l-Serine production using a resting cell system of Hyphomicrobium strains

Twenty-six strains of methylotrophic hyphomicrobia were examined for their ability to produce l-serine from methanol and glycine in a resting cell reaction. l-Serine productivity of over 5 mg/ml was observed in 7 strains, and Hyphomicrobium sp. NCIB10099 was found to exhibit the highest productivity. Under optimized conditions using this bacterium, 45 mg/ml l-serine was produced from 100 mg/ml glycine and 88 mg/ml methanol in 3 d. The high l-serine degrading activity of the bacterium was entirely suppressed by adding an appropriate amount of CdCl₂ (ca. 1 mM), resulting in an enhanced conversion ratio of glycine to l-serine (ca. 100% molar conversion).     

Efficient conversion of glycine to l-serine by a glycine-resistant mutant of a methylotroph using Co2+ as an inhibitor of l-serine degradation

A glycine-resistant mutant, no. 18, which was not lysed by glycine, was obtained from an l-serine-producing mutant, S395 (temperature-sensitive, O-methylserine-resistant), of a facultative methylotroph, Pseudomonas MS31. The mutant stably produced l-serine from glycine. The properties of the enzymes involved in the synthesis and degradation of l-serine were investigated in the wild-type strain MS31 and the l-serine-producing mutants. Mutant derivation had no effect on the activities of methanol dehydrogenase or serine hydroxymethyltransferase, which are involved in l-serine synthesis. On the other hand, the activity of l-serine dehydratase (SDH), which degrades l-serine, was reduced in the mutants. Cobalt (Co²⁺) inhibited SDH activity and its addition (6.5 mM) to the l-serine production culture significantly stimulated l-serine accumulation up to 14.9 mg/ml. The results suggest that blocking of SDH is important for the efficient production of l-serine from glycine by methylotrophs.     

Production of l-serine from methanol and glycine by resting cells of a methylotroph under automatically controlled conditions

Serine production from methanol and glycine was tried using frozen-thawed resting cells of a methylotroph, Protomonas extorquens NR-1 under multi-variable controlled conditions. The conditions for l-serine formation were optimized at 30°C. The production of l-serine in 0.4% CaCl₂ solution (initial pH 8.2) was the same as in 0.1 M Tris-HCl buffer (initial pH 8.3). Increasing the initial glycine concentration promoted l-serine formation. A high aeration rate decreased l-serine production. The optimum concentrations of dissolved oxygen and methanol were 0.5 ppm and 10 g/l, respectively. The highest l-serine, 24.9 g/l, was obtained at 24 h from 30.94 gl (as dry weight) resting cells using 100 g/l initial glycine with controlled pH. The relationship between the initial rate of l-serine formation and cell concentration indicated an unusual curve due to the effects of the added NaOH which was used for controlling the pH. In similar experiments without control of pH, a normal profile was observed with respect to the relationship between the initial rate of l-serine formation and cell concentration. The highest l-serine, 54.5 g/l, was obtained at 48 h by 36.4 g/l (as dry weight) resting cells. The yield (mol of l-serine/mol of added substrate) of l-serine from methanol and glycine were 8.3% and 39.3%, respectively. The selectivity of l-serine (mol of l-serine/mol of glycine consumed) was 67.9%. The stoichiometry of the maximum l-serine formation showed that the resting cells carried the highly active methanol dehydrogenase while serine transhydroxymethylase was rather low. Serine glyoxalate aminotransferase was not completely inhibited by the high concentration of glycine (about 68% of synthesized l-serine was detected in the supernatant.     

