Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (P_Hase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of P_Hase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.     

Effects of starvation and - or -alanine administration on the free - and -alanine levels in the muscle and hepatopancreas of the crayfish, Procambarus clarkii

Changes of - and -alanine and other free amino acids were examined in muscle and hepatopancreas of the crayfish Procambarus clarkii before and after a 1-month starvation in freshwater, 50% seawater, and seawater. Total free amino acids and - and -alanine in both tissues decreased during starvation. The largest decrease was found in the animals starved in freshwater. In 50% seawater and full-strength seawater, the ratio of -alanine to total alanine increased in both tissues, although the level was reduced a little. A large dose of -alanine administered into the tail muscle of freshwater crayfish was converted to -alanine and -alanine returned to the control level within a week. A large amount of -alanine was transported from muscle to hepatopancreas. -Alanine injected into freshwater crayfish was converted to -alanine, but was not transported to the hepatopancreas. In the case of -alanine injection into the muscle of crayfish acclimated to 50% seawater, large amounts of - and -alanine were maintained in the muscle during the experimental period of 4 days. These data indicate that -alanine is metabolized actively in crayfish tissues and accumulated as an important osmolyte during osmotic stress.     

Molecular characterization of alanine racemase from Bifidobacterium bifidum

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial alanine racemase, which is essential in the synthesis of -alanine as an essential component of the peptidoglycans. Alanine racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5′-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum alanine racemase possesses comparatively low affinities for both the coenzyme (9.1 μM for PLP) and substrates (44.3 mM for -alanine; 74.3 mM for -alanine). The alr gene encoding the alanine racemase was cloned and sequenced. The alr gene complemented the -alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the alanine racemase from B. bifidum NBRC 14252.     

Inhibitors of alanine racemase enzyme: a review

Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alafosfalin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.     

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC₅₀ = 3.3 μM, K_i = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

Tridec-1-ene-3,5,7,9,11-pentayne,an Ovicidal Substance from Xanthium canadense

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.     

Synthesis of acryloyl guar gum and its hydrogel materials for use in the slow release of l-DOPA and l-tyrosine

Acryloyl guar gum (AGG) and its hydrogel materials were synthesized for use as carriers and slow release devices of two pro-drugs, l-tyrosine and 3,4-dihydroxy phenylalanine (l-DOPA). To evaluate their structure-properties relationship, these were characterized by scanning electron micrography (SEM), FTIR spectroscopy and swelling studies. The hydrogel materials responded to the change of pH of the swelling medium, and exhibited reversible transitions in 0.9% saline solution. These were loaded with two pro-drugs, and their cumulative release behavior was studied at pH 2.2 and pH 7.4. The hydrogel materials exhibited structure-property relationship in the release of these pro-drugs. The % cumulative release of l-tyrosine was the maximum from the AGG-g-poly(methacrylic acid), while the maximum release of l-DOPA was observed from AGG-g-poly(AAc) in both the media. On the other hand, the AGG-g-poly(2-hydroxyethyl methacrylate) and AGG-g-poly(2-hydroxypropyl methacrylate) retained 42.33% and 49.05% of the drug even after 12 h.     

Diurnal variations in physiology and behaviour of the noble crayfish Astacus astacus and the signal crayfish Pacifastacus leniusculus

Heart rate, locomotor activity, and oxygen consumption were recorded simultaneously and continuously in seven individuals of the noble crayfish Astacus astacus (Linneus 1758) and seven individuals of the signal crayfish Pacifastacus leniusculus (Dana 1852). The recordings were made in the laboratory over 7 days at 15°C under a 12?:?12?h dark?:?light regime. Circadian rhythms in heart rate, locomotor activity and oxygen consumption were found both in A. astacus and P. leniusculus. Increased heart rate, locomotor activity, and oxygen consumption levels during night time in both A. astacus and P. leniusculus illustrated expression of nocturnal behaviour. No differences in oxygen consumption levels were observed between A. astacus and P. leniusculus. Also, no significant difference between heart rate levels or heart rate variances was found in A. astacus and P. leniusculus at night. During day, however, heart rate levels, heart rate variances and locomotor activity were higher in P. leniusculus than in A. astacus. The higher activity level in P. leniusculus than in A. astacus during daytime indicates that P. leniusculus is less strictly nocturnal than is A. astacus.     

Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning

The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.     

Occurrence of Alanine Racemase in Crustaceans and the Changes of the Properties During Seawater Acclimation of Crayfish

Alanine racemase activity was detected in the muscle and hepatopancreas of six macruran species. Optimal pH was around 8.5 irrespective of species and tissues. Apparent Michaelis constants ranged from 48 to 157 mM for muscle enzyme and 35 to 239 mM for hepatopancreas enzyme. The enzyme specifically catalyzed the racemization of d- and l-alanine. The enzyme did not require pyridoxal 5′-phosphate as well as FAD as a cofactor. Pyruvate and l-alanine showed strong inhibition in the direction of d to l.During seawater acclimation of crayfish Procambarus clarkii, alanine racemase activity increased about twice in muscle and 1.5 times in hepatopancreas. Michaelis constant, on the other hand, decreased 33% for muscle enzyme and 65% for hepatopancreas enzyme, suggesting the increase of substrate affinity during seawater acclimation. The activity in the physiological pH range (6.5–7.5) also increased with increasing salinity.     

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N^α-benzoyl-l-arginine, and N^ω-amino-l-arginine as substrates but not agmatine, l-homoarginine, N^α-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424–alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k₁ = 80 s⁻¹) and hydrolysis (k₂ = 35 s⁻¹) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k_cat = 2.6 s⁻¹), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N^ω-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Immobilization of Bacillus licheniformis L-Arabinose Isomerase for Semi-Continuous L-Ribulose Production

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific D-carbamoylase guarantee the total conversion from D,L-5-monosubstituted hydantoins with a low velocity of racemization to optically pure D-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55 degrees C with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni(2+) and Co(2+) and strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with Mn(2+), EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

β-1,3-glucanase activity in the stigma of healthy petunia flowers

β-1,3-Glucanase activity was detected in extracts of different tissues of healthy mature petunia flowers except the filament. The stigma was studied further as it had the highest enzyme activity and there is a paucity of information on the occurrence of this enzyme in this tissue. Specific activity of the enzyme was found to increase within the stigmatic tissue from early development until just before anthesis. Following non-denaturing polyacrylamide gel electrophoresis at pH 8.8, extracts of dehiscent stigma seem to contain three acidic isoforms of β-1,3-glucanase. Crude extracts of stigma was passed through a pachyman affinity column. A fraction of affinity-purified active β-1,3-glucanase enzyme was found to have no antifungal activity against Trichoderma viride, Phloma clematidina and Cladosporium fulvum.