Detoxification of cassava pulp using Brevibacterium sp. R312

Summary Brevibacterium sp. strain R312 has an endocellular -glucosidase, a nitrile hydratase and an amidase that can break down some cyanoglucosides. Nonsterile cassava pulp suspensions were fermented using this strain and 70%–80% reduction of nitrile compounds, in particular cyanoglucosides and -hydroxynitriles, was observed. This type of nitrile-hydratase-active microorganism could be a solution for the detoxification of cassava. Experiments conducted with the yeast Candida molischiana and C. wickkerhamii showed no improvement in detoxification. Offprint requests to: A. Arnaud     

Cloning vectors and antibiotic-resistance markers for Brevibacterium sp. R312

Replication of several cryptic plasmids from coryneform strains was investigated in Brevibacterium sp. R312. Only the Corynebacterium glutamicum pSR1 replicon was found to be suitable for establishing a host-vector system. Two pSR1 derivatives, pRPCG200 and pHYCG1, were used as cloning vectors. They carry a neomycin-resistance-encoding and a tetracycline-resistance-encoding gene, respectively.     

N-terminal amino acid sequence of Brevibacterium sp. R312 wide-spectrum amidase

Summary A wide-spectrum amidase from Brevibacterium sp. R312 was partially purified. The enzyme subunit was purified by reversed phase HPLC and the N-terminal amino acid sequence was found to be identical to that of Pseudomonas aeruginosa aliphatic amidase. Offprint requests to: A. Arnaud     

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli

Abstract The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli .     

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase.

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E. coli promoter and ribosome-binding site.     

A Study of the Mechanism of the Reactions Catalyzed by the Amidase Brevibacterium sp. R312

Besides its amide hydrolase activity, the amidase from Brevibacterium sp. R312 also exhibits an acyl-transferase activity.

The mechanism of the transfer reaction of the acyl from acetamide to hydroxylamine was studied. This is a “Bi Bi Ping Pong” type reaction. The kinetic parameters of the reaction were determined:

– Apparent V_m = 135 μmol · min ^–1 · mg^–1

– Acetamide K_m = 18.2 mM

– Hydroxylamine K_m = 131 mM     

Nitrile hydratase of Brevibacterium R312--purification and characterization

Nitrile hydratase was purified and crystallized from the crude extract of Brevibacterium R312 and found to be homogeneous by the results of disc gel electrophoresis, analytical ultracentrifuge and double diffusion in agarose. The enzyme has a molecular mass of about 85,000 Da and contains approximately 3 g atoms iron/mol enzyme. The enzyme was composed of two kinds of subunits, of which molecular masses were 26,000 Da and 27,500 Da. The concentrated solution of the enzyme had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 712 nm. The enzyme was active toward various aliphatic nitriles.     

Purification and Properties of Cyclodextrin Glucanotransferase from Brevibacterium sp. No. 9605

Cyclodextrin glucanotransferase (EC 2.4.1.19) from Brevibacterium sp. No. 9605 was purified to homogeneity by chromatography on butyl-Toyopearl 650M, γ-cyclodextrin-Sepharose 4B, and Toyopearl HW-55S. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the purified enzyme was 2.8. The optimum pH and temperature were pH 10 and 45°C, respectively. The enzyme was stable at the range of pH 6–8 and at temperatures 50°C or less in the presence of CaCl₂. The enzyme produced mainly γ-cyclodextrin from starch in the initial stage of reaction, but later, the proportion of β-cyclodextrin was increased.     

Brevibacterium sp. from poultry

Two isolates of methanethiol producing coryneform bacteria sharing morphological and physiological similarities with Brevibacterium are described. They were isolated from bumble-foot-like manifestations of poultry but proved to be non-pathogenic for experimental animals.     

The cellular location of nitrilase and amidase enzymes of Brevibacterium R312

Abstract Growth of Brevibacterium R312 on acetonitrile results in the appearance of acetate and ammonia in the medium, whereas incubation of harvested bacteria with acetonitrile results in appearance of acetamide, acetate and ammonia in the medium. Acetonitrilase and acetamidase activities were found to be located in the cytosolic fraction of the cell, suggesting that acetonitrile and acetamide readily enter and leave the bacterium. This was confirmed by the inability of these compounds to osmotically stabilise protoplasts, whereas acetate does so. The small size and neutral non-ionic structure of acetonitrile and acetamide suggest they probably permeate by simple diffusion.     

Electrotransformation of whole cells of Brevibacterium sp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10⁸ transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm⁻¹), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml⁻¹ and cell density (10¹⁰ cells ml⁻¹). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.     

The biotransformation of t-butylacetonitrile and its boron-containing analogue trimethylamine-cyanoborane by Brevibacterium R312

The ability of the nitrile hydratase/amidase system from Brevibacterium R312 to biotransform tert-butylacetonitrile was studied with a view to their utilisation in the production of novel amino acids from isostructural compounds. Brevibacterium R312 was able to transform nitriles with this structure; however, the wide spectrum amidase from this organism was unable to biotransform the corresponding amide to the carboxylic acid. Received: 8 December 1996 / Accepted: 25 April 1997     

Esterase Activities of Brevihacterium sp. R312 and Brevihacterium linens 62

Esterase activity of Brevihacterium linens 62 and Brevibacterium sp. R312 was detected. Each strain had esterase activities that hydrolyzed p-nitrophenyl acetate and α-naphthyl acetate. Biosynthesis and optimum pH and temperature of the two esterase activities showed that the latter were caused by different esterases. The influence of the culture medium and the growth substrate on biosynthesis of the esterase systems were studied. Hydrolysis of methylthioacetate and phenethyl acetate by cell extracts of the two strains was done. No enzymatic ester synthesis reaction was observed. However, transfer reactions by cell extracts of the two strains were done.     

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 orange-pigmented bacteria isolated from permafrost sediments, rice plots, and soils contaminated with wastes from the chemical and salt industries that 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-2118^T = UCM Ac-411^T), Brevibacterium aurantiacum (type strain VKM Ac-2111^T = NCDO 739^T = ATCC 9175^T), and Brevibacterium permense (type strain VKM Ac-2280^T = UCM Ac-413^T).     

Purification and properties of cyclomaltodextrinase from alkalophilic Bacillus sp.

An alkalophilic strain isolated from soil produced intracellular cyclomaltodextrinase on the culture medium at an initial pH of 10.6. The strain was identified as closely resembling Bacillus circulans. The enzyme was purified 252-fold from the cell extract by chitosan treatment, ammonium sulfate fractionation, DEAE-Toyopearl column chromatography, and gel filtration. The pH and temperature optima of the purified enzyme were 6.0 and 50°C. The molecular weight of the enzyme was 126,000, with two subunits of 67,000. The isoelectric point was pH 4.2. Enzyme activity was inhibited by Ag⁺, Hg²⁺, Cu²⁺, and p-chloromercuribenzoate. The enzyme hydrolyzed α-, β-, and γ-cyclodextrins, as well as linear maltodextrins, to yield maltooligosaccharides. Starch and maltose were not degraded by the enzyme.     

Purification and partial characterization of three extracellular cellulases from Cellulomonas sp.

Three extracellular cellulases have been purified from cultures of Cellulomonas. One was found in solution in the cell-free supernatant and two others were found to be bound to the cellulose added as a carbon source. The free enzyme and one of the cellulose-bound enzymes bind to Sephadex. The two cellulose-bound enzymes are glycosylated. The three enzymes behave as endocellulases towards soluble carboxymethyl-cellulose and have little activity on cellulose powder.