Screening of microorganisms producing α-methylserine hydroxymethyltransferase, purification of the enzyme, gene cloning, and application to the enzymatic synthesis of α-methyl-l-serine

Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85 kDa and contains 1 mol of pyridoxal-5′-phosphate per mol of the subunit. The K_m for α-methyl-l-serine and tetrahydrofolate was 0.54 mM and 73 μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278 bp, encoding a polypeptide with a calculated molecular mass of 46.0 kDa. Using E. coli cells as whole-cell catalysts, 9.7 mmol of α-methyl-l-serine was stereoselectively obtained from 15 mmol of d-alanine and 13.2 mmol of formaldehyde.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

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.     

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.     

Hydroxylation of l-proline to cis-3-hydroxy-l-proline by recombinant Escherichia coli expressing a synthetic l-proline-3-hydroxylase gene

A synthetic gene encoding a Streptomyces l-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l-proline to cis-3-hydroxy-l-proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy-l-proline.     

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.     

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.     

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.

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.     

Screening strains for directed biosynthesis of β-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml⁻¹ was obtained.     

Expression, purification and characterization of the chitinolytic β-N-acetyl-d-hexosaminidase from the insect Ostrinia furnacalis

Insect β-N-acetyl-d-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic β-N-acetyl-d-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic β-N-acetyl-d-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120 h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7 mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130 kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)₂ and release 4-methylumbelliferone (4MU) from substrates, 4MU-β-GlcNAc and 4MU-β-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect β-N-acetyl-d-hexosaminidases.     

Enzymatic resolution of methyl dl-β-acetylthioisobutyrate and dl-β-acetylthioisobutyramide using a stereoselective esterase from Pseudomonas putida IFO12996

Esterase (PpEST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-β-acetylthioisobutyrate (DL-MATI) and dl-β-acetylthioisobutyramide (DL-ATIA) to give d-β-acetylthioisobutyric acid (DAT). DAT is a key intermediate for the synthesis of a series of angiotensin converting enzyme inhibitors. To use enzyme for the DAT production, the PpEST gene of P. putida IFO12996 was cloned and expressed in Escherichia coli. PpEST with a molecular weight of 33 kDa could hydrolyze DL-MATI and DL-ATIA to give DAT with enantiometric excess value (e.e. value) about 97% and enantioselectivity value (E-value) >150, respectively. The kinetic constants of PpEST for DL-MATI and DL-ATIA were examined and they showed that DL-ATIA was a poorer substrate than DL-MATI for PpEST. However, DL-ATIA was 20-fold more soluble in water than DL-MATI, it was more stable than DL-MATI and it did not show substrate inhibition of the PpEST up to 780 mM. This result suggested that PpEST is an esterase but with amidase activity, which can kinetically resolve DL-ATIA to yield DAT and DL-ATIA is a better choice than DL-MATI for industrial production of DAT by the enzymatic resolution method.     

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.     

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.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation–reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2–7 were confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

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%.     

Isolation and purification of d-mannose from palm kernel

An economically viable procedure for the isolation and purification of d-mannose from palm kernel was developed in this research. The palm kernel was catalytically hydrolyzed with sulfuric acid at 100 °C and then fermented by mannan-degrading enzymes. The solution after fermentation underwent filtration in a silica gel column, desalination by ion-exchange resin, and crystallization in ethanol to produce pure d-mannose in a total yield of 48.4% (based on the weight of the palm kernel). Different enzymes were investigated, and the results indicated that endo-β-mannanase was the best enzyme to promote the hydrolysis of the oligosaccharides isolated from the palm kernel. The pure d-mannose sample was characterized by FTIR, ¹H NMR, and ¹³C NMR spectra.     

Molecular mass control using polyanionic cyclodextrin derivatives for the epsilon-poly-l-lysine biosynthesis by Streptomyces

To control the molecular mass of a natural polycationic, antimicrobial, Streptomyces-biosynthesized polymer, epsilon-poly-l-lysine, addition of polyanionic cyclodextrin derivatives to the culture medium was evaluated. Chemically modified cyclodextrins such as a sulfated cyclodextrin caused a notable shortening of the polymer length of epsilon-poly-l-lysine from 3.5 to 4.5 kDa to less than 2.5 kDa by the enforcing action of glycerol, which has a weak potential to inhibit polymer elongation by terminal esterification. Meanwhile, polyanionic cyclodextrin inhibited the shortening action with n-octanol, which has a strong ability to inhibit polymer elongation through an undetermined function. The design of chemical structures of polyanionic cyclodextrin, optimization of their addition concentrations, and selection of hydroxyl compounds coexisting with them are critical for this simple and easy method for polymer length control and terminal modification of epsilon-poly-l-lysine.     

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.