l-serine production from methanol and glycine with an immobilized methylotroph

l-Serine production from methanol and glycine was attempted using immobilized resting cells of a methylotroph, Protomonas extorquens NR 1, under automatically controlled conditions. A Ca-alginate system was selected. The conditions for l-serine formation were optimized at 30°C. A concentration of glycine 100 g·l⁻¹ which was the optimum concentration for l-serine production by free resting cells was used in the reaction mixture. The optimum concentrations of methanol and dissolved oxygen were 20 g·l⁻¹ and 5 ppm, respectively. Under the optimum conditions, 11.3 g·l⁻¹ of l-serine was produced within 36 h. The selectivities (mole of l-serine/mole of substrate consumed) of l-serine from methanol and glycine were 4.5% and 95.1%, respectively. The size of gel beads affected the l-serine formation rate. The initial rate of l-serine formation decreased with an increase in the size of beads. However, the l-serine formation rate increased at elevated concentrations of dissolved oxygen, even with large sized beads. This result implies that the oxygen diffusion inside the gel beads limited the l-serine formation rate. The observed effectiveness factor of the immobilized cells could be estimated by the theoretical effectiveness factor of the zero-order reaction with respect to the dissolved oxygen.Repeated use was not feasible without reactivation of the immobilized cells. Reusability was examined by reactivation of the immobilized resting cells in appropriate media for 12 h. The reactivated immobilized resting cells were used again in the next cycle. By this procedure, several cycles of l-serine formation were made possible.     

l-Serine Production by a Mutant of Sarcina albida Defective in l-Serine Degradation

For improved l-serine production, an l-serine dehydratase-defective mutant of Sarcina albida IAM 1012 was obtained. In the mutant, the activities of the enzymes responsible for l-serine production were as high as those in the parent strain, and, at a low glycine concentration, the mutant accumulated l-serine more efficiently than the parent. Under optimum conditions, 21 mg of l-serine per ml accumulated from 100 mg of glycine per ml. l-Serine was isolated from a reaction mixture as l-serine m-xylene-4-sulfonate, and free amino acid was obtained in high yields by use of an ion-exchange resin. Residual glycine was recovered at a yield of 61%.     

Production of l-serine from glycine by Corynebacterium glycinophilum and properties of serine hydroxymethyltransferase,a key enzyme in l-serine production

Fermentative production of l-serine from glycine by Corynebacterium glycinophilum AJ-3413, an auxotrophic mutant of Leu and Met with increased productivity of l-serine using a one liter jar fermentor was carried out and the properties of serine hydroxymethyltransferase (SHMT), a key enzyme in l-serine synthesis, of the parental strain AJ-3170 were investigated. SHMT was effectively induced by the addition of glycine to the medium at an early stage of cultivation. Under optimal conditions, AJ-3413 produced 16.0 g/l of l-serine from 30 g/l of glycine with a molar yield of 38%. The partially purified SHMT catalyzed the l-allo-threonine degradation in addition to l-serine degradation, but could not catalyze l-threonine degradation. This enzyme showed an absolute tetrahydrofolic acid requirement for l-serine degradation to glycine and formaldehyde, but not for l-allo-threonine degradation. Pyridoxal 5′-phosphate appeared to be required for enzyme activity. The K_m values for glycine and formaldehyde in l-serine synthesis, and for l-serine in l-serine degradation were 1.85, 0.29 and 1.64 mM, respectively.     

L-serine production by a methionine-auxotrophic mutant of methylotrophic Pseudomonas

A methionine-auxotropic mutant deficient in homocysteine transmethylation activity was induced from a methylotrophic L-serine-producing derivatives of Pseudomonas MS31. This mutant grown with limited L-methionine had more than 1.7-fold higher serine hydroxymethyltransferase (SHMT) activity than its parent strain. The elevated SHMT activity significantly contributed to the improvement of L-serine accumulation from glycine and methanol. Under the optimum conditions, this mutant accumulated up to 23.9 mg/ml of L-serine. The yield coefficient L-serine from consumed glycine was 89% (mol/mol). The maximum conversion rate of added glycine (19 mg/ml) to L-serine was 77% (mol/mol).     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional l-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of l-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (< 30 s) paired-tracer dilution technique. In the non-diabetic pancreas l-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 μU/ml). Transport of l-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of l-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

Effects of l-serine dehydratase activity on l-serine production by Corynebacterium glycinophilum and an examination of the properties of the enzyme

The results with Corynebacterium glycinophilum AJ-3170 and various mutants from AJ-3170 indicated that l-serine production was almost inversely proportional to l-serine degrading activity. The crude extract of the parental strain, AJ-3170, showed l-serine and l-threonine degrading activities. The 2 activities were completely separated from each other by gel-filtration, indicating that each activity comes from a different enzyme. The l-serine degrading enzyme, l-serine dehydratase (SD), was purified 30-fold from AJ-3170. Molecular weight of SD was 130,000. The enzyme was specific for l-serine, activated slightly by FeCl₂ and inhibited by MnCl₂. The double reciprocal plots of SD rate against substrate concentration gave an upwards-curved line. The value of [S]_0.5 was 35 mM.     

In vitro antimicrobial activity against Pseudomonas aeruginosa and acute oral toxicity of marine algae Gracilaria changii

Methanol extract of the Gracilaria changii has been screened for antimicrobial activity against Pseudomonas aeruginosa. Antimicrobial activities were carried out using disc diffusion assay and broth dilution method against P. aeruginosa. The methanol extract of G. changii showed a good antimicrobial activity against P. aeruginosa with MIC (Minimum Inhibitory Concentration) value of 6.25 mg/ml. Exposure of P. aeruginosa cells to 6.25 mg/ml of methanol extract of G. changii resulted in complete inhibition of the bacterial cells. The main abnormalities noted via SEM and TEM studies were the alterations in morphology and cytology of the bacterial cells. The main reason for this deterioration was discussed. The effect of the methanol extract on the growth profile for the bacteria was also done and confirmed the bactericidal effect of the G. changii methanol extract on P. aeruginosa by changing the normal growth profile of P. aeruginosa. In an acute toxicity study using mice, the median lethal dose (LD₅₀) of the extract was greater than 2000 mg/kg, and we found no pathological changes in macroscopic examination by necropsy of mice treated with extract. We conclude that G. changii might be safely used as an antimicrobial agent.     

Characterization,modification, and overexpression of 3-phosphoglycerate dehydrogenase in Corynebacterium glutamicum for enhancing l-serine production

The direct fermentative production of l-serine from renewable biomass using Corynebacterium glutamicum is attracting increasing attention. In this study, wild-type C. glutamicum SYPS-062 produced up to 6.65 ± 0.23 g/L l-serine; to further improve l-serine production, the serA gene was cloned, and the C-terminal domain of 3-phosphoglycerate dehydrogenase (PGDH) from this strain was truncated. When expressed in Escherichia coli, the resultant mutein SerAΔ197 showed a specific PGDH activity of 1.092 ± 0.05 U/mg protein, representing a decrease of 25.87 % from that encoded by serA, and was no longer sensitive to high concentrations of l-serine. When serA Δ591 was overexpressed in C. glutamicum SYPS-062, the activity of PGDH in C. glutamicum pJC1-tac-serA Δ591 increased by 47.72 %, and the resultant strain C. glutamicum pJC1-tac-serA Δ591 could accumulate 7.69 ± 0.22 g/L l-serine. Furthermore, when serA Δ591 was overexpressed in C. glutamicum SYPS-062ΔsdaA, the resultant strain could accumulate 8.84 ± 0.23 g/L l-serine at 102 h, and the yield of l-serine on cells (Y p/x) improved by 60 % when compared with that noted in the control. These results demonstrate that l-serine production in C. glutamicum SYPS-062 could be improved by overexpressing a C-terminal truncation of PGDH in combination with other genetic modifications.     

In vitro propagation of the medicinal plant Ziziphora tenuior L. and evaluation of its antioxidant activity

Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants.The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA.DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC₅₀ was 0.307 and 0.369 mg/ml, respectively, while the IC₅₀ of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity.     

Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species

The antimicrobial activity of 35 indigenous South African Helichrysum species was determined against six microorganisms. Seven of the 36 chloroform:methanol (1:1) extracts (leaf and stem extracts for all plants and an additional flower extract for H. rugulosum) exhibited minimum inhibitory concentration (MIC) values lower than 0.1 mg/ml against Bacillus cereus and/or Staphylococcus aureus. The in vitro cytotoxicity [against transformed human kidney epithelial (Graham) cells, MCF-7 breast adenocarcinoma and SF-268 glioblastoma cells] of these extracts was also determined at a concentration of 0.1 mg/ml using the sulforhodamine B (SRB) assay. For seven species less than 25% growth was observed for the Graham and MCF-7 cell lines at the test concentration.     

Effect of acute ethanol on serine biosynthesis in liver

The effect of an acute intraperitoneal dose of ethanol (1 g/kg), glucose (7.2 g/kg), or the combination of the two on the metabolite pattern of the biosynthetic pathway of l-serine has been determined in rabbit liver in vivo as has the effect of 10 mm ethanol on the glucose-, fructose-, or pyruvate-stimulated accumulation of l-serine in rabbit hepatocytes in vitro. In vivo, the 50% increase in l-serine and 80% increase in l-phosphoserine content of liver following glucose injection was completely prevented by ethanol. In fact, the l-phosphoserine content fell to only 6% of the control value. In spite of these and other significant changes in the metabolite pattern of the pathway of l-serine biosynthesis (d-3-phosphoglycerate dehydrogenase, l-phosphoserine aminotransferase (PSAT), and l-phosphoserine phosphatase), the mass action ratio of the combined reactions of the first two steps remained close to their equilibrium position. As a consequence it is estimated that the tissue content of phosphohydroxypyruvate fell to less than 2% of the control value, to approximately 0.3% of its K_m for the PSAT reaction. The conclusion that acute ethanol blocks l-serine biosynthesis (presumably by redox effects) was supported by the prevention or inhibition of l-serine accumulation in hepatocytes metabolizing glucose, fructose, or pyruvate. Because l-serine is an important source of one-carbon fragments, the inhibition of its biosynthesis may be another mechanism by which ethanol interferes with folate and one-carbon metabolism.     

Identification of phytochemical components from Aerva lanata (Linn.) medicinal plants and its in-vitro inhibitory activity against drug resistant microbial pathogens and antioxidant properties

This study aimed to determine the phytochemical components, microbial inhibitory effectiveness and antioxidant properties of Aerva lanata plant extracts. The whole plant showed various medicinal applications in folklore and traditional medicine in various parts of the world. The organic extracts such as ethanol, ethyl acetate, chloroform, acetone, water and methanol were subjected for various phytochemical analysis and confirmed for the existence of flavonoids, glycosides, terpenoids and alkaloid containing components. Alternatively, the extracts were performed for the antibacterial activities against the microbial pathogens and antioxidant properties. Results indicated that, the solvent extracts showed prominent activity against the tested strains. The MIC concentrations of plant were detected from 5 mg/ml to 40 mg/ml. The plant extract was highly effective against E. coli and E. aerogenes and the MIC was 5 mg/ml. In addition, the extracts noted promising antioxidant activities. The antioxidant activities were dose dependent manner. In conclusion, A. lanata extracts showed that significant major phytochemicals and effective antioxidant and anti-microbial properties.     

Evaluation of in vitro antioxidant,antimicrobial, genotoxic and anticancer activities of lichen Cetraria islandica

In this study, the antioxidant, antimicrobial, genotoxic and anticancer activities of Cetraria islandica methanol extract were determined by using free radical and superoxide anion scavenging activity, reducing power, determination of total phenolic compounds and flavonoid contents, broth microdilution minimal inhibitory concentration against five bacterial and five fungal species, cytokinesis block micronucleus (MN) assay on peripheral blood lymphocytes (PBLs) and the microculture tetrazolium test on FemX (human melanoma) and LS174 (human colon carcinoma) cell lines. As a result of the study, we found that C. islandica methanol extract exhibited moderate free-radical-scavenging activity with IC₅₀ values 678.38 μg/ml. Moreover, the tested extract had effective reducing power and superoxide anion radical scavenging. The minimal inhibitory concentration values against the tested microorganisms ranged from 0.312 to 5 mg/ml. The extract increased MN frequency in a dose dependent manner, but it was significant in higher tested concentrations (50, 100 and 200 μg/ml). No significant differences were observed between NDI values in all treatments and untreated PBLs. In addition, the tested extract had strong anticancer activity towards both cell lines with IC₅₀ values of 22.68 and 33.74 μg/ml. It can be concluded that the tested extract exhibited a certain level of in vitro antioxidant, antimicrobial, genotoxic and anticancer activities.     

