In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.
Aflatoxins* ; Agar ; Aspergillus flavus* ; Aspergillus* ; Bacillus megaterium* ; Bacillus* ; Germination ; Pseudomonas* ; Sphingomonas

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.
Aspergillus flavus ; Aspergillus fumigatus ; Aspergillus* ; Bacillus megaterium* ; Bacillus* ; Fungi ; Germination ; In Vitro Techniques ; Penicillium* ; Pseudomonas*

Bacillus megaterium ; pathogenicity ; Brain Abscess ; diagnosis ; microbiology ; Female ; Humans ; Middle Aged

This study aimed to further enhance 2-keto-L-gulonic acid (2-KLG) production efficiency. A strategy for enhancing Ketogulonigenium vulgare growth and 2-KLG production by improving B. megaterium growth with sucrose was developed based on the time course of osmolality during 2-KLG industrial scale fermentation and effects of osmolality on cells growth and 2-KLG production. Results showed that the accumulation of 2-KLG and the feeding of alkaline matter led to an osmolality rise of 832 mOsmol/kg in the culture broth. High osmotic stress (1 250 mOsmol/kg) made the growth ofB. megaterium and K. vulgare decreased 15.4% and 31.7%, respectively, and consequently the titer and productivity of 2-KLG reduced 67.5% and 69.3%, respectively. When supplement sucrose under high osmotic condition (1 250 mOsmol/kg), B. megaterium growth was significantly improved, with the result that 2-KLG production was increased 87%. Furthermore, by applying this sucrose addition strategy further to batch fermentation in 3 L fermentor, the productivity of 2-KLG increased 10.4%, and the duration of fermentation declined 10.8%. The results presented here provide a potential strategy for enhancing the target metabolites produced by mixed strains at environmental stress.
Bacillus megaterium ; genetics ; growth & development ; metabolism ; Fermentation ; Industrial Microbiology ; Osmosis ; Rhodobacteraceae ; genetics ; growth & development ; metabolism ; Stress, Physiological ; Sucrose ; pharmacology ; Sugar Acids ; metabolism

The aim of this study was to improve the 2-keto-L-gulonic acid (2-KLG) production efficiency by Ketogulonicigenium vulgare and Bacillus megaterium by using multi-stage pH control strategy. The effect of pH on the cell growths and 2-KLG production showed that the optimum pH for K. vulgare and B. megaterium cell growth were 6.0 and 8.0, respectively, while the optimum pH for 2-KLG production was 7.0. Based on the above results, we developed a three-stage pH control strategy: the pH was kept at 8.0 during the first 8 h, then decreased to 6.0 for the following 12 h, and maintained at 7.0 to the end of fermentation. With this strategy, the titer, productivity of 2-KLG and L-sorbose consumption rate were achieved at 77.3 g/L, 1.38 g/(L x h) and 1.42 g/(L x h), respectively, which were 9.7%, 33.2% and 25.7% higher than the corresponding values of the single pH (pH 7.0) control model.
Bacillus megaterium ; growth & development ; metabolism ; Culture Media ; chemistry ; Fermentation ; Hydrogen-Ion Concentration ; Rhodobacteraceae ; growth & development ; metabolism ; Sorbose ; metabolism ; Sugar Acids ; metabolism

A beta-amylase gene (amyG) was cloned from a Bacillus megaterium WS06 and expressed in the Escherichia coli. Nucleotide sequence anlysis showed the amyG gene is composed of 1638 bp (545 amino acid residues with a Mr of 60.194 kD). The AmyG shows 94.5% sequence homologies with beta-amylase from Bacillus megaterium DSM319 and presents a normal beta-amylase primary structure, constituted by three parts: the N-terminal signal sequence, the catalytic domain and the C-terminal starch binding domains. The deduced amino acid sequence revealed that several highly conserved regions of the glycosylhydrolase family 14. The amyG gene was overexpressed using the pET21a vector and Escherichia coli BL21(DE3). The recombinant enzyme was purified 7.4 fold to electrophoretic homogeneity and had a Mr of 57 kD (by SDS-PAGE). The enzyme was optimally active at pH 7.0 and 60 degrees C and showed stability at the temperature below 60 degrees C. This enzyme efficiently hydrolyzed starch to yield maltose from non-reducing chain ends by exo-cleavage mode.
Bacillus megaterium ; enzymology ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Sequence Analysis, Protein ; Sequence Homology, Amino Acid ; Temperature ; beta-Amylase ; biosynthesis ; genetics ; metabolism

Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl(3), induction at OD(578) (optical density measured at 578 nm), induction time and inoculum concentration. Initially, Plackett-Burman (PB) design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90x10(-3) U/ml by the best combinations of the physicochemical parameters at optimum levels of 0.12 mg/L FeCl(3), inoculum concentration of 2.10%, induction at OD(578) equal to 1.07, and with 6.05 h of induction.
Bacillus megaterium ; enzymology ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Biotechnology ; Cytochrome P-450 Enzyme System ; biosynthesis ; genetics ; Escherichia coli ; enzymology ; genetics ; Fermentation ; Mixed Function Oxygenases ; biosynthesis ; genetics ; NADPH-Ferrihemoprotein Reductase ; Recombinant Proteins ; biosynthesis ; genetics

We screened a strain NK13 for a certain extent asymmetric hydrolysis the rac-ketoprofen Chloroethyl ester to (S)-Ketoprofen. As identified, NK13 was Bacillus megaterium. Digested NK13 genomic DNA with Sau3AI partially and recovered the fragment from 2 kb to 6 kb, cleaved the plasmid of pUC18 with BamH I, ligated the 2-6 kb fragment of NK13 genomic DNA into pUC18 plasmid, and then transformed an Escherichia coli strain DH5alpha. We created the gene library of NK13 and obtained a positive clone, pUC-NK1 in the library from the tributyrin flat. The result of sequencing showed that there was a whole open read frame (ORF) of 633 bp lipase gene in the plasmid of pUC-NK1. To compare with the genes of GenBank, this lipase gene was reported firstly (GenBank Accession No. EU381317). The lipase gene was amplified by PCR, using pUC-NK1 plasmid as template, and subcloned into the high expression vector pET21b(+) under the control of T7 promoter. The recombinant plasmid, pET-NKest1, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant lipase protein. After 3 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), lipase was expressed. SDS-PAGE analysis showed that the relative molecular mass of the lipase protein was about 20 kDa. The result of high performance liquid chromatography (HPLC) showed that the conversion rate of the recombinant strain was fifty times than the wild strain NK13's. The (S)-Ketoprofen enantiomeric excess of the recombinant strain was 75.28%, which indicated that the lipase could hydrolyze (S)-Ketoprofen Chloroethyl ester firstly. If we research the conditions of the hydrolysis rac-ketoprofen Chloroethyl ester of this lipase further, maybe it could offer a foundation to product (S)-Ketoprofen industrially.
Amino Acid Sequence ; Bacillus megaterium ; genetics ; isolation & purification ; metabolism ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Ketoprofen ; analogs & derivatives ; chemistry ; isolation & purification ; Lipase ; biosynthesis ; genetics ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism