Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Biotransformation of l-ornithine from l-arginine using whole-cell recombinant arginase

A recombinant arginase was generated for a whole-cell biotransformation system to convert l-arginine to l-ornithine in Escherichia coli. The gene ARG1 coding arginase from Bos taurus liver was synthesized and expressed in E. coli BL21 (DE3) via pETDuet-1. The recombinant arginase was used to catalyze l-arginine to l-ornithine and urea. The reaction was optimal at pH 9.5 and 37 °C. Manganese (10^?5 M) and Emulsifier OP-10 [0.033 % (v/v)] could promote arginase activity. In a scale up study, l-arginine conversion rate reached 98 % with a final concentration of 111.52 g l-ornithine/l.     

Characterization of a recombinant l-rhamnose isomerase from Dictyoglomus turgidum and its application for l-rhamnulose production

A putative recombinant enzyme from Dictyoglomus turgidum was characterized and immobilized on Duolite A568 beads. The native enzyme was a 46 kDa tetramer. Its activity was highest for l-rhamnose, indicating that it is an l-rhamnose isomerase. The maximum activities of both the free and immobilized enzymes for l-rhamnose isomerization were at pH 8.0 and 75 °C in the presence of Mn²⁺. Under these conditions, the half-lives of the free and immobilized enzymes were 28 and 112 h, respectively. In a packed-bed bioreactor, the immobilized enzyme produced an average of 130 g l-rhamnulose l^?1 from 300 g l-rhamnose l^?1 after 240 h at pH 8.0, 70 °C, and 0.6 h^?1, with a productivity of 78 g l^?1 h^?1 and a conversion yield of 43 %. To the best of our knowledge, this is the first report describing the enzymatic production of l-rhamnulose.     

Characterization of a d-psicose-producing enzyme, d-psicose 3-epimerase, from Clostridium sp.

The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co²⁺ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K _m, turnover number (k _cat), and catalytic efficiency (k _cat/K _m) for d-psicose were 227 mM, 32,185 min^?1, and 141 min^?1 mM^?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l^?1 was produced from 500 g d-fructose l^?1 after 5 h.     

Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine

We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K _m: 19 μM, V _max: 270 μmol/min/mg), but not d-histidine, l-histidine, d-tyrosine, l-tyrosine, d-phenylalanine, or l-phenylalanine. The enzyme was activated by BaCl₂ and strongly inhibited by CuSO₄, ZnSO₄, and HgCl₂. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7–104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.     

l-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of ³H-l-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. ³H-l-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na⁺- and K⁺-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. ³H-l-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, l-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM ³H-l-leucine in both Na⁺- and K⁺-containing incubation media. The residual ³H-l-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an l-methionine- and cation-independent transport system. ³H-l-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [l-leucine], following the carrier-mediated Michaelis–Menten equation. In NaCl, ³H-l-leucine influx displayed a low apparent K _M (high affinity) and low apparent J _max, while in KCl the transport exhibited a high apparent K _M (low affinity) and high apparent J _max. l-methionine or l-phenylalanine (7 and 20 mM) were competitive inhibitors of ³H-l-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in ³H-l-leucine influx K _M, but no significant response in ³H-l-leucine influx J _max. Potassium was a competitive inhibitor of sodium co-transport with ³H-l-leucine, significantly (p < 0.01) increasing ³H-l-leucine influx K _M in the presence of sodium, but having negligible effect on ³H-l-leucine influx J _max in the same medium. These results suggest that shrimp BBMV transport ³H-l-leucine by a single l-methionine- and l-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na⁺ or K⁺ acting as co-transport drivers binding to shared activator sites.     

Optimization of Biotransformation of l-Tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 Using Response Surface Methodology

l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson’s disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l^?1 cell mass and 1.488 g l^?1 l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l^?1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R²) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg^?1 tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.     

Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for l-malate production

Fumarase is a key enzyme that catalyzes the reversible hydration of fumarate to l-malate in the tricarboxylic acid cycle. This reaction has been extensively utilized for industrial applications in producing l-malate. In this study, a fumarase C gene from Streptomyces lividans TK54 (slFumC) was cloned and expressed as a fused protein (SlFumC) in Escherichia coli. The molecular mass of SlFumC was about 49 kDa determined by SDS-PAGE. Kinetic studies showed that the K _m value of SlFumC for l-malate increased by approximately 8.5-fold at pH 6.5 (6.7 ± 0.81 mM) to 8.0 (57.0 ± 1.12 mM), which was higher than some known fumarases. The catalytic efficiency (k _cat) and the specific activity increased by about 9.5-fold at pH 6.5 (65 s^?1) to 8.0 (620 s^?1) and from 79 U/mg at pH 6.5 to 752 U/mg at pH 8.0, respectively. Therefore, SlFumC may acquire strong catalytic ability by increasing pH to partially compensate for the loss of substrate affinity. The enzyme also showed substrate inhibition phenomenon, which is pH-dependent. Specific activity of SlFumC was gradually enhanced with increasing phosphate concentrations. However, no inhibition was observed at high concentration of phosphate ion, which was distinctly different in case of other Class II fumarases. In industrial process, the reaction temperatures for l-malate production are usually set between 40 and 60 °C. The recombinant SlFumC displayed maximal activity at 45 °C and remained over 85 % of original activity after 48 h incubation at 40 °C, which was more thermostable than other fumarases from Streptomyces and make it an efficient enzyme for use in the industrial production of l-malate.     

A d-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for d-psicose production: cloning, expression, purification, and characterization

d-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for d-psicose production from d-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a d-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using d-psicose as the substrate, the apparent K _m, k _cat, and catalytic efficiency (k _cat/K _m) were 27.4 mM, 49 s^?1, and 1.78 s^?1 mM^?1, respectively. Under the optimal conditions, the equilibrium ratio of d-fructose to d-psicose was 69:31. For high production of d-psicose, 216 g/L d-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate d-fructose, indicating that the enzyme was a potential d-psicose producer for industrial production.     

Backbone and side-chain 1H, 15N and 13C assignment of apo- and imipenem-acylated l,d-transpeptidase from Bacillus subtilis

The d,d-transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel β-lactam resistance mechanism involving l,d-transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the l,d-transpeptidases. To date, only one class of the entire β-lactam family, the carbapenems, is able to inhibit the l,d-transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the l,d-transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain ¹H, ¹⁵N and ¹³C NMR assignment of the l,d-transpeptidase from Bacillus subtilis (Ldt_Bs) in the apo and in the acylated form with a carbapenem, the imipenem.     

d-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum l-arabinose isomerase

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum l-AI were used for production of d-tagatose from d-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of d-galactose to d-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L^?1 substrate and at 37.5 °C after 5 days. The d-tagatose production rate of 185 g L^?1 day^?1 was obtained at 300 g L^?1 galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial d-tagatose production rate was 290 g L^?1 day^?1 under these conditions.     

Purification, characterization and kinetic properties of extracellular l-asparaginase produced by Cladosporium sp.

l-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, l-asparaginase has greater affinity towards l-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of l-glutamine. l-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Na⁺, K⁺ and Zn²⁺ significantly affected enzyme activity whereas presence of Fe³⁺, Pb²⁺ and KI stimulated the activity. Detergents studied also enhanced l-asparaginase activity. In-vitro half-life of purified l-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.     

Polarized spectroscopic elucidation of N-acetyl-l-cysteine, l-cysteine, l-cystine, l-ascorbic acid and a tool for their determination in solid mixtures

Method of linear polarized vibrational (both IR- and Raman) spectroscopy of oriented colloids in nematic host is applied on N-acetyl-l-cysteine, l-cysteine, l-cystine and l-ascorbic acid with a view to obtain experimental bands assignment and local structural elucidation in solid-state. Structural results are compared with available crystallographic data for all of the systems studied. Scopes and limitations of the polarized method are shown. Discussion on the correlation between polarized spectroscopic data and the space group type as well as the number of the molecules in the unit cell (Z) is performed. Compounds with monoclinic space group P2₁, containing Z = 1 (N-acetyl-l-cysteine) and 2 (l-cysteine and l-ascorbic acid) are elucidated. One of the rare for organic molecules, hexagonal P6₁22 space group and Z = 6 (l-cystine) is also elucidated. Experimental assignment of the characteristics frequencies is obtained, explaining the typical for the crystals Fermi-resonance, Fermy–Davydov and Davydov splitting effects. For first time in the literature we are reported the orientation of the solid-mixture in nematic host, using the trade product ACC (Hexal, Germany), containing mainly N-acetyl-l-cysteine and l-ascorbic acid. Quantitative IR-spectroscopic approach for determination of solid mixtures is presented as well. The intensity ratio between 1,716 cm^?1 (characteristic for N-acetyl-l-cysteine) and 990 cm^?1, (attributed N-acethyl-cysteine and vitamin C) is used. Linear regression analysis between content and the peak ratio data for ten solid-binary mixtures, leads to straight-line plot y = 1.08₂ (±0.04₉) + (?0.11₄ ± 0.01₁)x, where x = 1/X _i . Factor r of 0.9641 and a reliability of 98.85% are obtained. The analysis of ACC 200 (Hexal, Germany) show that the IR measurements leads to standard deviation of 0.010 and 0.011 at P about 0.0500 for the systems and a confidence of >98.77₁%.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

