Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Characterization of salt-tolerant mutant for enhancement of l-threonine production in Escherichia coli

 Escherichia coli strain HS3, metabolically engineered to have Met^–, AHV^r, Ile^L and AEC^r characteristics, produced 58.0 g/l of l-threonine, but it was neither salt-tolerant nor osmotolerant; and the growth and threonine production of the strain were severely inhibited both by the addition of NaCl with a concentration higher than 2% and by the presence of glucose with a concentration higher than 10%. Therefore, salt-tolerant mutants were isolated. The salt-tolerant mutants, HS454 and HS528 which were derived from strain HS3, were both tolerant to salt (2%) and hyperproductive. The growth and l-threonine production by the mutant strain HS454 were almost unaffected by a glucose concentration lower than 10%, but gradually reduced with increasing glucose concentration, up to 15%. However, the mutant strain HS528 showed slightly enhanced growth and l-threonine production with increasing glucose concentration, up to 10–12.5%. Strains HS454 and HS528 produced 69.8 g/l and 74.0 g/l of l-threonine, respectively in a 5-l jar fermentor. Received: 21 January 2000 / Received revision: 31 March 2000 / Accepted: 5 May 2000     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Friable embryogenic callus and somatic embryo formation from cotyledon explants of African marigold (Tagetes erecta L.)

Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l^–1 2,4-D and 0.2 mg l^–1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l^–1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic callus was transferred to MS medium supplemented with 3 mg l^–1 ABA and 60 g l^–1 sucrose. The addition of 10–30 mM l-glutamine improved embryo development. Received: 13 May 1997 / Revision received: 24 February 1998 / Accepted: 28 March 1998     

Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system

The effects of l-arginine, and its analogues N ^ω-nitro-l-arginine methyl ester and N ^ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation. l-Arginine, at 10^–8 mol · l^–1induced a slight vasodilatory effect (max 10%). N ^ω-nitro-l-arginine methyl ester and N ^ω-Nitro-l-arginine in the range 10^–8–10^–4 mol · l^–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10^–4 mol · l^–1 N ^ω-nitro-l-arginine or N ^ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l^–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced a strong vasoconstriction (already significant at 10^–5 mol · l^–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine induced a biphasic response with vasodilation at low concentration (max 15% at 10^–8 mol · l^–1). Serotonin displayed a dose-response vasodilation in the range 10^–8–10^–4 mol · l^–1 (max 20%). These vasodilative effects were reduced or abolished by 10^–4 mol · l^–1 l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system. Accepted: 1 July 1996     

Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose

In order to improve the production rate of l-lysine, a mutant of Corynebacterium glutamicum ATCC 21513 was cultivated in complex medium with gluconate and glucose as mixed carbon sources. In a batch culture, this strain was found to consume gluconate and glucose simultaneously. In continuous culture at dilution rates ranging from 0.2 h⁻¹ to 0.25 h⁻¹, the specific l-lysine production rate increased to 0.12 g g⁻¹ h⁻¹ from 0.1 g g⁻¹ h⁻¹, the rate obtained with glucose as the sole carbon source [Lee et al. (1995) Appl Microbiol Biotechnol 43:1019–1027]. It is notable that l-lysine production was observed at higher dilution rates than 0.4 h⁻¹, which was not observed when glucose was the sole carbon source. The positive effect of gluconate was confirmed in the shift of the carbon source from glucose to gluconate. The metabolic transition, which has been characterized by decreased l-lysine production at the higher glucose uptake rates, was not observed when gluconate was added. These results demonstrate that the utilization of gluconate as a secondary carbon source improves the maximum l-lysine production rate in the threonine-limited continuous culture, probably by relieving the limiting factors in the lysine synthesis rate such as NADPH supply and/or phosphoenolpyruvate availability. Received: 16 May 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Regeneration of Ceratozamia euryphyllidia (Cycadales, Gymnospermae) plants from embryogenic leaf cultures derived from mature-phase trees

