Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

GABA and glutamate specifically induce contractions in the sponge <Emphasis Type="Italic">Tethya wilhelma</Emphasis>

Sponges (Porifera) are nerve- and muscleless. Nevertheless, they react to external stimuli in a coordinated way, by body contraction, oscule closure or stopping pumping activity. The underlying mechanisms are still unknown, but evidence has been found for chemical messenger-based systems. We used the sponge Tethya wilhelma to test the effect of γ-aminobutyric acid (GABA) and glutamate (l-Glu) on its contraction behaviour. Minimal activating concentrations were found to be 0.5 μM (GABA) and 50 μM (l-Glu), respectively. Taking maximum relative contraction speed and minimal relative projected body area as a measure of the sponge’s response, a comparison of the dose–response curves indicated a higher sensitivity of the contractile tissue for GABA than for l-Glu. The concentrations eliciting the same contractile response differ by about 100-fold more than the entire concentration range tested. In addition, desensitising effects and spasm-like reactions were observed. Presumably, a GABA/l-Glu metabotropic receptor-based system is involved in the regulation of contraction in T. wilhelma. We discuss a coordination system for sponges based on hypothetical chemical messenger pathways. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. K. Ellwanger and A. Eich contributed equally and designed and performed experiments, analysed data and revised the paper, M. Nickel designed the study and experiments, analysed data, prepared the figures, wrote and revised the paper.     

Growth inhibitory activity of ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranosyl ester and ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase

Exogenously applied ABA-β-d-glucopyranosyl ester (ABA-GE) inhibited shoot growth of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), Digitaria sanguinalis L., timothy (Pheleum pratense L.) and ryegrass (Lolium multiflorum Lam.) seedlings at concentrations greater than 0.1 μM. The growth inhibitory activity of ABA-GE on these shoots was 26–40% of that of (+)-ABA. ABA-β-d-glucosidase activities in these seedlings were 11–31 nmol mg⁻¹ protein min⁻¹. These results suggests that exogenously applied ABA-GE may be absorbed by plant roots and hydrolyzed by ABA-β-d-glucosidase, and liberated free ABA may induce the growth inhibition in these plants. Thus, although ABA-GE had been thought to be physiologically inactive ABA conjugate, ABA-GE may have important physiological functions rather than an inactive conjugated ABA form.     

Effects of methylammonium and ofL-methionine-DL-sulfoximine on the growth and nitrogen metabolism ofPhaeodactylum tricornutum

Phaeodactylum tricornutum Bohlin grew well withL-methionine-DL-sulfoximine (MSX) as sole nitrogen source. Such growth helps to explain the lack of effect of MSX on ammonium assimilation by this organism. Methylammonium inhibited growth with nitrate or MSX as sole nitrogen source but not growth on ammonium. Methylammonium could not be metabolised byP. tricornutum but was accumulated in the cells, the concentration factor sometimes approaching 25,000. Ammonium addition, but not that of MSX or nitrate, displaced methylammonium from the cells and this displacement was followed by resumption of growth. Both methylammonium and ammonium inhibited the uptake of nitrate and nitrite by the cells but inhibition by methylammonium, in comparison with that by ammonium, required a higher concentration and a longer time to develop. Inhibition by methylammonium is shown to be associated with its accumulation by the cells. Methylammonium also prevented the disappearance of nitrate from the interior of the cells (presumably by nitrate assimilation) whereas ammonium did not. It is concluded that methylammonium and ammonium differ in the ways in which they inhibit nitrate metabolism inP. tricornutum.Abbreviation MSX L-methionine-DL-sulfoximine     

Effect of polyamines and L-ornithine on the development of proembryogenic masses of Cryptomeria japonica

We examined the effects of polyamines, namely, putrescine, spermidine and spermine, and of amino acids, such as l-arginine and l-ornithine, as part of our efforts to identify factors that stimulate the development of proembryogenic masses (PEMs) of Cryptomeria japonica. We maintained two distinct types of PEM designated PEMs A, which consisted of normal embryogenic cells as single embryos with elongated suspensor cells, and PEMs B, which consisted of abnormal embryogenic cells with coalesced embryos on modified Campbell and Durzan medium (mCD) supplemented with individual polyamines at 0–100 μM or amino acids at 0–16.4 mM. All additives had a stimulatory/suppressive effect. Microscopy and image-processing techniques revealed that the regions of authentic embryos of PEMs that were treated with l-ornithine were remarkably enlarged and that the suspensor cells had elongated in the same direction. When all PEMs A were transferred to maturation medium (mCD that contained abscisic acid and maltose at various concentrations), only PEMs that had been treated with l-ornithine matured into somatic embryos and were able to germinate on hormone-free mCD. Our results indicate that l-ornithine is an important stimulator of the development of PEMs to the pre-filamentous stage in C. japonica.     

Increased Lipid Peroxidation and Ascorbic Acid Utilization in Testis and Epididymis of Rats Chronically Exposed to Lead

The hypothesis has been recently presented that lead may exert its negative effect at least partially through the increase of reactive oxygen species (ROS) level in tissues. However, little is known about the influence of lead intoxication on equilibrium between generation and elimination of ROS in the male reproductive system. Sexually mature male Wistar rats were given ad libitum 1% of aqueous solution of lead acetate (PbAc) for 9 months. Significantly higher lead concentrations were found in blood [median 7.03 (Q25–Q75: 2.99–7.65) versus 0.18 (0.12–0.99) μg dl⁻¹, P < 0.01], caput epididymis [median 5.51 (Q25–Q75: 4.31–7.83) versus 0.51 (0.11–0.80) μg g⁻¹ d.m., P < 0.001], cauda epididymis [median 5.88 (Q25–Q75: 4.06–8.37) versus 0.61 (0.2 – 1.08) μg g⁻¹ d.m., P < 0.001] and testis [median 1.81 (Q25–Q75: 0.94–2.31) versus 0.17 (0.03–0.3) μg g⁻¹ d.m., P < 0.01] of lead-intoxicated rats when compared to the control. The concentration of ascorbyl radical, generated in vitro from l-ascorbic acid (present in tissues in vivo) was measured by means of Electron Paramagnetic Resonance (EPR) spectroscopy. The EPR signal of ascorbyl radical in caput epididymis, cauda epididymis, testis and liver of lead acetate-treated animals revealed a significant decrease by 53%, 45%, 40% and 69% versus control tissues, respectively. Plasma l-ascorbic acid content measured by high performance liquid chromatography (HPLC) method and total antioxidant status (TAS) measured by means of spectrophotometry were also significantly lower in the intoxicated versus control animals (28% and 21%, respectively). In the group exposed to lead the concentration of lipid peroxide in homogenates of the reproductive system organs was significantly elevated versus control group. It can be assumed that the lower EPR signal was caused by decreased tissue concentrations of l-ascorbic acid. The latter may have resulted from consumption of ascorbic acid for scavenging of ROS excess in tissues of animals chronically exposed to lead.     

A preliminary study of the bioremediation potential of Codium fragile applied to seaweed integrated multi-trophic aquaculture (IMTA) during the summer

In integrated multi-trophic aquaculture (IMTA), seaweeds have the capacity to reduce the environmental impact of nitrogen-rich effluents in coastal ecosystems. To establish such bioremediation systems, selection of suitable seaweed species is important. The distribution and productivity of seaweeds vary seasonally based on water temperature and photoperiod. In Korea, candidate genera such as Pophyra, Laminaria, and Undaria grow from autumn to spring. In contrast, Codium grows well at relatively high water temperatures in summer. Thus, aquaculture systems potentially could capitalize on Codium’s capacity for rapid growth in the warm temperatures of late summer and early fall. In this study, we investigated ammonium uptake and removal efficiency by Codium fragile. In laboratory experiments, we grew C. fragile under various water temperatures (10, 15, 20, and 25°C), irradiances (dark, 10, and 100 μmol photons m⁻² s⁻¹), and initial ammonium concentrations (150 and 300 μM); in all cases, C. fragile exhausted the ammonium supply for 6 h. At 150 μM of , ammonium removal efficiency was greatest (99.5 ± 2.6%) when C. fragile was incubated at 20°C under 100 μmol photons m⁻² s⁻¹. At 300 μM of , removal efficiency was greatest (86.3 ± 2.1%) at 25°C under 100 μmol photons m⁻² s⁻¹. Ammonium removal efficiency was significantly greater at 20 and 25°C under irradiance of 100 μmol photons m⁻² s⁻¹ than under other conditions tested.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan

Antigen 85 (ag85) is a complex of acyltransferases (ag85A–C) known to play a role in the mycolation of the d-arabino-d-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-β-d-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl α-d-arabinofuranoside (2), methyl β-d-arabinofuranoside (3), and methyl 2-O-β-d-arabinofuranosyl-α-d-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-α-d-arabinofuranoside (10), methyl 5-O-octanoyl-β-d-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-β-d-arabinofuranosyl)-5-O-octanoyl-α-d-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that d-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its α,γ-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH₃SeH from seleno-l-methionine (l-SeMet) by the α,γ-elimination reaction. The l-SeMet α,γ-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The K _m value of the enzyme for the catalysis of l-SeMet was 15.5 m M, and the V _max was 0.29 units/mg protein. Pyridoxal 5′-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50°C; it catalyzed the α,γ-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the α,β-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inerts. Therefore, the purified enzyme was different from the bacterial l-methionine γ-lyase that metabolizes l-SeMet to CH₃SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the α,γ-elimination reaction of l-SeMet and immediately converts it to CH₃SeH, an important metabolite of Se.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

Development of medium for enhanced production of glutaminase-free <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase from <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> MTCC 1428

Glutaminase-free l-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of l-asparaginase using the Plackett–Burman experimental design. The medium components, viz., glucose, l-asparagine, KH₂PO₄, and MgSO₄·7H₂O, were screened based on their high confidence levels (P < 0.04). The optimum levels of glucose, l-asparagine, KH₂PO₄, and MgSO₄·7H₂O were found to be 2.076, 5.202, 1.773, and 0.373 g L⁻¹, respectively, using the central composite experimental design. The maximum specific activity of l-asparaginase in the optimized medium was 27.88 U mg⁻¹ of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.