Characterization of a novel GH2 family α-l-arabinofuranosidase from hyperthermophilic bacterium Thermotoga thermarum

The 2,367-bp ORF of TtAFase from Thermotoga thermarum DSM 5069 encodes a calculated 90-kDa α-l-arabinofuranosidase (TtAFase), which does not belonging to any reported glycosyl hydrolase families α-l-arabinofuranosidases in the database and represents a novel one of glycosyl hydrolase family 2. The purified recombinant TtAFase produced in Escherichia coli BL21 (DE3) had optimum activity at pH 5.5 and at 80 °C. It was stable up to 80 °C and from pH 4.5–8.5. Kinetic experiments at 80 °C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a K _m of 0.77 mM, V _max of 2.3 μmol mg^?1 min^?1 and k _cat of 4.5 s^?1. The enzyme had no apparent requirement of metal ions for activity, and its activity was significantly inhibited by Cu²⁺ or Zn²⁺.     

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities

The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, ¹H NMR, ¹³C NMR, ¹H–¹H COSY, ¹H–¹³C HMBC) and Marfey’s method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(l-Pro-l-Trp), cyclo(l-Leu-l-Val), cyclo(d-Pro-d-Met), and cyclo(d-Pro-d-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(l-Leu-l-Val). Cyclo(l-Leu-l-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(l-Pro-l-Trp) against S. aureus (4 μg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 μg/ml by cyclo(l-Pro-l-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete’s foot, jock itch, and ringworm. The activity of cyclo(l-Pro-l-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 μg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial secondary metabolites, which may receive greater benefit as potential sources of new drugs in the pharmaceutical industry.     

Enantioselective N-acetylation of 2-phenylglycine by an unusual N-acetyltransferase from Chryseobacterium sp.

The demand for d-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the l-form of racemic d,l-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50 % and an enantiomer excess of >99.5 % under optimal culture conditions, consequently resulting in 99 % pure d-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by l-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates l-2-phenylglycine, d-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of d,l-2-phenylglycine.     

Characterization of a serine hydroxymethyltransferase for l-serine enzymatic production from Pseudomonas plecoglossicida

Pseudomonas plecoglossicida, a bacterium strain that exhibits high Serine hydroxymethyltransferase (SHMT) activity, was isolated from the seawater. A full-length glyA encoding SHMT was obtained by a modified thermal asymmetric interlaced-PCR (TRIL-PCR), which consisted of 1,254 bp, encoded a 417 amino acid polypeptide, and shared the highest identity (75 %) with a glyA gene from Acinetobacter radioresistens CMC-1. Recombinant glyA gene was expressed in Escherichia coli BL21 (DE3) and purified by electrophoretic homogeneity. The enzyme showed the optimal activity at pH 8.0 and 40 °C, and remained stable in high alkali conditions. Using SHMT to produce l-serine by catalyzing the reaction of glycine and tetrahydrofolate is one of the most promising routes to synthesize l-serine, achieving 33.4 mM l-serine at the 12th h of the enzymatic reaction with the substrates of glycine (133 mM) and formaldehyde (13.3 mM). The properties make the SHMT a candidate for further enzymatic studies and industrial applications.     

Gender-related effects on urine l-cystine metastability

Cystinuria is an autosomal recessive disease that causes l-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare l-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with l-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the l-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence l-cystine solubility. l-cystine solubility Z score was +0.44 ± 1.1 and ?0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the l-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro l-cystine precipitation assays confirmed that these molecules induce higher rates of l-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate l-cystine solubility and may represent new targets for therapy in cystinuria.     

Synthesis of β-alanine from l-aspartate using l-aspartate-α-decarboxylase from Corynebacterium glutamicum

β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC _C.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. The purified ADC _C.g was optimal at 55 °C and pH 6 with excellent stability at 16–37 °C and pH 4–7. A pH–stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6–7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g l-aspartate/l and another three feedings of 0.5 % (w/v) l-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.