An efficient biocatalytic synthesis of imidazole-4-acetic acid

Objective

To develop a new and efficient biocatalytic synthesis method of imidazole-4-acetic acid (IAA) from l-histidine (l-His).

Results

l-His was converted to imidazole-4-pyruvic acid (IPA) by an Escherichia coli whole-cell biocatalyst expressing membrane-bound l-amino acid deaminase (ml-AAD) from Proteus vulgaris firstly. The obtained IPA was subsequently decarboxylated to IAA under the action of H₂O₂. Under optimum conditions, 34.97 mM IAA can be produced from 50 mM l-His, with a yield of 69.9%.

Conclusions

Compared to the traditional chemical synthesis, this biocatalytic method for IAA production is not only environmentally friendly, but also more cost effective, thus being promising for industrial IAA production.

     

Effect of high hydrostatic pressure treatment on isoquercetin production from rutin by commercial α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase

Objectives

To optimize conversion of rutin to isoquercetin by commercial α-l-rhamnosidase using high hydrostatic pressure (HHP).

Results

The de-rhamnosylation activity of α-l-rhamnosidase for isoquercetin production was maximal at pH 6.0 and 50 °C using HHP (150 MPa). The enzyme showed high specificity for rutin. The specific activity for rutin at HHP was 1.5-fold higher than that at atmospheric pressure. The enzyme completely hydrolysed 20 mM rutin in tartary buckwheat extract after 2 h at HHP, with a productivity of 10 mM h^?1. The productivity and conversion were 2.2- and 1.5-fold higher at HHP than at atmospheric pressure, respectively.

Conclusions

This is the first report concerning the enzymatic hydrolysis of isoquercetin in tartary buckwheat at HHP.

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

Production of tartaric acid using immobilized recominant <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objective

To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of l-(+)-tartaric acid.

Results

E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg^?1. The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U^?1 h^?1 during the continuous enzymatic production process.

Conclusion

ESH with a histidine tag was immobilized and used for the continuous production of l-(+)-tartaric acid.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

Identification of serum metabolic markers for diagnosis of women with dormant genital tuberculosis

Introduction

Genital tuberculosis (GTB) in women is one of the common causes of infertility in emerging countries. As an intracellular pathogen, Mycobacterium tuberculosis in the endometrium significantly alters the host metabolism in dormant GTB cases. Nuclear magnetic resonance (NMR) based metabolic profiling has emerged as a useful tool for identification of biomarkers in biological fluids.

Objective

To investigate NMR based serum metabolic profile of dormant GTB women as compared to controls.

Methods

Dormant GTB women (n = 26) and unexplained infertile women (controls; n = 26), healthy proven fertile women undergoing voluntary sterilization (n = 25) and women undergoing recurrent spontaneous miscarriage (RSM) (n = 27) were included in the study. 700 MHz proton NMR spectra of serum collected from these patients were recorded. Multivariate analysis including principal component analysis, partial least squares discriminant analysis and orthogonal projection to latent structure-discriminant analysis was applied to all the spectra. Association of dysregulated serum metabolites with our earlier findings related to altered endometrial tissue metabolites in dormant GTB women was studied using multiple correlation analysis.

Results

This study indicates a clear metabolic differentiation between women with dormant GTB and controls. Metabolites including 3-hydroxybutyrate, succinate, citrate, acetate, l-glutamine, l-lysine, glutamate, l-threonine and 1-methyl histidine were found to be significantly upregulated in serum of women with dormant GTB compared with controls. Pearson’s correlation analysis showed a significant correlation between the expression of endometrial tissue and serum metabolites.

Conclusions

The set of identified metabolites may be considered as candidate markers for the diagnosis of dormant GTB and help clinicians in early therapeutic management.

     

Highly selective and efficient biotransformation of linarin to produce tilianin by naringinase

Objectives

To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin.

Results

Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-l-rhamnosidase activity while the β-d-glucosidase activity was abrogated. The addition of Mg²⁺, Fe²⁺ and Co²⁺ was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and ¹H-NMR.

