Brevibacterium Sp Research Articles

New cyclic tetrapeptide from the coral-derived endophytic bacteria Brevibacterium sp. L-4 collected from the South China Sea

One new cyclic tetrapeptide cyclic-(Tyr-Ala-Leu-Ser) (1) along with four natural compounds firstly obtained 3H-imidazole-4-carboxylic acid (2), 2-methyl-3H-imidazole-4-carboxylic acid (3), 3-ethylidene-6-isopropyl-piperazine-2,5-dione (4), and 3-isobutylidene-6-methyl piperazine-2,5-dione (5) have been isolated from the coral derived endophytic bacteria Brevibacterium sp. L-4 collected from the South China Sea. Their structures were elucidated through spectroscopic techniques including NMR (1D and 2D), MS, and EA, and their relative configurations were also assigned by NMR analysis.

Bioremediation of hexavalent chromate using permeabilized Brevibacterium sp. and Stenotrophomonas sp. cells

Bioremediation has been found to be a useful method for removing hexavalent chromium (Cr(VI)), which is very toxic, from wastewater. Two strains of bacteria that were able to reduce Cr(VI) effectively were isolated from Cr(VI) contaminated soil samples and identified as Brevibacterium sp. K1 and Stenotrophomonas sp. D6, respectively, based on 16S rRNA gene sequence analyses. Brevibacterium sp. K1 and Stenotrophomonas sp. D6 could grow in Luria–Broth medium containing K2Cr2O7 at 1000 and 1600 mg/L, respectively, and they completely reduced the Cr(VI) in LB medium containing K2Cr2O7 at 200 mg/L within 72 h. Further analyses revealed that permeabilized K1 and D6 cells reduced Cr(VI) more effectively than did the resting cells. Triton X-100 was the best permeabilizing agent that was tested. The permeabilized cells of both strains could completely reduce Cr(VI) in industrial wastewater twice before needing to be replenished. The results suggested that these chromate-reducing bacteria are potential candidates for practical use biotreating industrial effluents containing Cr(VI) with Stenotrophomonas sp. D6 being the more effective bacterium.

Run-off studies demonstrate parallel transport behaviour for a marker of poultry fecal contamination and Staphylococcus aureus.

To determine whether poultry litter marker gene LA35 is correlated with pathogens and fecal indicator bacteria (FIB) in run-off from poultry litter-amended plots. A rainfall simulator with various vegetative filter strip lengths was employed to evaluate the correlation of a microbial source tracking (MST) marker for poultry feces/litter (the 16S rRNA gene of Brevibacterium sp. LA35 [LA35] measured by quantitative PCR) with pathogens and FIB in run-off. LA35 was correlated with Staphylococcus aureus, Escherichia coli, Enterococcus spp. and Bacteroidales levels. Salmonella was present at low concentration in litter, but became undetectable by qPCR in run-off. Escherichia coli, LA35 and Staph.aureus exhibited mass-based first flush behaviour in the run-off. Correlation of LA35 with FIB and pathogens in run-off from poultry litter-amended fields suggest comparable transport mechanisms and that LA35 is a useful tracer for harmful bacteria in the environment released from poultry litter. To protect human health, an effective marker for poultry fecal contamination should exhibit similar fate and transport characteristics compared to pathogens. This study is among the first to demonstrate such a relationship in run-off for a MST marker.

Biosynthesis of Benzoylformic Acid from Benzoyl Cyanide with a New Bacterial Isolate of Brevibacterium sp. CCZU12-1

Brevibacterium sp. CCZU12-1 with high nitrilase activity could effectively hydrolyze benzoyl cyanide into benzoylformic acid. After the culture optimization, the preferred carbon sources, nitrogen sources, and inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (2.5 g/L), and ε-caprolactam (2.0 mM), respectively. After the reaction optimization, the optimum reaction temperature, reaction pH, organic cosolvent, and metal ion were 30 °C, 7.0, ethanol (2%, v/v), and Ca(2+) (0.1 mM), respectively. At biotransformation of 120-mM benzoyl cyanide for 24 h, the yield of benzoylformic acid reached 91.8%. Moreover, the microbial nitrilase from Brevibacterium sp. CCZU12-1 could hydrolyze various nitriles, and it significantly exhibited high nitrilase activity against benzoyl cyanide, 3-cyanopyridine, and α-cyclohexyl-mandelonitrile.

Growth enhancement of the halotolerant Brevibacterium sp. JCM 6894 by methionine externally added to a chemically defined medium.

