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.     

Antioxidative defense organization in retroperitoneal white adipose tissue during acclimation to cold—The involvement of l-arginine/NO pathway

1. Retroperitoneal white adipose tissue (RpWAT) antioxidative defense was investigated in untreated, l-arginine-treated and N^ω-nitro-l-arginine methyl ester (l-NAME)-treated rats kept at 4±1 °C (1, 3, 7, 12, 21 and 45 days) and compared to control rats at 22±1 °C.

2. Cold-acclimation-induced RpWAT weight decrease was accompanied by a decline in glutathione level and increased activity of manganese superoxide dismutase (MnSOD), glutathione S-transferase (GST), catalase, glutathione peroxidase and glutathione reductase at different time-points.

3. l-arginine accelerated RpWAT weight decrease, the increase in MnSOD and GST activities and the prolonged increase of catalase, MnSOD and GST activities. l-NAME delayed cold-induced catalase activity increase and tissue weight decrease. Prolonged l-NAME-treatment had a similar effect on RpWAT as l-arginine.

4. Results suggest the involvement of l-arginine/NO pathway in RpWAT oxidative metabolic augmentation induced by cold-acclimation.

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.     

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.     

Biodegradation pathway of l-glutamatediacetate by Rhizobium radiobacter strain BG-1

An aerobic bacterium was isolated from activated sludge in a medium containing l-glutamate-N,N-diacetate (l-GLDA) as sole carbon and energy source. The isolate was identified as a Rhizobium radiobacter species. Besides l-GLDA, the strain utilized nitrilotriacetate (NTA) and proposed intermediates in l-GLDA metabolism such as glyoxylate and l-glutamate. l-GLDA-grown cells oxidized l-GLDA, l-glutamate but not iminodiacetate (IDA), and trans-ketoglutaconate, indicating removal of a carboxymethyl group as an initial degradation reaction. The removal of the first carboxymethyl group of l-GLDA is catalyzed by an NADH-dependent mono-oxygenase. The oxidative deamination of l-glutamate by a dehydrogenase resulting in the formation of oxoglutarate was also detected in cell-free extracts of R. radiobacter sp. A pathway for the metabolism of l-GLDA R. radiobacter sp. is proposed: First, l-GLDA leads to l-glutamate-N-monoacetate (l-GLMA) which in turn leads to l-glutamate. Then, l-glutamate leads to oxoglutarate, an intermediate of the TCA cycle.     

Unique transglycosylation potential of extracellular α-d-galactosidase from Talaromyces flavus

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.     

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.     

Encapsulation of an Agrobacterium radiobacter extract containing d-hydantoinase and d-carbamoylase activities into alginate–chitosan polyelectrolyte complexes: Preparation of the biocatalyst

Alginate–chitosan polyelectrolyte complexes (PECs) have been used for the first time as a suitable matrix for coimmobilisation of enzymes to reproduce a multistep enzymatic route for production of d-amino acids. Encapsulation of a crude cell extract from Agrobacterium radiobacter containing d-hydantoinase and d-carbamoylase activities into the PECs with negligible leakage from the formed capsules was accomplished. All results in this study indicate that the preparation of the biocatalyst (preparation method and chitosan characteristics) play a key role in the biocatalyst's properties. The most suitable biocatalysts were prepared using a chitosan with a medium molecular weight (600 kDa) and a degree of deacetylation of 0.9. For all of the preparation conditions under study, an encapsulation yield of around 60% was achieved and the enzymatic activity yields ranged from 30 to 80% for d-hydantoinase activity and from 40 to 128% for d-carbamoylase activity relative to the activities of the soluble extract. All of the biocatalysts were able to hydrolyze l,d-hydroxyphenylhydantoin into p-hydroxyphenylglycine with yields ranging from 30 to 80%.     

Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids

d-Amino acid oxidase is a FAD-dependent enzyme that catalyses the conversion of the d-enantiomer of amino acids into the corresponding α-keto acid. Substrate specificity of the enzyme from the yeast Rhodotorula gracilis was investigated towards aromatic amino acids, and particularly synthetic α-amino acids.A significant improvement of the activity (V_max,app) and of the specificity constant (the V_max,app/K_m,app ratio) on a number of the substrates tested was obtained using a single-point mutant enzyme designed by a rational approach. With R. gracilis d-amino acid oxidase the complete resolution of d,l-homo-phenylalanine was obtained with the aim to produce the corresponding pure l-isomer and to use the corresponding α-keto acid as a precursor of the amino acid in the l-form.     

The immobilization of d-hydantoinase and characterization under classic condition and microwave irradiation

d-Hydantoinase was covalently immobilized onto polystyrene anion exchange resin via glutaraldehyde. Immobilization conditions were optimized: the carrier as D-92 type polystyrene anion exchange resin, temperature as 25 °C, immobilization time as 12 h, and initial concentration of protein as 6 mg/ml. Under the optimized reaction conditions the activity of the free and immobilized d-hydantoinase was determined. The free and immobilized d-hydantoinase samples were characterized with their kinetic parameters, thermal, and storage stability. The K_m and V_max values were 14.985 mM and 0.6 mM/min for the free, and 27.030 mM and 1.187 mM/min for the immobilized, respectively. Operational stability of the immobilized d-hydantoinase was also detected in a circulating packed-bed reactor. The half-time of the immobilized d-hydantoinase was 11 days. Nearly 90% of activity of the immobilized d-hydantoinase was reserved for 100 days stored at 4 °C. The free and immobilized d-hydantoinases were also characterized under microwave irradiation. Results shown that the reactions catalyzed by both free and immobilized d-hydantoinase were accelerated under microwave irradiation. The half-time of the immobilized d-hydantoinase reduced to 16 min under microwave irradiation.     

