Isolation and properties of the natural and the recombinant sialidase fromClostridium septicum NC 0054714

The natural sialidase ofClostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed byEscherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 °C in 60mm sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having (2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the (2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. (2-3) Linkages are more rapidly hydrolysed than (2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.Abbreviations BSM bovine submandibular gland mucine - CMM cooked meat medium - EDTA ethylenediaminetetraacetic acid - FPLC fast performance liquid chromatography - LB Luria-Bertani - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4,5Ac₂ N-acetyl-4-O-acetylneuraminic acid - pI isoelectric point - SDS sodium dodecyl sulfate     

Chromate tolerance in strains of Rhodosporidium toruloides modulated by thiosulfate and sulfur amino acids

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 g ml^– (0.2 mm) Cr(VI) as K₂Cr₂O₇. DBVPG 6662 was inhibited by 100 g ml^– (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 g mg^– cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 g ml^– Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at m levels increased tolerance up to 10 g ml^–, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.     

Cell wall polymers of Actinomadura carminata INA 4281

The cell walls of Actinomadura carminata INA 4281 were found to contain peptidoglycan, teichoic acid, and nonpeptidoglycan amino acids. The peptidoglycan was of the A1 type and contained a small amount of ll-DAP in addition to m-DAP. The teichoic acid was an 1,3-poly(glycerol phosphate) chain composed of about eight glycerophosphate units, two of which had a 2-acetamido-2-deoxy--d-galactopyranosyl substituent and one, a 3-O-methyl--d-galactopyranosyl-(1 3)-2-acetamido-2-deoxy--d-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was identified by chemical analysis and ¹³C-NMR spectroscopy. The teichoic acid contained 3-O-methyl-d-galactose (madurose) — the first ever finding of this compound within a teichoic acid. The nonpeptidoglycan amino acids made up some 30% of the cell wall's dry weight, about a quarter of the amino acids being removable with sodium dodecyl sulfate. Further treatment of the cell walls with LiCl and guanidine hydrochloride caused only a small loss of the amino acids and slight changes in their molar ratio.Abbreviations Gro glycerol - GroP monophosphate glycerol - GroP₂ diphosphate glycerol - Gro2P -monophosphate glycerol - P_TA phosphorus of teichoic acids - P_NA phosphorus of nucleic acids - TA teichoic acid     

Evidence for a relationship between malate metabolism and activity of 1-sinapoylglucose: L-malate sinapoyltransferase in radish (Raphanus sativus L.) cotyledons

The control of malate metabolism and stimulation of 1-sinapolyglucose: L-malate sinapoyltransferase (SMT) activity in radish (Raphanus sativus L. var. sativus) cotyledons has been studied. The light-induced and nitrate-dependent activity of SMT catalyzes the formation of O-sinapoly-L-malate via 1-O-sinapoyl--D-glucose. When dark-grown radish seedlings, cultivated in quartz sand with nutrient solution containing NO ₃ ^- as the sole N source, were treated with light, SMT activity increased concomitantly with free malate in the cotyledons. This light effect was suppressed in seedlings grown in a culture medium which contained in addition to NO ₃ ^- also NH ₄ ⁺ . However, treatment with methionine sulfoximine neutralized this ammonium effect, resulting again in both rapid accumulation of malate and rapid increase in SMT activity. When seedlings grown on NO ₃ ^- nitrogen were subsequently supplied with NH ₄ ⁺ nitrogen, the accumulated level of L-malate rapidly dropped and the SMT increase ceased. The enzyme activity decreased later on, reaching the low activity level of plants which were grown permanently on NO ₃ ^- /NH ₄ ⁺ -nitrogen. An external supply (vacuum infiltration) of malate to excised cotyledons and intact seedings, grown on NO ₃ ^- /NH ₄ ⁺ -nitrogen medium, specifically promoted a dose-dependent increase in the activity of SMT. In summary these results provide evidence indicating that the SMT activity in cotyledons of Raphanus sativus might be related to the metabolism of malic acid.Abbreviation MSO L-methionine sulfoximine - SinGlc 1-O-sinapoyl--D-glucose - SinMal O-sinapoyl-L-malate - SMT 1-O-sinapoyl--D-glucose:L-malate sinapolytransferase     

