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.     

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na⁺/K⁺-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na⁺/K⁺- and Mg²⁺-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na⁺/K⁺-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na⁺/K⁺-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na⁺/K⁺-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na⁺/K⁺-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.     

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.     

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.     

Properties of the sugar carrier in Baker's yeast

Incubation ofSaccharomyces cerevisiae cells withd-galactose induced the formation of galactose-utilizing enzymes, among them a monosaccharide carrier, apparently synthesized as a proteinde novo. The synthesis of the carrier preceded that of galactokinase by as much as 2 h. The inducible carrier shows a preference for monosaccharides with an axial hydroxyl group at carbon 4 of theC1 chair conformation or at carbon 2 of the1C chair conformation. Through its mediation, some sugars normally poorly transported (d-galactose,d-fucose,l-xylose,l-arabinose) can enter into the entire cell water, occupying then one more kinetic (and morphological ?) compartment than before induction. Some other monosaccharides, readily transported even by a constitutive carrier system (e.g.l-sorbose,d-xylose,d-arabinose) share the newly synthesized carrier.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

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.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

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.     

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium

The l-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of d-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting l-alanine to the germination inhibitor d-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of l- to d-alanine. Unlike ATCC 14579 spores, l-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.     

Effects of l-lysine and d-lysine on ɛ-Poly-l-lysine biosynthesis and their metabolites by Streptomyces ahygroscopicus GIM8

?-Poly-l-lysine (?-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of l-lysine, which is used as a safe food preservative. In this study, the effects of l-lysine and its isomer, d-lysine, on ?-PL biosynthesis and their metabolites by the ?-PL-producing strain Streptomyces ahygroscopicus GIM8 were determined. The results indicated that l-lysine added into the fermentation medium in the production phase mainly served as a precursor for ?-PL biosynthesis during the flask culture phase, leading to greater ?-PL production. At an optimum level of 3 mM l-lysine, a ?-PL yield of 1.16 g/L was attained, with a 41.4% increment relative to the control of 0.78 g/L. Regarding d-lysine, the production of ?-PL increased by increasing its concentrations up to 6 mM in the initial fermentation medium. Interestingly, ?-PL production (1.20 g/L) with the addition of 3 mM d-lysine into the initial fermentation medium in flasks was higher than that of the initial addition of 3 mM L-lysine (1.06 g/L). The mechanism by which d-lysine improves ?-PL biosynthesis involves its utilization that leads to greater biomass. After S. ahygroscopicus GIM8 was cultivated in the defined medium with L-lysine, several key metabolites, including 5-aminovalerate, pipecolate, and l-2-aminoadipate formed in the cells, whereas only l-2-aminoadipate was observed after d-lysine metabolism. This result indicates that l-lysine and d-lysine undergo different metabolic pathways in the cells. Undoubtedly, the results of this study are expected to aid the understanding of ?-PL biosynthesis and serve as reference for the formulation of an alternative approach to improve ?-PL productivity using l-lysine as an additional substrate in the fermentation medium.     

Metabolic engineering of Escherichia coli to produce (2S, 3R, 4S)-4-hydroxyisoleucine

The stereo-specific l-isoleucine-4-hydroxylase (l-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to “shunt” destroyed TCA, thereby coupling l-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of l-isoleucine into 4-HIL with an 82% yield.     

Is l-arabinose important for the endophytic lifestyle of Pseudomonas spp.?

Twenty endophytic bacteria were isolated from surface-sterilized stems and roots of cucumber plants. After removal of potential siblings and human pathogens, the remaining seven strains were identified based on their 16S rDNA as Pseudomonas fluorescens (2 strains) and P. putida (5 strains). Three strains, namely P. fluorescens CS1, P. fluorescens CR2 and P. putida CR3, were able to suppress tomato foot and root rot (TFRR). Special attention was paid to the characterization of the BIOLOG carbon oxidation profiles of the isolated pseudomonads in order to identify nutrients which might be important for their endophytic lifestyle. Comparative analysis of the profiles of these seven strains with those of seven rhizospheric Pseudomonas spp. revealed that endophytes were able to oxidize l-arabinose and 2,3-butanediol significantly more often than the rhizospheric group. An independent growth experiment performed in tubes using l-arabinose and 2,3-butanediol as sole carbon sources showed the same results as seen using BIOLOG for l-arabinose, but not for 2,3-butanediol. Since l-arabinose is one of the most abundant sugars in xylem of cucumber plants and was not detected in their rhizosphere, our data suggest that utilization of l-arabinose might be a trait contributing to the endophytic lifestyle of the isolated Pseudomonas endophytes.     

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.     

Biochemical and kinetic characterization of GH43 β-d-xylosidase/α-l-arabinofuranosidase and GH30 α-l-arabinofuranosidase/β-d-xylosidase from rumen metagenome

The present study focuses on characterization of two hemicellulases, RuXyn1 and RuXyn2, from rumen bacterial metagenome and their capabilities for degradation of xylans. Glycosyl hydrolase (GH) family?43 ??-d-xylosidase/??-l-arabinofuranosidase RuXyn1 can hydrolyze p-nitrophenyl-??-d-xylopyranoside (pNPX), p-nitrophenyl-??-l-arabinofuranoside (pNPA), and xylo-oligosaccharide substrates, while GH30 1,5-??-l-arabinofuranosidase/??-d-xylosidase RuXyn2, the first ??-l-arabinofuranosidase assigned to this GH family, shows activities towards 1,5-??-l-arabinobiose and pNPX substrates but no activity for pNPA. Kinetic analysis for aryl-glycosides revealed that RuXyn2 had higher catalytic efficiency than RuXyn1 toward pNPX substrate. RuXyn1 shows high synergism with endoxylanase, elevating by 73% the reducing sugars released from brichwood xylans, and converted most intermediate xylo-oligosaccharide hydrolysate into xylose. The high xylose conversion capability of RuXyn1 suggests it has potential applications in enzymatic production of xylose and improvement of hemicellulose saccharification for production of biofuels. RuXyn2 shows no obviously synergistic effect in the endoxylanase-coupled assay for enzymatic saccharification of xylan. Further cosmid DNA sequencing revealed a neighboring putative GH43 ??-l-arabinofuranosidase RuAra1 and two putative GH3 ??-xylosidase/arabinosidases, RuXyn3 and RuXyn5, downstream of RuXyn2, indicating that this hemicellulase gene cluster may be responsible for production of end-product, xylose and arabinose, from hemicellulose biomass.     

d-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation

Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L^?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g^?1 and 1.01 g L^?1 h^?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h^?1 and product formation rate 0.026 h^?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.