Embryogenic cultures were induced from pinnae removed from young leaf flushes of mature-phase trees of the endangered cycad species, Ceratozamia euryphyllidia. Induction media consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l^–1 glutamine, 100 mg l^–1 asparagine, 100 mg l^–1 arginine, 60 g l^–1 sucrose, 2 g l^–1 gellan gum, 4.65–13.94 μm kinetin and 4.52–9.05 μm 2,4-dichlorophenoxyacetic acid. Cultures were maintained in darkness. Embryogenic cultures were comprised of precotyledonary somatic embryos that proliferated by somatic polyembryogenesis following subculture onto medium without plant growth regulators. Somatic embryo development and maturation occurred spontaneously from proliferating cultures on medium without plant growth regulators. Somatic embryos were monocotyledonous and mature somatic embryos germinated on semisolid medium without growth regulators. Subsequent development, which included the elongation of the first leaves, occurred only after subculture onto semisolid medium without plant growth regulators containing 0.5% (wt/vol) activated charcoal and under low light intensity. The time period from explanting to plant recovery was approximately 3 years. Received: 25 September 1997 / Revision received: 16 December 1997 / Accepted: 29 December 1997     

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.     

Cytochemische Darstellung von Peroxisomen in Pilzzellen

Zusammenfassung Mit Hilfe der cytochemischen Methode vonFahimi (1968) wurde in den Zellen der PilzeNeurospora crassa, Rhizopus nigricans undSaccbaromyces cerevisiae das Enzym Katalase in distinkten Granula nachgewiesen. Die Spezifität der Färbereaktion wurde durch ihre Hemmbarkeit mit 3-Amino-1,2,4-triazol gezeigt.

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Cytochemical localization of peroxisomes in fungal cells

Summary In the cells of the fungiNeurospora crassa, Rhizopus nigricans, andSaccharomyces cerevisiae catalase was detected in distinct granula with the cytochemical procedure ofFahimi (1968). The specificity of the staining reaction was demonstrated by the inhibitory action of 3-amino-1,2,4-triazole.

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Frau Prof. Dr. B.Haccius danke ich für die Überlassung der Pilzkulturen.     

Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10^–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10^–5 m), kinetin (Kn) (5×10^–6 m), 1-indolebutyric acid (IBA) (2×10^–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l^–1), or MS with 2,4-D (1×10^–5 m), 6-benzylaminopurine (BAP) (1×10^–5 m), and soluble PVP (250 mg l^–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10^–6 m), Kn (5×10^–6 m) and soluble PVP (250 mg l^–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10^–6 m) and IBA (2.5×10^–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced. Received: 16 April 1998 / Revision received: 25 September 1998 / Accepted: 10 October 1998     

Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars

Corynebacterium glutamicum strains CRA1 and CRX2 are able to grow on l-arabinose and d-xylose, respectively, as sole carbon sources. Nevertheless, they exhibit the major shortcoming that their sugar consumption appreciably declines at lower concentrations of these substrates. To address this, the C. glutamicum ATCC31831 l-arabinose transporter gene, araE, was independently integrated into both strains. Unlike its parental strain, resultant CRA1-araE was able to aerobically grow at low (3.6 g·l⁻¹) l-arabinose concentrations. Interestingly, strain CRX2-araE grew 2.9-fold faster than parental CRX2 at low (3.6 g·l⁻¹) d-xylose concentrations. The corresponding substrate consumption rates of CRA1-araE and CRX2-araE under oxygen-deprived conditions were 2.8- and 2.7-fold, respectively, higher than those of their respective parental strains. Moreover, CRA1-araE and CRX2-araE utilized their respective substrates simultaneously with d-glucose under both aerobic and oxygen-deprived conditions. Based on these observations, a platform strain, ACX-araE, for C. glutamicum-based mixed sugar utilization was designed. It harbored araBAD for l-arabinose metabolism, xylAB for d-xylose metabolism, d-cellobiose permease-encoding bglF ^317A , β-glucosidase-encoding bglA and araE in its chromosomal DNA. In mineral medium containing a sugar mixture of d-glucose, d-xylose, l-arabinose, and d-cellobiose under oxygen-deprived conditions, strain ACX-araE simultaneously and completely consumed all sugars.     

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.     

Low salt medium for lactate and ethanol production by recombinant Escherichia coli B

Individual nutrient salts were experimentally varied to determine the minimum requirements for efficient l(+)-lactate production by recombinant strains of Escherichia coli B. Based on these results, AM1 medium was formulated with low levels of alkali metals (4.5 mM and total salts (4.2 g l⁻¹). This medium was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18–19 mmol l⁻¹ h⁻¹ for both (initial 48 h of fermentation).     

Regeneration of Syngonium podophyllum ‘Variegatum’ through direct somatic embryogenesis

A simple and effective method of regenerating Syngonium podophyllum ‘Variegatum’ via direct somatic embryogenesis has been established. Leaf and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with either α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos directly formed at one or two sides of petiole explants on MS medium supplemented 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA or 2.0 mg l⁻¹ TDZ with 0.2 mg l⁻¹ NAA or with 0.2 and 0.5 mg l⁻¹ 2,4-D, respectively. The frequency of petiole explants with somatic embryos produced was as high as 86% when cultured on medium containing 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA. Up to 85% of somatic embryos were able to germinate after transferring onto medium containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ NAA. Approximately 50–150 plantlets were regenerated from a single petiole explant. However, there was no somatic embryo formation from leaf explants regardless of growth regulator combinations used. Regenerated plantlets from petiole explants were stable and grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.     

Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes

We derived l-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selected the potent l-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg l-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The l-methionine biosynthetic enzymes, cystathionine γ-synthase and cystathionine β-lyase, of the l-methionine-overproducing mutants were little repressed by l-methionine. To analyse the mechanism of l-methionine overproduction in the mutant strains, the metJ gene coding for the E. colimet repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser → Asn) at position 54 was observed in four independent l-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the l-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the l-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in l-methionine overproduction, probably because of the derepression of l-methionine biosynthetic enzymes. Received: 6 January 1999 / Received last revision: 19 February 1999 / Accepted: 26 February 1999     

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l⁻¹ xylan and 10.2 g l⁻¹ yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml⁻¹ after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9 (21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of 78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast. Received: December 10, 1999 / Accepted: March 23, 2000     

<Emphasis Type="Italic">Thermincola ferriacetica</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a new anaerobic,thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction

A moderately thermophilic, sporeforming bacterium able to reduce amorphous Fe(III)-hydroxide was isolated from ferric deposits of a terrestrial hydrothermal spring, Kunashir Island (Kurils), and designated as strain Z-0001. Cells of strain Z-0001 were straight, Gram-positive rods, slowly motile. Strain Z-0001 was found to be an obligate anaerobe. It grew in the temperature range from 45 to 70°C with an optimum at 57–60°C, in a pH range from 5.9 to 8.0 with an optimum at 7.0–7.2, and in NaCl concentration range 0–3.5% with an optimum at 0%. Molecular hydrogen, acetate, peptone, yeast and beef extracts, glycogen, glycolate, pyruvate, betaine, choline, N-acetyl-d-glucosamine and casamino acids were used as energy substrates for growth in presence of Fe(III) as an electron acceptor. Sugars did not support growth. Magnetite, Mn(IV) and anthraquinone-2,6-disulfonate served as the alternative electron acceptors, supporting the growth of isolate Z-0001 with acetate as electron donor. Formation of magnetite was observed when amorphous Fe(III) hydroxide was used as electron acceptor. Yeast extract, if added, stimulated growth, but was not required. Isolate Z-0001 was able to grow chemolithoautotrophicaly with molecular hydrogen as the only energy substrate, Fe(III) as electron acceptor and CO₂ as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H₂ and CO₂, requiring the presence of 0.2 g l⁻¹ of acetate as the carbon source. The G+C content of strain Z-0001^T DNA G+C was 47.8 mol%. Based on 16S rRNA sequence analyses strain Z-0001 fell into the cluster of family Peptococcaceae, within the low G+C content Gram-Positive bacteria, clustering with Thermincola carboxydophila (98% similarity). DNA–DNA hybridization with T. carboxydophila was 27%. On the basis of physiological and phylogenetic data it is proposed that strain Z-0001^T (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov. The GenBank accession number for the sequence reported in the paper is AY 631277.     

Characterization of a recombinant cellobiose 2-epimerase from <Emphasis Type="BoldItalic">Caldicellulosiruptor saccharolyticus</Emphasis> and its application in the production of mannose from glucose

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^–1 mg^–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l^–1 and fructose at 47.5 g l^–1 were produced from 500 g l^–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme.     

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.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.