Effect of hemX gene deletion on heme synthesis in Bacillus amyloliquefaciens / 生物工程学报

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Aislamiento e identificación de microorganismos potencialmente amilolíticos y celulolíticos de suelos de humedales de Bogotá / Isolation and identification of potential amylolytic and cellulolytic microorganisms from soil of Bogota wetlands

RESUMEN Las amilasas y celulasas de origen microbiano se han utilizado desde hace más de tres décadas en la industria. El aislamiento de microorganismos nativos con capacidad amilolítica y celulolítica es el punto de partida para aprovechar la biodiversidad microbiana en la producción de amilasas y celulasas con características específicas que permitan obtener nuevos productos y optimizar procesos industriales donde estas sean aplicables. El objetivo de este trabajo fue aislar, a partir de suelo de cinco humedales en Bogotá, cepas microbianas productoras de enzimas amilolíticas y celulolíticas. Se realizó la medición de halos de hidrólisis en agar almidón y agar carboximetilcelulosa. Se evaluó la actividad enzimática por medio de la producción de azúcares reductores, determinados mediante la técnica del ácido 3,5 dinitrosalicílico. Se seleccionaron cuatro aislamientos amilolíticos diferentes, todos identificados como Bacillus amyloliquefaciens, con actividades entre 480±35 y 752±33 U/mL a 60°C. Cinco aislamientos celulolíticos diferentes fueron seleccionado, dos identificados como Bacillus amyloliquefaciens, dos como Yersinia massiliensis y uno como Stenotrophomonas nitritireducens, con actividades enzimáticas entre 13.82 ± 2.5 y 19.11 ± 2.3 U/mL a 50°C. Estos resultados demuestran que dentro de la biodiversidad de los suelos de humedales de Bogotá existen microrganismos productores de amilasas y celulasas que podrían ser aplicadas en procesos industriales.

ABSTRACT The amylases and cellulases obtained from microorganisms have been used since more than three decades in industry. The isolation of native microbial strains with amylolytic and cellulolytic ability is the starting point to make the best of microbial biodiversity and support the production of amylases and cellulases with novel characteristics to obtain new products and optimize industrial processes where these enzymes can be applied. The objective of this work was to isolate microbial strains with the capacity to produce amylolytic and cellulolytic enzymes from the soil of five wetlands in Bogotá. Hydrolysis halos measurements in starch agar and carboxymethylcellulose agar were performed. The enzymatic activity was determined through the production of reducing sugars which were determined by 3,5-dinitrosalicylic acid method. Four different amylolytic isolations were selected and all of them were identified as Bacillus amyloliquefaciens. The amylolytic activity was between 480 ± 35 and 752±33 U/mL at 60°C. Five different cellulolytic strains were selected and two of them were identified as Bacillus amyloliquefaciens, two as Yersinia massiliensis and one as Stenotrophomonas nitritireducens. Their cellulolytic activities were from 13.82 ± 2.51 to 19.11 ± 2.3 U/mL at 50°C. These results demonstrate that as a part of the Bogota wetlands soil biodiversity there are microorganisms producing amylases and cellulases which might be applied in industrial processes.

A wide spectrum of antibacterial activity of secondary metabolites from Bacillus amyloliquefaciens ELI149 / Um amplo espectro de atividade antibacteriana dos metabólitos secundários de Bacillus amyloliquefaciens ELI149

A highly potent secondary metabolites-producing Bacillus strain was isolated from Mexican soil (Puebla State), together with other fifty strains. The fifty-one strains were subjected for metabolites extraction and evaluated as antibacterial against several bacteria. The active metabolites of these strains were extracted using amberlite XAD16 absorbent resin. The antibacterial activity of crude extracts of all strains was performed by disk diffusion method against some pathogenic gram positive and gram-negative bacteria. Among all Bacillus strains tested, the most potent strain ELI149 (NRB) was selected for molecular characterization. The nucleotide sequence of the 16S rRNA gene (1.5 Kb) of this strain evidenced a 94% similarity with Bacillus amyloliquefaciens strain IIHR-Ba-2, which showed the highest inhibition against the most bacteria probed even greater inhibition than the standard antibiotic. In conclusion, secondary metabolites extracted from Bacillus amyloliquefaciens strain are highly potent as antibiotic against the most bacteria probed. Identification of which metabolites extracted from amberlite are the responsible of the bacteria growth inhibition will be a challenge

