluorimetric and gas liquid chromatographic methods. The urine is extracted with ethyl acetate from a highly acidic sample, purified by extraction into acetate buffer and the diazo colour reaction developed. All stages of the procedure are carried out using the Brown and semi-automatic extractor equipped with partition tube. The within and between assay coefficient of variation obtained on pooled freeze-dried urine samples was 6.7% and 7.7% respectively.

In vitro biosynthesis of oöthecal sclerotization agents from tyrosine by haemolymph of Periplaneta Americana

Whole haemolymph of the adult female American cockroach was homogenized in distilled water and the extract subjected to column chromatography on Sepharose-6B. The partially purified enzyme, when incubated with the appropriate cofactors, was capable of catalyzing several reactions. With pyridoxal-phosphate and NAD but without the addition of copper, l- 14C(U)-tyrosine was metabolized to tyramine, octopamine, p- hydroxymandelic acid, p- hydroxybenzaldehyde , p- hydroxybenzylalcohol , p- hydroxybenzoic acid, p- hydroxyphenylpyruvic acid, p- hydroxyphenylacetic acid, p- hydroxyphenyllactic acid, and p- hydroxyphenylpropionic acid. The major metabolites of l-2′- 14C-tyramine were octopamine, p- hydroxymandelic acid and p- hydroxybenzoic acid. Further experiments using the crude homogenate and unlabeled substrates showed that octopamine was metabolized to p- hydroxymandelic acid and that it produced p- hydroxybenzaldehyde , p- hydrosybenzoic acid, and 3,4-dihydroxybenzoic acid (protocatechuic acid). These results suggest the following pathway exists for the synthesis of protocatechuic acid: tyrosine → tyramine → octopamine → p- hydroxymandelic acid → p- hydroxyphenylglyoxylic acid and/or p- hydroxybenzylalcohol → p- hydroxybenzaldehyde → p- hydroxybenzoic acid→protocatechuic acid. Hydroxylation of the p- hydroxy compounds occurred in the presence of copper but at different rates. The initial transamination and/or deamination of tyrosine to the p- hydroxypyruvate is apparently not instrumental in the synthesis of protocatechuic acid although the results of this investigation do not offer conclusive evidence concerning this question.

Mandelic acid-4-hydroxylase, a new inducible entyme from [formula omitted

A soluble fraction of Pseudomonas convexa catalyzed the hydroxylation of mandelic acid to p -hydroxymandelic acid. The enzyme had a pH optimum of 5.4 and showed an absolute requirement for Fe 2+, tetrahydropteridine, NADPH. p -Hydroxymandelate, the product of the enzyme reaction was identified by paper chromatography, thin layer chromatography, UV and IR-spectra.

Studies of catecholamine excretion by children with neural tumors.

ABSRACT The urinary excretion of dopamine, norepinephrine, epinephrine, metanephrine-normetanephrine and 3-methoxy-4- hydroxymandelic acid was measured in 17 children with tumors of neural crest origin; in 13 alterations in catecholamine metabolism were demonstrated. There were no apparent abnormalities in catecholamine excretion in 4 patients with “cured” neuroblastoma and in 7 with a variety of tumors not of neural crest origin. A preliminary biochemical classification of patients with neural tumors is suggested, based on the urinary excretion of catecholamines and metabolites.