Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

The Effect of Salinity Pretreatment on Cd Accumulation and Cd-Induced Stress in <Emphasis Type="Italic">BADH</Emphasis><Emphasis Type="Italic">-</Emphasis>Transgenic and Nontransgenic Rice Seedlings

The influence of betaine aldehyde dehydrogenase (BADH) and salinity pretreatment on oxidative stress under cadmium (Cd) toxicity was investigated in rice cv. Xiushui 11 and its BADH-transgenic line Bxiushui 11. The results showed that plants previously treated with 4.25 and 8.5 mM NaCl, respectively, for 5 days each had higher Cd concentrations in both roots and shoots of the two rice genotypes compared with the controls. Malondialdehyde (MDA) content in both leaves and roots was increased by salinity pretreatment and was significantly lower in the salinity-pretreatment plants than in the controls when the plants were consequently exposed to Cd stress. Salinity pretreatment also increased proline content and the activities of superoxide dismutase (SOD) and peroxidase (POD) in both leaves and roots. It can be assumed that salinity pretreatment enhances the defensive ability of plants against oxidative stress through increasing activities of antioxidative enzymes. The BADH-transgenic line (Bxiushui 11) had lower Cd and MDA content, higher SOD and POD activities, and higher proline content than its wild type (Xiushui 11). The current results suggest that betaine, a product of BADH expression, improves the tolerance of rice plants to Cd stress through increasing the activities of antioxidative enzymes and osmoprotectant content.     

Purification and properties of the formate dehydrogenase and characterization of the<Emphasis Type="Italic"> fdhA</Emphasis> gene of<Emphasis Type="Italic"> Sulfurospirillum multivorans</Emphasis>

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene (fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe₄/S₄ cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.Abbreviations Fdh Formate dehydrogenase - PCE Tetrachloroethene     

Purification and characterization of xylanases from<Emphasis Type="Italic">Aspergillus giganteus</Emphasis>

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 degrees C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50 degrees C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

<Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> as a novel biocatalyst for pyruvate production from<Emphasis Type="SmallCaps"> DL</Emphasis>-lactate

Pseudomonas stutzeri SDM oxidized dl-lactic acid (25.5 g l^-1) into pyruvic acid (22.6 g l^-1) over 24 h. Both NAD⁺-independent d-lactate dehydrogenase and NAD⁺-independent l-lactate dehydrogenase were found for the first time in the bioconversion of lactate to pyruvate based on the enzyme activity assay and proteomic analysis. Jianrong Hao and Cuiqing Ma contributed equally to this work     

Early perception responses of<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> cells in response to lipopolysaccharides from<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis>

Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe- / pathogen-associated molecular patterns, have diverse roles in plant–microbe interactions, e.g. LPS are able to promote plant disease tolerance through activation of induced or acquired resistance. However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans. The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. The purified LPS_B.cep. was found to trigger a rapid influx of Ca²⁺ into the cytoplasm of aequorin-transformed tobacco cells. An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence. These early perception responses were accompanied by K⁺/H⁺ exchange and alkalinization of the extracellular medium. Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS_B.cep. elicitation was dissected, elucidated and compared to that induced by a yeast elicitor. These results suggest that LPS_B.cep. interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.Abbreviations DDC Diethyldithiocarbamate - DMSO Dimethyl sulfoxide - DPI Diphenylene iodonium - H ₂ DCF-DA 2,7-Dihydrodichlorofluorescein-diacetate - LPS Lipopolysaccharides - NAC N-Acetyl-l-cysteine - PTIO 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - ROS Reactive oxygen species - YE Yeast elicitor     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

Cloning and characterization of the lactate dehydrogenase genes from<Emphasis Type="Italic">Lactobacillus</Emphasis> sp. RKY2

Lactic acid is an environmentally benign organic acid that could be used as a raw material for biodegradable plastics if it can be inexpensively produced by fermentation. Two genes (IdhL andIdhD) encoding the L-(+) and D-(−) lactate dehydrogenases (L-LDH and D-LDH) were cloned fromLactobacillus sp., RKY2, which is a lactic acid hyper-producing bacterium isolated from Kimchi. Open reading frames ofIdhL for andIdhD for the L and D-LDH genes were 962 and 998 bp, respectively. Both the L(+)- and D(−)-LDH proteins showed the highest degree of homology with the L- and D-lactate dehydrogenase genes ofLactobacillus plantarum. The conserved residues in the catalytic activity and substrate binding of both LDHs were identified in both enzymes.     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

A betaine aldehyde dehydrogenase gene from <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis> enhances tolerance of <Emphasis Type="Italic">Arabidopsis</Emphasis> to high salt and drought stresses

YUCCA is an important enzyme which catalyzes a key rate-limiting step in the tryptophan-dependent pathway for auxin biosynthesis and implicated in several processes during plant growth and development. Genome wide analyses of YUCCA genes have been performed in Arabidopsis, rice, tomato, and Populus, but have never been characterized in soybean, one of the most important oil crops in the world. In this study, 22 GmYUCCA genes (GmYUCCA1-22) were identified and named based on soybean whole-genome sequence. Phylogenetic analysis of YUCCA proteins from Glycine max, Arabidopsis, Oryza sativa, tomato, and Populus euphratica revealed that GmYUCCA proteins could be divided into four subfamilies. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that GmYUCCA genes have diverse expression patterns in different tissues and under various stress treatments. Compared to the wild type (WT), the transgenic GmYUCCA5 Arabidopsis plants displayed downward curling of the leaf blade margin, evident apical dominance, higher plant height, and shorter length of siliques. Our results provide a comprehensive analysis of the soybean YUCCA gene family and lay a solid foundation for further experiments in order to functionally characterize these gene members during soybean growth and development.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Characteristics of fungal phytases from<Emphasis Type="Italic"> Aspergillus fumigatus</Emphasis> and<Emphasis Type="Italic"> Sartorya fumigata</Emphasis>

Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these phytases displayed 2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 (Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.Some of the data presented here, in particular the amino acid sequences of the phytases from different A. fumigatus and S. fumigata isolates, were first presented at the workshop on "The biochemistry of plant phytate and phytases", Copenhagen, Denmark, 25–28 October 1997     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.