Brevibacillus Agri Research Articles

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and l- N-carbamoylase activities as biocatalyst for the production of l-homophenylalanine

A dihydropyrimidinase gene ( pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for d, l- p-hydroxyphenylhydantoin and d, l-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for d, l-homophenylalanylhydantoin ( d, l-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn 2+, with a maximal activity at 60 °C and pH 8.0. This enzyme was completely thermostable at 50 °C for 20 days. A whole cell biocatalyst for the production of l-homophenylalanine ( l-HPA) from d, l-HPAH by coexpression of the pydB gene and a thermostable l- N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and l- N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-β- d-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for l-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10 mM d, l-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for l-HPA production.

Predominant Culturable Bacillus Species in Japanese Arable Soils and Their Potential as Biocontrol Agents

To reveal predominant culturable Bacillus species in Japanese arable soils, aerobic spore-forming bacteria were isolated from different soils in Japan and their phylogenic positions were estimated from the 16S rDNA sequences. Bacillus species were also isolated from tomato roots grown in soils for 4 weeks to estimate the difference in phylogenic positions between soil and root isolates. The number of culturable Bacillus species ranged from 6.4 to 7.4 log cfu g-1 soil and 4.7 to 6.7 log cfu g-1 root in soils and tomato roots, respectively, and the proportion of Bacillus species among total culturable bacteria ranged from 3.8% to 17.9% and 0.1% to 6.7% in soils and roots, respectively. The differences in the number and percentage of culturable Bacillus species between the soils and roots were highly significant (P=0.0000 and 0.0023, respectively), indicating that Bacillus species were more predominant in soil than in root. A total of 108 isolates were characterized using the restriction fragment length polymorphism (RFLP) method and partial 16S rDNA sequences. They affiliated to 22 species including the genus Bacillus, Brevibacillus and Paenibacillus. No nitrogen fixers were isolated. The predominant species was Bacillus megaterium to which 42 isolates (39%) belonged. Some of the species, such as Bacillus luciferensis and Brevibacillus agri, were isolated only from the tomato roots and considered to be more adapted to the rhizosphere than soil. Among 108 isolates, only one strain, N2S6, belonging to Bacillus megaterium, showed consistent antagonistic activity against the bacterial wilt of tomato caused by Ralstonia solanacearum.

Molecular Characterisation of Bacterial Contamination in Semi-Final Gelatine Extracts, Using Denaturing Gradient Gel Electrophoresis

Contamination of gelatine may affect the safety and/or quality of its applications. Characterisation of bacterial isolates from semi-final gelatine batches revealed thermotolerant, aerobic, endosporeforming contaminants. In this paper, bacterial contamination in gelatine batches is analysed without previous isolation, by means of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA sequences. V9 and V6-V8 regions of the 16S rDNA gene were found more suitable for this purpose than V1 or V3 regions. Bacillus fumarioli, Bacillus licheniformis, members of the 'Bacillus cereus group', Bacillus subtilis, Bacillus shackletonii, Brevibacillus borstelensis and Brevibacillus agri were detected.

Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp. nov..

Thirty-three clinical, dairy and industrial isolates of aerobic endospore-forming bacteria which were unreactive in routine identification tests were characterized genotypically by using amplified rDNA restriction analysis (ARDRA), 16S rDNA sequencing and DNA-DNA reassociation, and phenotypically by using fatty acid methyl ester (FAME) analysis, SDS-PAGE of whole-cell proteins, API Biotype 100 assimilation tests and 16 other routine phenotypic tests. Three isolates were identified as strains of Bacillus badius, 12 as Brevibacillus agri, including 3 strains associated with an outbreak of waterborne illness, 4 as Brevibacillus centrosporus and 2 as Brevibacillus parabrevis; 12 strains contaminating an antibiotic production plant were recognized as members of a new species, for which the name Brevibacillus invocatus is proposed, with the type strain LMG 18962T (= B2156T = CIP 106911T = NCIMB 13772T).

Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process.

The methanotrophic bacterium Methylococcus capsulatus (Bath) grows on pure methane. However, in a single cell protein production process using natural gas as methane source, a bacterial consortium is necessary to support growth over longer periods in continuous cultures. In different bioreactors of Norferm Danmark A/S, three bacteria consistently invaded M. capsulatus cultures growing under semi-sterile conditions in continuous culture. These bacteria have now been identified as a not yet described member of the Aneurinibacillus group, a Brevibacillus agri strain, and an acetate-oxidiser of the genus Ralstonia. The physiological roles of these bacteria in the bioreactor culture growing on natural, non-pure methane gas are discussed. The heterotrophic bacteria do not have the genetic capability to produce either the haemolytic enterotoxin complex HBL or non-haemolytic enterotoxin.

Reclassification of "Bacillus pulvifaciens" group II as Brevibacillus agri.

Organisms formerly identified as strains of Paenibacillus pulvifaciens group II were reclassified as members of the species Brevibacillus agri. The reclassification was based on phylogenetic analysis of 16S rRNA gene sequences from selected Bacillaceae. The analysis strongly supported placement of group II strains in the genus Brevibacillus. High DNA relatedness of 87-100% and close phenotypic similarity demonstrated that group II strains were members of the species Brevibacillus agri.