Resolution of dl-hydantoins by d-hydantoinase from Vigna angularis: Production of highly enantioenriched N-carbamoyl-d-phenylglycine at 100% conversion

Summary. d-Hydantoinase from Vigna angularis hydrolyzed rac-5-monosubstituted-hydantoins with polar and aromatic side chains and dihydrothymine but rac-5,5-disubstituted-hydantoins were not substrates of this enzyme. 5-Phenylhydantoin was the best substrate. By using this substrate, N-carbamoyl-d-phenylglycine was obtained in quantitative yield and over 98% ee. Received February 17, 2000; Accepted April 4, 2000     

Comparative study of cellular structures implicated in gravisensing in statocytes of primary and lateral roots of Vigna angularis

Summary. The cellular structures of statocytes implicated in gravisensing in primary and lateral roots of Vigna angularis were compared. The statocytes of lateral roots already had small amyloplasts immediately after they emerged from the primary root. Although these amyloplasts sedimented, the lateral roots showed much weaker gravitropism than primary roots, at least until they reached a length of about 30 mm. The nuclei were usually positioned in the upper end of the statocytes in both types of roots. Electron microscopic surveys showed that many tubular elements of endoplasmic reticulum (ER) were frequently localized in the lower end of the statocyte and they sometimes diverged or curved, suggesting that the ER forms a large reticulate complex. It is worth noting that statocytes with a large ER complex were found much more frequently in primary roots than in lateral roots. The amyloplasts were not always settled on this complex but were very frequently under it, especially in the primary roots. In lateral roots, they were usually localized under the ER complex when they were present. Thus, it is suggested that the differential development and organization of the amyloplast-ER complex system is involved in the differential gravitropism of the two types of roots. Correspondence and reprints: Department of Biology, Faculty of Science, Ehime University, Bunkyo-cho, Matsuyama 790-8577, Japan.     

Benzyladenine-induced adventitious shoot regeneration from hypocotyls of adzukibean (Vigna angularis {Willd.} Ohwi & Ohashi)

A complete in vitro plant regeneration systemvia adventitious shoot-bud formation from seedlinghypocotyls of adzukibean was developed. Cotyledonarynode (CN) and root explants from 4-d-old invitro-germinated seedlings of 4 cultivars werecultured on agar-solidified R medium consisting of MSsalts, B5 vitamins, 3.0% (w/v) sucrose and 4.4 µM6-benzyladenine (BA). Shoot buds arose adventitiouslyat the basipetal cut of the hypocotyl in 40 to 85%of type 1 CN (8–10 mm) explants 6 weeks later with cvsBansei aki azuki and Tanbadainagon azuki exhibitingsignificantly higher response. Shorter type 2 (4–5 mm) CN explants responded poorly (0–6%) in culturewhile intact roots were regenerative but at lowerfrequencies (5 to 31%). In cv Bansei aki azuki,optimum shoot initiation (78.7%) was achieved whentype 1 CN explants from BA-preconditioned seedlingswere re-cultured in R medium (SC0). BA supplied at8.8 µM during SC0 significantly reduced the shootinitiation response to 46.7%. In the same manner,substituting equimolar concentration (4.4 µM) ofkinetin (KN), zeatin (Z) or thidiazuron (TDZ) to BAduring SC0 yielded no or significantly lowerresponses. Mean shoot production ranged from 6.2 to9.8 shoots per explant 6 weeks after subculture (SC1)and varied significantly among the 4 cultivars used. Efficient rooting (93–96%) was achieved on excisedshoots transferred to MS salts + B5 vitamins basalmedium alone. The tissue culture system covered only16 weeks to completion. Regenerated plantlets weretransferred to soil with 86–100% survival rate andall surviving plants were morphologically normal andseed-fertile.     

A genetic linkage map of azuki bean constructed with molecular and morphological markers using an interspecific population (Vigna angularis x V. nakashimae)

A genetic linkage map of azuki bean (Vigna angularis) was constructed with molecular and morphological markers using an F₂ population of an interspecific cross between azuki bean and its wild relative, V. nakashimae. In total, 132 markers (108 RAPD, 19 RFLP and five morphological markers) were mapped in 14 linkage groups covering 1250 cM; ten remained unlinked. The clusters of markers showing distorted segregation were found in linkage groups 2, 8 and 12. By comparing the azuki linkage map with those of mungbean and cowpea, using 20 RFLP common markers, some sets of the markers were found to belong to the same linkage groups of the respective maps, indicating that these linkage blocks are conserved among the three Vigna species. This map provides a tool for markerassisted selection and for studies of genome organization in Vigna species.     

Identification of a digalactosyl ononitol from seeds of adzuki bean (Vigna angularis)

A digalactosyl ononitol was isolated from seeds of adzuki bean (Vigna angularis [Willd.] Ohwi et Ohasi). Analysis of hydrolysis products and NMR spectroscopy established its structure as O-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->3)-4-O-methyl-D-myo-inositol.     

n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

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

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

Liberation of amino acids by heterotrophic nitrogen fixing bacteria

Summary. Large amounts of amino acids are produced by nitrogen-fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Mesorhizobium and Sinorhizobium when growing in culture media amended with different carbon and nitrogen sources. This kind of bacteria live in close association with plant roots enhanced plant growth mainly as a result of their ability to fix nitrogen, improving shoot and root development suppression of pathogenic bacteria and fungi, and increase of available P concentration. Also, it has been strongly evidenced that production of biologically substances such as amino acids by these rhizobacteria are involved in many of the processes that explain plant-grown promotion. This paper reviews literature concerning amino acids production by nitrogen-fixing bacteria. The role of amino acids in microbial interactions in the rhizosphere and establishment of plant bacterial association is also discussed.     

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N′-diethylene-N″-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen–phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25–2500 ng/ml for monochloroTEPA and 25–5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at −80°C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.     

Histological study of callus formation and root regeneration from mung bean (Vigna radiata W.)

We have established a reproducible culture system for callus formation and root development from juvenile stem segments of mung bean(Vigna radiata). In particular, we have studied the influence of plant growth regulators. Induction of calli from young stem explants was very effective on MS inorganic salts supplemented with 0.5 mg/L 2,4-D and 1.0 mg/L kinetin. In regenerating adventitious roots from callus tissues, we found that a combination of 0.75 mg/L NAA, 1.5 mg/L kinetin, and MS salts resulted in 20% efficiency. Histological examination showed that callus tissues originated from out-growths of the cambium rings through de-novo meristematic activity. Those rings were localized outside the vascular cambium. Adventitious roots that developed from root primordia originated from the center of the Callus masses. These primordia produced tracheid-like cells, which then became meristemoid cells for the cambium. Newly formed adventitious roots had the typical tetrarche actinostele type.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.     

Enzymatic production of N-acetyl- -glucosamine from chitin. Degradation study of N-acetylchitooligosaccharide and the effect of mixing of crude enzymes

N-Acetyl- -glucosamine (GlcNAc) was produced from chitin by use of crude enzyme preparations. The efficient production of GlcNAc by cellulases derived from Trichoderma viride (T) and Acremonium cellulolyticus (A) was observed by HPLC analysis compared to lipase, hemicellulase, and pectinase. β-Chitin showed higher degradability than α-chitin when using cellulase T. The optimum pH of cellulase T was 4.0 on the hydrolysis of β-chitin. The yield of GlcNAc was enhanced by mixing of cellulase T and A.     

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B₅-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 μM TDZ or 5.0 μM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 μM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB₅ medium supplemented with 4.0 μM GA₃, 12.5 μM AgNO₃ and 0.4 μM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 μM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.     

Purification and Properties of a Ribonuclease from Cowpea Cotyledons

The isolation and characterisation of cotyledonary ribonucleases (RNase; EC 3.1.27.1), are basic steps to understand the physiology and biochemistry of RNA turnover and mobilisation during seed germination and seedling establishment, as well as how environmental stresses affect them. RNase was isolated and purified 928-fold, to apparent electrophoretic homogeneity from 5-d-old seedlings of Vigna unguiculata. It is a protein with an apparent molecular mass of 16 kDa having three major isoforms. Its optimum pH is 5.8, which decreases to 5.2 in presence of KCl. It has an apparent K_m of 0.80 mg RNA cm^-3 and retains 40 % of its activity when heated to 80 °C. It is completely inhibited by Cu²⁺, Hg²⁺ and Zn²⁺ and is almost insensitive to Mg²⁺, Ca^2+- and EDTA. Urea, Fe²⁺, Co²⁺ and 2-mercaptoethanol partially inhibit its activity. Its amino acid composition shows a resem lance to that of other plant RNases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Library screening for <Emphasis Type="SmallCaps">D</Emphasis>-amino-acid oxidase gene: Application of real-time PCR

Summary. Quantitative real-time PCR shows the quantity in addition to the presence of the target sequence. This property seemed very useful for library screening. Then, real-time PCR was employed to screen for phages carrying D-amino-acid oxidase gene from mouse genomic library. Using stepwise dilution screening combined with real-time PCR, positive phages were isolated in a short time.     

Synthesis, characteristics and biological activity of pentacoordinated spirophosphoranes derived from amino acids

Summary. The reactions of phosphorus trichloride with various amino acids afford the pentacoordinated spirophosphoranes. The reaction procedures were traced by ³¹P NMR spectra techniques. A new crystal structure of alanine derivative was characterized, which is a slightly distorted TBP structure. Besides, this kind of spirophosphoranes are potent inhibitors to tyrosinase.     

Effects of hypoxic and osmotic stress on the free D-aspartate level in the muscle of blood shell Scapharca broughtonii

Summary. Blood shell, Scapharca broughtonii, contains large quantities of free D-aspartate comparable to free L-aspartate in its tissues. When the shell was reared in hypoxic seawater, D-aspartate as well as L-aspartate in the foot muscle decreased rapidly, and their total level became about one-fourth within 24hr. None of the other amino acids examined showed a similar behavior, but many of them rather increased during the same period. The increase in L-alanine was especially remarkable and was almost equal to the sum of the decrease in aspartate enantiomers. When the shell that had been acclimated to hypoxic seawater for 96hr was transferred to normoxic seawater, all the amino acid levels mostly returned to the control levels within 96hr. In contrast to these effects of hypoxic stress, hyperosmotic stress of 150% seawater had no effect on the D- and L-aspartate levels in the same tissue. These results suggest that D-aspartate is involved in anaerobic energy metabolism of this bivalve as well as L-aspartate, whose vital role in anoxia-tolerant bivalves is well known.     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.