A note on the enzymic action and biosynthesis of a nitrile-hydratase from a Brevibacterium sp.

The effect of different compounds on the enzymic action of the nitrile-hydratase used for the bioconversion of nitriles was studied. An excess of acrylonitrile as a substrate was shown to inhibit the activity of the enzyme. This inhibition occurred only at relatively high substrate concentrations (0.2 mol/l or more). The nitrile bioconversion products (acrylamide, propionamide) and their structural analogues (acrylic acid, thioacetamide) were shown to inhibit the enzyme competitively. The most important inhibition found was that of cyanide (K_i= 0.004 mol/l), a break down product of some nitriles. By using an acetamidase-negative mutant, amides were shown to inhibit biosynthesis of nitrile-hydratase. An identical result was obtained with thioacetamide, a non-substrate compound for acetamidase. This compound repressed the biosynthesis of nitrile-hydratase by both the wild type and the acetamidase-negative mutant to the same extent.     

Regulation of lysine biosynthesis in Brevibacterium flavum

L-lysine synthesis pathway enzyme activities: β-aspartate kinase (EC.2.7.2.4), diaminopimelate decarboxylase (EC.4.1.1.20) for two L-lysine producing strains Brevibacterium flavum 22LD and RC-115 were studied. It has been found that β-aspartate kinase and diaminopimelate decarboxylase in the Br. flavum RC-115 are less sensitive to feed-back inhibition by lysine and threonine. It is supposed that desensitized β-aspartate kinase in the Br. flavum RC-115 can be determined by genetical changes of the regulatory properties of the β-aspartate kinase. Auxotrophity in the locus of homoserine dehydrogenase was tested and no homoserine dehydrogenase (EC.1.1.1.3) activity was found in either strain. The combination of these both types of mutation supplemented by the lack of catabolic repression in the RC-115 strain makes it an active lysine producer in the medium with high carbohydrates content.     

Regulation of Aspartate Family Amino Acid Biosynthesis in Brevibacterium flavum

Dihydrodipicolinate (DDP)* synthetase and DDP-reductase were partially purified about 30 and 15 folds, respectively, from sonic extracts of Brevibacterium flavum.

In contrast with DDP-synthetase from Escherichia coli, the B. flavum enzyme was only slightly inhibited by α, ε-diaminopimelate, a precursor of lysine, but not by lysine itself. Single or simultaneous addition of any other amino acid(s) of aspartate family did not affect the activity significantly. Optimum pH for DDP-synthetase was 8.4 with Tris-HCl buffer. Kms for aspartic-β-semialdehyde and pyruvate at pH 7.5 were 2×10^?4m and 1×10^?4m, respectively. The formation of DDP-synthetase was not significantly repressed by lysine.

DDP-reductase of B. flavum required NADH or NADPH as the cofactor. This enzyme was not inhibited by single or simultaneous addition of aspartate family amino acid(s).

From the above results, the regulation mechanism of lysine biosynthesis in B. flavum was discussed.     

应用乙酰短杆菌酶法合成2'-脱氧腺苷

胸苷和腺嘌呤在乙酰短杆菌作用下转化成胸腺嘧啶和目标产物2’-脱氧腺苷。50mL的三角瓶装液10mL,40mmol/L的底物,50mmol/L的磷酸盐缓冲液(pH7)进行反应,5%游离乙酰短杆菌在55℃下,经1h转化率就可达到65.6%。     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Optimization of ectoine synthesis through fed-batch fermentation of Brevibacterium epidermis

A production process for ectoine has been developed, using Brevibacterium epidermis DSM20659 as the producer strain. First, the optimal conditions for intracellular synthesis of ectoine were determined. The size of the intracellular ectoine pool is shown to be dependent on the external salt concentration, type of carbon source, and yeast extract concentration. Under the optimized conditions of 1 M NaCl, 50 g/L monosodium glutamate, and 2.5 g/L yeast extract, a maximum concentration of intracellular ectoine of 0.9 g/L was obtained in shake flask cultures. After optimizing the batch fermentation parameters of temperature, pH, agitation, and aeration, the yield could be further increased by applying the fed-batch fermentation principle in 1.5- to 2-L fermentors. Glutamate and yeast extract were fed to the bacterial cells such that the total glutamate concentration in the broth remained constant. A total yield of 8 g ectoine/L fermentation broth was obtained with a productivity of 2 g ectoine/L/day. After the bacterial cells were harvested from the culture broth, the ectoine was recovered from them by a two-step extraction with water and ethanol. Crystallization of the product was obtained after concentration of the extract via evaporation under reduced pressure. After this downstream process, 55% of the ectoine produced in the fermentor could be crystallized in four fractions. The first fractions were of very high purity (98%). This production process can compete with other described production processes for ectoine in productivity and simplicity. Further advantages are the relatively low amounts of NaCl needed and the absence of hydroxyectoine, often a byproduct, in the final product.     

Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity

The growth of a halotolerant Brevibacterium sp. JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives. The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity. Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl. The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer. The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl). The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.     

Influence of substrate on the amino acid profile and macromolecular composition of a Brevibacterium species

Brevibacterium JM98A (ATCC 29895) was grown aerobically in carbon-limited continuous culture on the following substrates: gluconic acid, galactose, fumaric acid, glutamic acid, and aspapagine. Both whole and trichloacetic acid(TCA)-ex-tracted cells were analyzed for their amino acid compositions. No significant variations of amino acid profile were induced by change of substrate. Only the valine content varied significantly with growth rate. Some significant variations were observed between whole and extracted cell samples, primarily in the levels of the essential amino acids threonine, cystine, and valine.     

Three new species of brevibacteria--Brevibacterium antiquum sp. nov., Brevibacterium aurantiacum sp. nov. and Brevibacterium permense sp. nov

This work deals with the taxonomic study of 12 orange-pigmented bacteria isolated from permafrost sediments, rice plots, and soils contaminated with wastes from the chemical and salt industries, which were assigned to the genus Brevibacterium on the basis of phenotypic characteristics, as well as of some strains described previously as Brevibacterium linens. The study revealed three genomic species, whose members and the type strains of the closest species of Brevibacterium had DNA similarity levels between 24 and 59%. The strains of the genomic species differed from each other and from the known species of Brevibacterium in some physiological and biochemical characteristics, as well as in the sugar and polyol composition of their teichoic acids. The 16S rDNA sequence analysis confirmed the assignment of the environmental isolates to the genus Brevibacterium and showed the phylogenetic distinction of the three genomic species. The results obtained in this study allow three new Brevibacterium species to be described: Brevibacterium antiquum (type strain VKM Ac-2118T = UCM Ac-411T), Brevibacterium aurantiacum (type strain VKM Ac-2111T = NCDO 739T = ATCC 9175T), and Brevibacterium permense (type strain VKM Ac-2280T = UCM Ac-413T).