We examined amino acid requirements for the growth of the halotolerant Brevibacterium sp. JCM 6894 in the absence and presence of 1.2 M NaCl in a chemically defined medium. The experiment was also carried out in the presence of 1.2 M KCl. As a result, growth was highly enhanced by methionine in the absence and presence of KCl as well as NaCl up to 1.2 M. However, growth in the presence of 150 mM methionine was repressed by leucine (up to 100 mM）and valine (up to 100 mM). Concentration-dependent growth inhibition was observed in the presence of isoleucine (up to 150 mM) and threonine (up to 300 mM). When the cells were incubated in the absence of externally added K+, growth was strongly repressed, even in the presence of 150 mM methionine. The growth, however, recovered drastically by the addition of 1 mM KCl, regardless of the presence and absence of 1.2 M NaCl. These results indicate that methionine, which seems to be symported into cytoplasm with K+, plays an important role in the growth of the strain under salt stress.

Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp.

Chicken feces commonly contain human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a previously developed SYBR green qPCR assay (LA35) to a novel TaqMan qPCR assay (CL) for the environmental detection of poultry-associated fecal pollution. We tested both assays against chicken litter (n = 40), chicken fecal samples (n = 186), non-chicken fecal sources (n = 484), and environmental water samples (n = 323). Most chicken litter samples (i.e., ≥98%) were positive for both assays with relatively high signal intensities, whereas only 23% and 12% of poultry fecal samples (n = 186) were positive with the LA35 and the CL assays, respectively. Data using fecal samples from non-target animal species showed that the assays are highly host-associated (≥95%). Bayesian statistical models showed that the two assays are associated with relatively low probability of false-positive and false-negative signals in water samples. The CL marker had a lower prevalence than the LA35 assay when tested against environmental water samples (i.e., 21% vs. 31% positive signals). However, by combining the results from the two assays the detection levels increased to 41%, suggesting that using multiple assays can improve the detection of chicken-fecal pollution in environmental waters.

Chemically Modified Sepharose as Support for the Immobilization of Cholesterol Oxidase

Because the cholesterol oxidase from Brevibacterium sp. M201008 was not as stable as the free enzyme form, it had been covalently immobilized onto chemically modified Sepharose particles via N-ethyl-N'-3-dimethylaminopropyl carbodiimide. The optimum immobilization conditions were determined, and the immobilized enzyme activity obtained was 12.01 U/g Sepharose-ethylenediamine. The immobilization of the enzyme was characterized by Fourier transform infrared spectroscopy. The immobilized enzyme exhibited the maximal activity at 35°C and pH 7.5, which was unchanged compared with the free form. After being repeatedly used 20 times, the immobilized enzyme retained more than 40.43% of its original activity. The immobilized enzyme showed better operational stability, including wider thermal and pH ranges, and retained 62.87% activity after 20 days of storage at 4°C, which was longer than the free enzyme.

Production of Maltooligosaccharides from Black Potato (Coleus tuberosus) Starch by α-amylase from a Marine Bacterium (Brevibacterium sp.)

High quality maltooligosaccharides were produced from indigenous Indonesian black potato starch by making use of an amylase from Brevibacterium sp. Optimal production was achieved at 2.5% (w/v) substrate concentration, an enzyme-substrate ratio of 1:5 (w/v) and hydrolysis time of 4 h. Under such conditions the yield of reducing sugars was 14 240 ppm with a polymerization degree of 16. Thin layer chromatography (TLC) revealed the formation of glucose, maltose, and maltotriose with Rf values of 0.60, 0.52, and 0.37, respectively. HPLC analysis of freeze-dried samples disclosed Rf values of 0.60, 0.50, 0.37, and 0.12. Maltooligosaccharide profile analysis both using TLC and HPLC showed that the enzymatically hydrolyzed samples contained glucose, maltose, and maltotriose. Thus, black potato starch can be randomly converted into simple sugars and maltooligosaccharides applying by amylolytic enzymes from the marine microbe Brevibacterium sp.

Production of Maltooligosaccharides from Black Potato (Coleus tuberosus) Starch by α-amylase from a Marine Bacterium (Brevibacterium sp.)

High quality maltooligosaccharides were produced from indigenous Indonesian black potato starch by making use of an amylase from Brevibacterium sp. Optimal production was achieved at 2.5% (w/v) substrate concentration, an enzyme-substrate ratio of 1:5 (w/v) and hydrolysis time of 4 h. Under such conditions the yield of reducing sugars was 14 240 ppm with a polymerization degree of 16. Thin layer chromatography (TLC) revealed the formation of glucose, maltose, and maltotriose with Rf values of 0.60, 0.52, and 0.37, respectively. HPLC analysis of freeze-dried samples disclosed Rf values of 0.60, 0.50, 0.37, and 0.12. Maltooligosaccharide profile analysis both using TLC and HPLC showed that the enzymatically hydrolyzed samples contained glucose, maltose, and maltotriose. Thus, black potato starch can be randomly converted into simple sugars and maltooligosaccharides applying by amylolytic enzymes from the marine microbe Brevibacterium sp.