Chemical composition, antioxidant and antimicrobial activities of essential oil of Thymus algeriensis wild-growing in Libya

The composition of essential oil isolated from Thymus algeriensis growing wild in Libya was analyzed by GC and GC-MS. The essential oil was characterized with thymol (38.50%) as the major component. The oil was screened for antioxidant activity using DPPH assay, and compared to thymol and carvacrol. Antioxidant activity was high, with the IC₅₀ of 0.299 mg/ml, compared to 0.403 and 0.105 mg/ml for thymol and carvacrol, and 0.0717 mg/ml for BHA. In addition, antimicrobial activity was tested against eight bacteria and eight fungi. T. algeriensis oil showed inhibitory activity against tested bacteria at 0.001–0.05 mg/ml, while bactericidal activity (MBC) was achieved at 0.0025–0.05 mg/ml. For antifungal activity MICs ranged 0.0005–0.025 mg/ml and MFC 0.001–0.05 mg/ml. High antimicrobial activity against the fungi in particular suggests that the essential oil of Thymus algeriensis could have a useful practical application.     

Production of Pyrroloquinoline Quinone by Using Methanol-Utilizing Bacteria

A large number of methanol-utilizing bacteria were screened for extracellular production of pyrroloquinoline quinone (PQQ) by using methanol as the carbon and energy sources. Of the bacteria selected, Hyphomicrobium sp. strain TK 0441 was examined for PQQ production by using a jar fermentor. The amount of PQQ in the broth and the level of methanol dehydrogenase activity in the cells were increased by simply decreasing the amount of Fe added to the medium. On the other hand, extracellularly produced protein which interfered with the purification of PQQ was decreased by simply increasing the amount of Mg added to the medium. A suitable medium that contained 1 μg of Fe per ml, 150 μg of Mg per ml, and trace elements was developed. In this medium, the production of PQQ reached approximately 1 mg/ml and protein formation was low.     

Bioanalytical method for the simultaneous determination of d- and l-serine in human plasma by LC/MS/MS

d-Serine is an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors. Plasma concentrations of d-serine and the ratio of d-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the d- and l-isomers of serine in human plasma. Since d- and l-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. d- and l-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. d-Serine (m/z 106.1 > 60.0), l-serine (m/z 106.1 > 60.1) and dl-serine-d₃ (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of d- and l-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01–10 μg/mL (d-serine) and 0.1–100 μg/mL (l-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of d- and l-serine in human plasma were 0.124–0.199 and 7.97–13.1 μg/mL, respectively.     

Reversible Activation of Halophilic β-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart

Effects of a water-miscible organic solvent, methanol, on the structure and activity of halophilic β-lactamase derived from Chromohalobacter sp.560 (HaBla), were investigated by means of circular dichroism (CD) measurement and enzymatic activity determination. Beta-lactamase activity was enhanced about 1.2-fold in the presence of 10–20% methanol. CD measurement of HaBla revealed different structures depending on the methanol concentration: native-like active form (Form I) in 10–20% methanol and methanol-induced inactive form at higher concentration (Form II in 40–60% and Form III in 75–80% methanol). Incubation of HaBla with 40% methanol led to the complete loss of activity within ~80 min accompanied by the formation of Form II, whose activity was recovered promptly up to ~80% of full activity upon dilution of the methanol concentration to 10%. In addition, when the protein concentration was sufficiently high (e.g., 0.7 mg/ml), HaBla activity of Form III in 75% methanol could be recovered in the same way (with slightly slower recovery rate), upon dilution of the methanol concentration. In contrast, non-halophilic β-lactamase from Escherichia coli K12 strain MG1655 (EcBla) was irreversibly denatured in the presence of 40% methanol. HaBla showed remarkable ability to renature from the methanol-induced inactive states.