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid     

The effects of reduced nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot

The addition of l-glutamine, -alanine or l-glutamic acid strongly stimulates somatic embryo formation in carrot, not only in the number of somatic embryos formed but also with respect to their development. The effects of the amino acids on somatic embryogenesis were stronger than that of ammonium ion. In particular, l-glutamine strongly stimulated the development of somatic embryos. To clarify the different effects of amino acids and ammonium ion, the activity of glutamine synthetase (GS; EC 6.3.1.2), a key enzyme involved in nitrogen assimilation, was measured. Its activity decreased during the later stages of embryo development.Abbreviations -Ala -alanine - Glu l-glutamic acid - Gln l-glutamine - 2,4-D 2, 4-dichlorophenoxyacetic acid - -GHA l-glutamic acid -monohydroxamate - GS glutamine synthetase - MS medium Murashige & Skoog (1962) medium - MS-NH₄ medium MS medium without NH₄NO₃ - MS+NH₄ medium MS-NH₄ medium with 10 mM NH₄Cl - MS+ala medium MS-NH₄ medium with 10 mM -alanine - MS+GLU medium MS-NH₄ medium with 10 mM l-glutamic acid - MS+GLN medium MS-NH₄ medium with 10 mM l-glutamine - NIR nitrite reductase - NR nitrate reductase     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Preliminary studies of micropropagation of Hopea odorata,a dipterocarp tree

Plantlet development from in vitro cultures of Hopea odorato Roxb. is described. Embryos excised from seeds and cultured on Gamborg's B5 or modified Murashige and Skoog (MS) medium with benzyladenine (BA, 2.2–22.2 M) produced axillary shoots at cotyledonary and/or stem nodes. Shoot production was greatest in germinated embryos on modified MS medium with 8.9 M BA. Excised axillary shoots formed few buds when cultured on medium with BA and limited root development occurred on Woody Plant Medium with naphthaleneacetic acid. Nodal explants from aseptically grown plantlets sprouted axillary shoots in modified MS medium with BA.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - PVPP polyvinylpolypyrrolidone     

Biosynthesis of poly (γ-glutamic acid) from l-glutamine,citric acid and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. PGA was only slightly produced from medium (100 ml) containing 2 g citric acid and 0.5 g ammonium sulfate in B. subtilis IFO3335. When 0.01 g/100 ml l-glutamine was added to this medium, a large amount of PGA (0.45 g/100 ml), without any by-products such as polysaccharides, was produced. The changes in cell growth, and PGA, glutamic acid, citric acid and ammonium sulfate concentrations in this medium during cultivation were investigated. It was found that PGA was effectively produced for the short time of 20 h after an induction period and that glutamic acid was scarcely excreted during PGA production. PGA could be effectively produced using this medium containing l-glutamine, citric acid and ammonium sulfate. It is suggested that a small amount of l-glutamine added to the medium activated enzymes in the pathway of PGA synthesis in B. subtilis IFO3335. It can be presumed that the enzyme catalyzing the reaction from 2-oxoglutaric acid to l-glutamic acid was glutamate synthase in this bacterium.     

High-frequency somatic embryogenesis from small suspension-cultured clusters of cells of an interspecific hybrid of Oryza

Suspension cultures in which cell clusters were small and had a high capacity for somatic embryogenesis (about 60%) were established from immature panicles of F₁ plants from a cross between Oryza sativa and Oryza latifolia The cells were subcultured at seven-day intervals in a modified N6 medium. The cell clusters were quite small (approximately 30–200m in diameter) after culture for two months in this medium. When small clusters of cells were transferred to N6 medium, that had been diluted with an equal volume of water and supplemented with -naphthalenacetic acid (53 nM), 4-pyridylurea (2.2 M) and sucrose (30 gl^-1), somatic embryogenesis occurred at high frequency (about 60%). The system established in the present work is useful for biochemical and molecular biological research of the somatic embryogenesis in the Gramineae.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthalenacetic acid - 4-PU 4-pyridylurea - MS Murashige and Skoog (1962)     

Improved Microbial Production of Colominic Acid,a Homopolymer of N-Acetylneuraminic Acid

A culture medium has been devised for producing colominic acid in improved yields. Major improvements were obtained by using sorbitol as a source of carbon, by adding phosphate in high concentrations, and by supplementing a limited amount of yeast extract. E. coli O 16: Kl: HNM produced approximately 3000 µg/ml of colominic acid on cultivation at 37°C for 46 hr with a liquid medium consisting of sorbitol (2.0%), (NH₄)₂SO₄ (0.5%), K₂HPO₄ (1.4%), MgSO₄·7H₂O (0.05%), and yeast extract (0.05%).

Isolation and purification by deproteinization with ammonium sulfate, precipitation with ethanol, and by column chromatography on anion exchange resins resulted in a pure colominic acid preparation devoid of internal ester linkages.

In producing colominic acid, strains forming S-type colonies were more active than those forming R-type colonies.     

Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii

Summary Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 mol NH ₄ ⁺ · mg chlorophyll (Chl)^-1, with an average production rate of 14 mol NH ₄ ⁺ · mg Chl^-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 mol NH ₄ ⁺ · mg Chl^-1 and 6.7 mol NH ₄ ⁺ · mg Chl^-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.     

Continuous microbial production ofl-leucine with cell retention

Summary Continuous production ofl-leucine was carried out withCorynebacterium glutamicum, strain ATCC 13032 starting from-ketoisocaproic acid as the precursor, glucose as the carbon source and ammonium sulphate as the nitrogen source, with biotin in a mineral medium. By means of cross-flow microfiltration or centrifugal separation for cell retention in continuous fermentation an increase in cell density was achieved and the product solution was obtained cell-free. The cells were concentrated to over 70 g cell dry mass/1. In experiments of up to 42 days, conversion rates of 85%–99% andl-leucine yields of 85%–93% were achieved. With a substrate residence time of 3.6 h, 114 mmol/1l-leucine was produced with a space-time yield of 97 g/1 per day. A scale-up of the fermentation volume from 4 to 1001 provided comparable results.     

Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton

Improvement in somatic embryogenesis has been achieved in several cotton lines (Gossypium hirsutumL.) from the Georgia and Pee Dee germplasm with culture media containing various Putrescine concentrations. The best results were obtained with the -naphthalene acetic acid (NAA)-based treatments, S15 g.05 NAA and EMMS₂, as compared to the 2,4-dichlorophenoxyacetic acid (2,4-D)-based culture medium, EMMS₄. Inclusion of 0.5 mg l^–1 Putrescine improved somatic embryo (SE) induction for most treatments and lines tested. An 8-and 2-fold improvement was achieved in SE production on the EMMS₂-0.5 Putrescine treatment as compared to EMMS₂ alone for cotton lines PD 97019 and GA 98033, respectively. A significant increase in SE number (53-fold) was obtained with the addition of 0.5 mg l^–1Putrescine to EMMS₂ for PD 97021, which was essentially recalcitrant without Putrescine treatment. Conversion of SEs into plants was both genotype- and culture medium-dependent.     

Lysine catabolism inStreptomyces ambofaciens producer of macrolide antibiotic,spiramycin

Spiramycin production was highly stimulated when lysine was used as the sole nitrogen source. This amino acid was catabolized by the -transaminase pathway characterized by dosage of cadaverine aminotransferase (CAT) enzyme. The Km_cadaverine was of 57mM. CAT was highly induced by lysine (634% in comparison with ammonium). Addition of 40mm of ammonium in a culture begun with 20mm of lysine as the sole initial nitrogen source repressed CAT biosynthesis by 24% but did not affect spiramycin production seriously. Addition of 20mm of lysine in a culture started with 40mm ammonium induced CAT biosynthesis of 425%, but did not allow spiramycin production. In these two cases, spiramycin production seems to be conditioned by the nitrogen source initially present in the culture medium. CAT activity was inhibited by ammonium ions (33% at 20mm), whereas lysine had no effects.     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).