Conclusion

A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-l-rhamnosidase activity of naringinase and blocked its β-d-glucosidase activity.

     

Structure elucidation of metabolite x17299 by interpretation of mass spectrometric data

Introduction

A major bottleneck in metabolomic studies is metabolite identification from accurate mass spectrometric data. Metabolite x17299 was identified in plasma as an unknown in a metabolomic study using a compound-centric approach where the associated ion features of the compound were used to determine the true molecular mass.

Objectives

The aim of this work is to elucidate the chemical structure of x17299, a new compound by de novo interpretation of mass spectrometric data.

Methods

An Orbitrap Elite mass spectrometer was used for acquisition of mass spectra up to MS⁴ at high resolution. Synthetic standards of N,N,N-trimethyl-l-alanyl-l-proline betaine (l,l-TMAP), a diastereomer, and an enantiomer were chemically prepared.

Results

The planar structure of x17299 was successfully proposed by de novo mechanistic interpretation of mass spectrometric data without any laborious purification and nuclear magnetic resonance spectroscopic analysis. The proposed structure was verified by deuterium exchanged mass spectrometric analysis and confirmed by comparison to a synthetic standard. Relative configuration of x17299 was determined by direct chromatographic comparison to a pair of synthetic diastereomers. Absolute configuration was assigned after derivatization of x17299 with a chiral auxiliary group followed by its chromatographic comparison to a pair of synthetic standards.

Conclusion

The chemical structure of metabolite x17299 was determined to be l,l-TMAP.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Furosemide enhances the sensitivity of urinary metabolomics for assessment of kidney function

Introduction

The ability of urinary metabolomics to detect meaningful, tissue-specific, biological effects (i.e., toxicity, disease) is compounded by high background variability. We hypothesize that sensitivity can be enhanced by imposing a tissue-targeted metabolic stressor.

Objective

We tested whether the sensitivity of metabolomics to assess kidney function is improved under the diuretic stress of furosemide.

Methods

To mildly compromise kidney, rats were given a sub-acute dose of d-serine. Then at 24 h postdose, we administered vehicle solution (control) or the diuretic drug, furosemide, and conducted NMR-based urinary metabolomics.

Results

Principal Components and OPLS discriminant analyses showed no effects on urinary profiles in rats receiving d-serine alone. However, the effects of d-serine were observable under furosemide-induced stress, as urinary profiles classified separately from rats receiving furosemide alone or vehicle-treated controls (p?<?0.001). Furthermore, this profile was uniquely different from a co-treatment effect observed following co-administration of d-serine?+?furosemide. We identified 24 metabolites to classify the effects of furosemide in normal rats vs. d-serine-compromised rats. Most notably, a furosemide-induced increase in urinary excretion of α-ketoglutarate, creatinine, trigonelline, and tryptophan in control rats, was significantly reduced in d-serine exposed rats (p?<?0.05). Interestingly, increased tryptophan metabolism has been shown to correlate with the severity of kidney transplant failure and chronic kidney disease.

Conclusions

We attribute these effects to differences in kidney function, which were only detectable under the stress imposed by furosemide. This technique may extend to other organ systems and may provide improved sensitivity for assessment of tissue function or early detection of disease.

     

Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from d-mannose by two-step fermentation pathway in Escherichia coli.

Results

E. coli BL21(DE3) harboring pET-22b(+) vectors with six genes, i.e., glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (WcaG), and α-1,2-fucosyltransferase (Fuct) were co-inoculated, and the multienzyme synthetic pathway was constructed to produce fucosyloligosaccharide using d-mannose as substrate. The product, analyzed by LC/MS, fucosyloligosaccharide was formed under the catalysis of Fuct using GDP-fucose as donor substrate and lactose as acceptor substrate. Fucosyloligosaccharides reached 22 mM by a two-step fermentation compared to 3.7 mM with a one-pot fermentation.

Conclusions

Fucosyloligosaccharide was produced by a two-step fermentation to avoid the inhibitory effect of GDP-fucose on Gmd. Two-step fermentation is a rational synthetic pathway for accumulating fucosyloligosaccharide.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Increased productivity of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid and total turnover number of NAD<Superscript>+</Superscript>/NADH in a one-pot system through enhanced thermostability of <Emphasis Type="SmallCaps">l</Emphasis>-threonine deaminase

Objective

To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).

Results

l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD⁺/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.

Conclusions

By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.

     

Unexpected differential metabolic responses of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> to the abundant presence of glutamate and fucose

Introduction

Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity.

Objectives

For instance, the C. jejuni 11168 strain can utilize both l-fucose and l-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of l-fucose and l-glutamate in the growth medium.

Methods

To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h.

Results

This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon l-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy.

Conclusions

Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon l-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.

     

Co-expression of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase and catalase in <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce α-ketoglutaric acid by whole-cell biocatalyst

Objectives

To improve the production of α-ketoglutaric acid (α-KG) from l-glutamate by whole-cell biocatalysis.

Results

A novel and highly active l-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg^?1 at pH 6.0, 35 ^oC. The apparent K_m and V_max values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg^?1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H₂O₂, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l^?1 with a conversion rate from l-glutamate of 98.5% after 12 h.

Conclusions

An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.

     

Serum metabolomic study for detecting biomarkers of non-traumatic osteonecrosis of the femoral head

Introduction

Non-traumatic osteonecrosis of the femoral head (NTONFH) is a progressive disease, always leading to hip dysfunction if no early intervention was applied. The difficulty for early diagnosis of NTONFH is due to the slight symptoms at early stages as well as the high cost for screening patients by using magnetic resonance imaging.

Objective

The aim was to detect biomarkers of early-stage NTONFH, which was beneficial to the exploration of a cost-effective approach for the early diagnose of the disease.

Methods

Metabolomic approaches were employed in this study to detect biomarkers of early-stage NTONFH (22 patients, 23 controls), based on the platform of ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and the uses of multivariate statistic analysis, putative metabolite identification, metabolic pathway analysis and biomarker analysis.

Results

In total, 33 serum metabolites were found altered between NTONFH group and control group. In addition, glycerophospholipid metabolism and pyruvate metabolism were highly associated with the disease.

Conclusion

The combination of LysoPC (18:3), l-tyrosine and l-leucine proved to have a high diagnostic value for early-stage NTONFH. Our findings may contribute to the protocol for early diagnosis of NTONFH and further elucidate the underlying mechanisms of the disease.

     

Functional expression of a human GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose transporter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.

Results

The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of ³H-GDP-l-fucose with a V_max of 8 pmol/min mg with a K_m of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.

Conclusions

The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Characterization of a novel asparaginase from soil metagenomic libraries generated from forest soil

Objectives

To screen soil metagenomic libraries for novel enzymes with enhanced activities.

Results

To screen soil metagenomic libraries for novel enzymes with enhanced activities. A novel l-asparaginase was identified from forest soil metagenome and its characteristics were studied. The purified protein had a specific activity of 696 IU mg^?1 and optimum activity at pH 7 and 35 °C. Enhanced enzyme activities were observed in the presence of Mg²⁺, Ca²⁺ and K⁺. The K_m value, 2 mM, and enzyme specificity constant 7.7 mM^?1s^?1 indicated that the recombinant enzyme has good substrate affinity to l-asparagine compared with commercially-available Escherichia coli asparaginase. The IC₅₀ value of 0.78 µg ml^?1 (0.47 IU ml^?1) was observed with HL60 cell line and 0.39 µg ml^?1(0.23 IU ml^?1) with MOLT-3 and MOLT-4 cell lines, which is better than that of commercially-available drugs.

Conclusion

The soil metagenome derived l-asparaginase with enhanced activities could be a potential candidate to develop as a drug in Acute Lymphoblastic Leukemia (ALL) therapy.