Synthesis and glycogen phosphorylase inhibitory activity of N-(β-d-glucopyranosyl)amides possessing 1,4-benzodioxane moiety

A series of N-(β-d-glucopyranosyl)amides 5d–i were synthesized by PMe₃ mediated Staudinger reaction of O-peracetylated β-d-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d–i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure–activity relationships of these compounds are also discussed in this paper.     

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.     

Control of water activity in lipase catalysed esterification of chiral alkanoic acids

An enzymatic method for ready access to d-sedoheptulose-7-phosphate on a preparative scale was developed, based on the irreversible transketolase-catalyzed reaction: β-hydroxypyruvate + d-ribose-5-phosphate → d-sedoheptulose-7-phosphate. d-Sedoheptulose-7-phosphate disodium salt was obtained in 81% overall yield determined using a standard curve obtained by LC/MS/MS.     

Evaluation of l-glutamine for cryopreservation of boar spermatozoa

The aim of the present study was to evaluate the protective effect of l-glutamine (l-Gln) against cryopreservation injuries on boar sperm. In Experiment 1, l-Gln from 20 to 80 mM was evaluated as a supplement for a standard freezing extender (egg yolk – EY – 20%, and glycerol 3%). No significant improvement (P > 0.05) was obtained for any post-thaw sperm parameter assessed (objective sperm motility – CASA system – and flow cytometric analysis of plasma and acrosomal membrane integrity −SYBR14/PI/PE-PNA− and plasma membrane stability −M540/YoPro1−). In Experiment 2, l-Gln was evaluated as a partial glycerol substitute in the freezing extender. Significant (P < 0.05) enhancement of post-thaw sperm motion parameters was achieved in sperm frozen in the presence of 2% glycerol and 80 mM l-Gln compared to control (3% glycerol). In Experiment 3, l-Gln was evaluated as an EY substitute in the freezing extender, and no functional sperm were recovered after thawing sperm frozen in the presence of l-Gln and the absence of EY. In conclusion, l-Gln has the ability to cryoprotect boar sperm when it is used as a partial glycerol substitute in the freezing extender.     

A pyrroloquinoline quinine-dependent membrane-bound d-sorbitol dehydrogenase from Gluconobacter oxydans exhibits an ordered Bi Bi reaction mechanism

A membrane-bound pyrroloquinoline quinine (PQQ)-dependent d-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of d-sorbitol to l-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by d-sorbitol), while products are released in sequence (first l-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for d-sorbitol, in addition to PQQ-binding and Mg²⁺-binding sites in the catalytic center.     

Development of an interference-free biosensor for l-glutamate using a bienzyme salicylate hydroxylase/l-glutamate dehydrogenase system

An amperometric biosensor was developed for the interference-free determination of l-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l-glutamate consuming NAD⁺. The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of l-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate with a detection limit of 3.0 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and electroactive substances.     

l-Carnitine enhances resistance to oxidative stress by reducing DNA damage in Ataxia telangiectasia cells

In this study, the modulating effect of l-carnitine on tert-butyl-hydroperoxide-induced DNA damage was compared with that of mannitol, a well known scavenger of hydroxyl radicals, both in normal and Ataxia telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines established from A. telangiectasia (A-T) patients. The alkaline version of the comet assay was employed to measure the frequency of single-strand breaks (SSBs) and alkali-labile sites induced by t-butyl-OOH immediately after treatment and at different recovery times in normal and A-T cell lines, with and without pre-treatment with l-carnitine. In addition, both the yield of induced chromosomal damage and the effect on cell proliferation were evaluated. Our results show that pre-treatment of cells with l-carnitine produced an enhancement of the rate and extent of DNA repair in A-T cell lines at early recovery time; furthermore, in samples pre-treated with l-carnitine a reduction of all types of chromosomal aberration was observed, both in A-T and in wild-type cell lines. The reducing effect of l-carnitine pre-treatment on oxidative DNA damage was more prominent than that of pre-treatment with mannitol. In conclusion, we demonstrated a protective effect of l-carnitine on oxidative stress-induced DNA damage in A-T cells, suggesting its possible role in future pharmacological applications in A-T therapy.     

Enzymatic synthesis of d-xylulose 5-phosphate from hydroxypyruvate and d-glyceraldehyde-3-phosphate

An enzymatic method for obtaining d-xylulose 5-phosphate has been developed, based on the irreversible reaction catalyzed by transketolase: hydroxypyruvate + d-glyceraldehyde-3-phosphate → d-xylulose 5-phosphate. The preparations of sodium d-xylulose 5-phosphate, obtained using this approach, were 88% pure and contained no aldehyde admixtures.     

Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol

The effects of d-limonene concentration, enzyme loading, and pH on ethanol production from simultaneous saccharification and fermentation (SSF) of citrus peel waste by Saccharomyces cerevisiae were studied at 37 °C. Prior to SSF, citrus peel waste underwent a steam explosion process to remove more than 90% of the initial d-limonene present in the peel waste. d-Limonene is known to inhibit yeast growth and experiments were performed where d-limonene was added back to peel to determine threshold inhibition amounts. Ethanol concentrations after 24 h were reduced in fermentations with initial d-limonene concentrations greater than or equal to 0.33% (v/v) and final (24 h) d-limonene concentrations greater than or equal to 0.14% (v/v). Ethanol production was reduced when enzyme loadings were (IU or FPU/g peel dry solids) less than 25, pectinase; 0.02, cellulase; and 13, beta-glucosidase. Ethanol production was greatest when the initial pH of the peel waste was adjusted to 6.0.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.