d-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl-d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl-d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl-d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.     

Optimization of human d-amino acid oxidase expression in Escherichia coli

Human d-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAAO are indeed required for the investigation of its structure–function relationships and for the screening of new inhibitors to be used in the treatment of schizophrenia. A recombinant hDAAO has been over-expressed in BL21(DE3)Star E. coli cells. By alternating screenings of medium components at flask level and investigating physiological parameters in 2 L controlled batch fermentations, an improved, robust and scalable microbial process was set up giving almost a 40- and 4-fold improvement in volumetric productivity and specific activity, respectively. Under these conditions 770 U/L culture hDAAO with a specific activity of 0.4 U/mg protein and a specific productivity of 24.9 U/g biomass were produced. Optimization of medium ingredients, of the time and the amount of inducer’s addition, pH control at the moment of induction and harvest, low mechanical shear stress regime during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is higher than any previously described production of hDAAOs. A yield of 100 mg of pure hDAAO/L culture thus became available in comparison to the 1–10 mg/L previously reported.     

Free l-amino acids and d-aspartate content in the nervous system of Cephalopoda. A comparative study

We have determined the content of free l-amino acids and d-aspartate in the nervous tissue of three representative cephalopods: Sepia officinalis, Octopus vulgaris, and Loligo vulgaris, and the optic lobes of adult and embryo Sepia officinalis. Taurine is the most abundant amino acid in the cephalopod nervous tissue. Its content amounts to more than 50% of the total free amino acids. The other most concentrated amino acids are Glu, Ala, Asp, and GABA. High concentrations of d-aspartate were found in the nervous tissue of all cephalopods examined (7–12 μmol/g wet tissue) which represents 50–80% of the total aspartate (d + l), depending on the animal. Among the various regions of the brain of Octopus vulgaris, d-aspartate was found to be evenly distributed in the various regions of the brain. In nerve tissue of Sepia officinalis, there is no significant difference in the pattern of free l-amino acids, in particular of the d-aspartate concentration, between adults and embryos, except for GABA, Gly, His and Thr. This suggests that d-aspartate in nerve tissue of the Cephalopoda is of endogenous origin and not a product of accumulation from exogenous sources. From a comparative study of the content of d-aspartate in the nervous tissue of different animals, we found that protostomia contain a significantly higher amount than deuterostomia. Thus, d-aspartate could be a criterion to distinguish the protostomia phyla from the deuterostomia phyla.     

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.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Modification of optimal pH in l-arabinose isomerase from Geobacillus stearothermophilus for d-galactose isomerization

l-Arabinose isomerase from Geobacillus stearothermophilus (GSAI; EC 5.3.1.4) has been genetically evolved to increase the reaction rate toward d-galactose, which is not a natural substrate. To change the optimal pH of GSAI for d-galactose isomerization (pH optimum at 8.5), we investigated the single point mutations influencing the activity based on the sequences of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val⁴⁰⁸ and Asn⁴⁷⁵ were determined to be highly influential mutation points for d-galactose isomerization activity. A random mutation was introduced into sites Val⁴⁰⁸ and Asn⁴⁷⁵ (X408V and X475N), and candidates were screened based on non-optimal pH conditions. Among the mutations of X408V and X475N, mutations of Q408V and R408V were selected. The optimal pH of the both mutations Q408V and R408V was shifted to pH 7.5. At the shifted optimal pH, the d-galactose isomerization activities of Q408V and R408V were 60 and 30% higher than that of the wild type at pH 8.5, respectively.     

Enzymatic conversion of N carbamoyl-d-amino acids to d-amino acids

An enzyme isolated from Agrobacterium radiobacter was shown to catalyse the following reaction: H₂O + N-carbamoyl-d-amino acid→ d-amino acid + NH₃ + CO₂ Some properties of this new enzyme, N-carbamoyl-d-amino acid amidohydrolase, are presented in this paper. The potential application of this enzyme for the preparation of some d-amino acids used as pharmaceutical intermediates is discussed.     

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.     

Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli

To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml⁻¹ was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l⁻¹. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml⁻¹ and GL-7-ACA acylase activity of 950 U l⁻¹ were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly.     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.     

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%.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].     

Production of -amino acids by N-acyl- -amino acid amidohydrolase and its structure and function

-Amino acids have been widely used as synthetic materials for various compounds such as pharmaceuticals and agrochemicals. The manufacture of -amino acids by fermentation is difficult, and enzymatic methods are mainly employed. At present, the optical resolution method using N-acyl- -amino acid amidohydrolase is the most useful and convenient. In this review, the application of N-acyl- -amino acid amidohydrolase to the production of -amino acids and recent progress in the study of structure–function relationships from the standpoint of improving this enzyme for industrial application are discussed.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4–9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1–3, 5–8, 10 and 12–15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

Production of quinoprotein d-glucose dehydrogenase in the culture medium of Acinetobacter calcoaceticus

On addition of low concentrations (0.005%) of Triton X-100 to a mineral medium supplemented with 0.5% heptadecane, a marked stimulation of growth rate was observed for Acinetobacter calcoaceticus strains able to grow on alkanes while appreciable amounts of soluble quinoprotein d-glucose dehydrogenase [d-glucose: (pyrroloquinoline-quinone) 1-oxidoreductase, EC 1.1.99.17] were found in the culture medium. At higher Triton X-100 concentrations (0.04%), still larger amounts of d-glucose dehydrogenase and also cytoplasmic enzyme activities appeared in the culture medium. Although combinations of other carbon sources plus non-ionic detergents also produced these enzymes in the medium, the combination of heptadecane and Triton X-100 gave higher levels and had a stabilizing effect on d-glucose dehydrogenase. Therefore, by using this combination and culturing within certain pH limits, a stable enzyme solution, having already a high specific activity, is produced while the cell harvesting and disruption steps can be circumvented. The results indicate that d-glucose dehydrogenase in this organism is a periplasmic enzyme, coupled to a cytochrome b.