A new method for tracking poultry litter in the Potomac Basin headwaters of West Virginia

To validate the distribution of a poultry litter-specific marker gene in faecally contaminated environmental waters of an intensive poultry litter rearing region. A TaqMan(®)-based qPCR assay for Brevibacterium sp. LA35 16S rRNA (LA35 gene), which was previously shown to be associated with poultry litter and faeces, was tested on 126 nontarget faecal samples and 28 poultry litter and faecal samples. The TaqMan assay was sensitive (76%) and specific (100%) to the LA35 gene and exhibited a detection limit for poultry litter in water samples that is sufficiently low (2.5 × 10(-2) mg litter l(-1)) to be applicable for environmental monitoring. The LA35 gene was detected in 43% of water samples (n = 30) collected in an intensive poultry rearing region of West Virginia which drains to the Chesapeake Bay. The poultry-specific TaqMan qPCR method for the LA35 gene is more specific than previously published methods and can be used to identify regions impacted by poultry rearing activities. The LA35 gene appears to have a broad geographical distribution as it has been found in poultry litter and faeces from Delaware and West Virginia, in this study and from Arkansas, Georgia, Florida, Minnesota, Oklahoma and Utah previously.

Effect of Oxidative Stress Induced by Brevibacterium sp. BS01 on a HAB Causing Species-Alexandrium tamarense

Harmful algal blooms occur all over the world, destroying aquatic ecosystems and threatening other organisms. The culture supernatant of the marine algicidal actinomycete BS01 was able to lysis dinoflagellate Alexandrium tamarense ATGD98-006. Physiological and biochemical responses to oxidative stress in A. tamarense were investigated to elucidate the mechanism involved in BS01 inhibition of algal growth. Transmission electron microscope analysis revealed that there were some chloroplast abnormalities in response to BS01 supernatant. The decrease in cellular-soluble protein content suggested that cell growth was greatly inhibited at high concentration of BS01 supernatant. The increase in the levels of reactive oxygen species (ROS) and malondialdehyde contents following exposure to BS01 supernatant indicated that algal cells suffered from oxidative damage. The content of pigment was significantly decreased after 12 h treatment, which indicated that the accumulation of ROS destroyed pigment synthesis. Moreover, the decrease of Fv/Fm ratio suggested that in the photosynthetic system, the dominant sites producing ROS were destroyed by the supernatant of the BS01 culture. The activities of the antioxidant enzymes including superoxide dismutase and peroxidase increased in a short time and decreased slightly with increasing exposure time. A real-time PCR assay showed changes in the transcript abundances of two photosynthetic genes, psbA and psbD. The results showed that BS01 supernatant reduced the expression of the psbA gene after 2 h exposure, but the expression of the psbD gene was increased at concentrations of 1.0 and 1.5%. Our results demonstrated that the expression of the psbA gene was inhibited by the BS01 supernatant, which might block the electron transport chain, significantly enhancing ROS level and excess activity of the antioxidant system. The accumulation of ROS destoryed pigment synthesis and membrane integrity, and inhibited or ultimately killed the algal cells.

Immobilization and stabilization of cholesterol oxidase on modified sepharose particles

For the cholesterol oxidase from Brevibacterium sp. M201008 was not stable as free enzyme form, it had been covalently immobilized onto functionalized sepharose particles that were activated with N-ethyl-N′-3-dimethylaminopropyl carbodiimide (EDC).The optimal conditions of enzyme immobilization were determined, and the immobilized enzyme activity was 18.03U/g of support. The surface morphology and thermal behavior of the immobilized enzyme were observed by scanning electron microscopy and differential scanning calorimetry, respectively. The binding of the enzyme to support was confirmed using Fourier transform infrared spectroscopy. The results demonstrated that the thermal, pH, and storage stabilities of cholesterol oxidase were increased by immobilization. More than 90% of the initial activity of the immobilized enzyme was remained at the points of pH 4.0 and 9.0, and that was much more stable than it was free enzyme form. Under same storage conditions, the free enzyme lost 97.8% of its initial activity after 45h, whereas the immobilized enzyme only lost its 35.8% activity.

Biodegradation of bensulfuron-methyl and its effect on bacterial community in paddy soils

Bensulfuron-methyl (BSM) is a new kind of sulfonylurea herbicide widely used to control broad-leaf weeds in rice paddies. The aim of this work was to study BSM biodegradation in paddy soils with BSM-degrading bacteria Bacillus megaterium L1 and Brevibacterium sp. BH and its effect on the structures of soil bacterial community. More than 90 % of BSM could be degraded in paddy soils with 0.0355 mg kg⁻¹ BSM concentration. Addition of BSM-degrading bacterial strains Bacillus megaterium L1 into BSM contaminated paddy soil could have the half-life time of BSM compared to treatment without Bacillus megaterium L1 inoculation. Denaturing gradient gel electrophoresis and principle component analysis indicated that the diversity of the soil microbial community structure changed along with the addition of BSM, which recovered at the end of the experiment (5 weeks). Addition of BSM-degrading bacteria Bacillus megaterium L1 enriched the diversity of soil microbial community structure in paddy soils. This study provides information on the biodegradation of BSM and BSM's influences on the soil bacteria microbial community structures.

Biodegradation of Sewage Wastewater Using Autochthonous Bacteria

The performance of isolated designed consortia comprising Bacillus pumilus, Brevibacterium sp, and Pseudomonas aeruginosa for the treatment of sewage wastewater in terms of reduction in COD (chemical oxygen demand), BOD (biochemical oxygen demand) MLSS (mixed liquor suspended solids), and TSS (total suspended solids) was studied. Different parameters were optimized (inoculum size, agitation, and temperature) to achieve effective results in less period of time. The results obtained indicated that consortium in the ratio of 1 : 2 (effluent : biomass) at 200 rpm, 35°C is capable of effectively reducing the pollutional load of the sewage wastewaters, in terms of COD, BOD, TSS, and MLSS within the desired discharge limits, that is, 32 mg/L, 8 mg/L, 162 mg/L, and 190 mg/L. The use of such specific consortia can overcome the inefficiencies of the conventional biological treatment facilities currently operational in sewage treatment plants.

Expression and comparison of recombinant cholesterol oxidases (COD) in Escherichia coli with native cholesterol oxidase expressed in Brevibacterium sp.

The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of the natural enzyme from Brevibacterium sp. was 17.5 ± 0.2 U/mg, while the activities of the exogenously expressed forms were 16 ± 0.3 U/mg for non-tagged enzyme from E. coli , 12 ± 0.1 U/mg for the N-terminal his-tagged enzyme, and 4 ± 0.3 U/mg for C-terminal his-tagged enzyme. Circular dichroism revealed that the added histidine residues altered the natural folding of the enzyme. The natural cholesterol oxidase was composed of 39% α-helix, 40% β-sheet, and 20% random coil, while the non-tagged enzyme was composed of 40% α-helix, 35% β-sheet, and 24% random coil. In contrast, the N-terminal his-tagged enzyme was composed of 45% α-helix, 29% β-sheet, and 25% random coil, and the C-terminal his-tagged enzyme was composed of 55% α-helix, 16% β-sheet, and 28% random coil. Hydrophobic fluorescence analysis revealed that the hydrophobicity of the enzyme was reduced by his-tags. Coenzyme-like fluorescent probe binding analysis indicated that the coenzyme binding site should be blocked by his-tags. The his-tag method for protein isolation can disrupt the catalytic activity of the cholesterol oxidase. Key words : Cholesterol oxidase; Brevibacterium sp.; Escherichia coli; structural disruption, His-tags.

Improvement of the thermostability and enzymatic activity of cholesterol oxidase by site-directed mutagenesis

Site-directed mutagenesis was applied to enhance the thermostability and enzymatic activity of cholesterol oxidase (ChOx) isolated from Brevibacterium sp. Three amino acid residues (Q153E, F128L, and S143H) located near the FAD-binding site of the enzyme were substituted based on structural analysis. The specific activity of the two-sites mutant Q153E/F128L increased by 11.6% and the relative activity increased by 47% when grown for 2 h at 50 °C. This mutant is a potential industrial strain for producing ChOx.

Correlation of quantitative PCR for a poultry-specific brevibacterium marker gene with bacterial and chemical indicators of water pollution in a watershed impacted by land application of poultry litter.

The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

Acceptor Specificity of Cyclodextrin Glucanotransferase from Brevibacterium sp. No. 9605 to Monosaccharides and Phenolic Compounds

Acceptor Specificity of Cyclodextrin Glucanotransferase from Brevibacterium sp. No. 9605 to Monosaccharides and Phenolic Compounds

Brevibacterium sp. No. 9605株の生産するγ-CGTaseの性質と利用

Brevibacterium sp. No. 9605株の生産するγ-CGTaseの性質と利用

Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay

To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571-bp product was detected in 76% of poultry-associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 10(7) -10(9) gene copies per gram in soiled litter, up to 10(5) gene copies per gram in spread-site soils, and 10(7) gene copies per litre in field run-off water. Results were corroborated by a blinded study conducted by a second laboratory. The poultry-specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land-applied poultry litter. This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.