Um altamente potent metabolitos secundários-produzindo tensão de Bacillus esteve isolada de terra mexicana (Puebla Estatal), junto com outras cinquenta tensões. As cinquenta e uma tensões estiveram submetidas para extracção de metabolitos e avaliado como antibacterial contra várias bactérias. Os metabolitos ativos destas tensões estiveram extraídos utilizando amberlite XAD16 resina absorbente. O antibacterial actividade dos extractos crus de todas as tensões esteve actuado por método de difusão do disco na contramão alguns a grama patogénica positivo e grama-bactérias negativas. Entre todas tensões de Bacillus provaram, a maioria de potent tensão ELI149 (NRB) esteve seleccionado para caracterização molecular. A sequência de nucleótido do 16S rRNA gene (1.5 Kb) desta tensão evidenced uma 94% semelhança com Bacillus amyloliquefaciens tensão IIHR-Ba-2, o qual mostrou a inibição mais alta na contramão as mais bactérias probed inclusive inibição maior que o antibiótico regular. Em conclusão, os metabolitos secundários extraíram de Bacillus amyloliquefaciens a tensão é altamente potent tão antibiótico na contramão as mais bactérias probed. Identificação do qual os metabolitos extraíram de amberlite é o responsável pela inibição de crescimento das bactérias será um repto.

A comparative study on enzyme production ability of microorganism producing γ-aminobutyric acid in processing of Sojae Semen Praeparatum at different pH and temperatures / 中草药

Objective: To lay a foundation for elucidating the mechanism of microbial regulation of γ-amino butyric acid (GABA) formation in the processing of Sojae Semen Praeparatum. Methods: The ability of glutamic acid decarboxylase and protease (neutral protease, alkaline protease and acid protease) production of Bacillus subtilis, Enterococcus avium, Enterococcus faecium, Bacillus amyloliquefaciens, Aspergillus niger, Aspergillus flavus, Aspergillus tamarii, Penicillium citrinum, Cladosporium tenuissimum, Phanerochaete sordida, Rhizopus oryzae, Sporidiobolus salmonicolor in different pH and temperature conditions were determined by Berthelot colorimetry and Folin phenol method. Results: The GAD and protease activities of the twelve strains were stronger at pH 5-7 and temperature 28-37 ℃. The highest GAD enzyme activity from A. flavus was 41.97 U/h, the optimum pH was 7 and the temperature was 28 ℃, followed by S. salmonicolor, A. niger, R. oryzae, and B. subtilis, respectively. The enzyme activities were 29.04, 25.78, 22.42 and 19.43 U/h. The highest neutral protease activity of B. subtilis was 24.80 U/mL, the optimum pH was 7 and the temperature was 37 ℃, followed by B. amyloliquefaciens, R. oryzae and E. avium. The enzyme activities were 16.86, 12.51 and 9.18 U/mL. The highest alkaline protease activity of B. amyloliquefaciens was 13.29 U/mL, the optimum pH was 7 and the temperature was 34 ℃, followed by B. subtilis and R. oryzae. The enzyme activities were 8.86 and 6.20 U/mL, respectively. The ability of 12 kinds of microorganisms to produce acid protease was generally poor. Conclusion: The optimal pH and temperature of the 12 kinds of microorganisms selected for this experiment are basically consistent with the natural processing of Sojae Semen Praeparatum. Among them, fungi have stronger GAD production capacity and bacteria have stronger neutral protease production capacity. The high GABA concentration of Sojae Semen Praeparatum is caused by the joint action of multiple strains.

Mutation and fermentation optimization of Bacillus amyloliquefaciens for acetoin production / 生物工程学报

As a platform chemical, acetoin has a great potential of application in medicine and food industries. In order to improve the efficiency of acetoin production, Bacillus amyloliquefaciens was treated by atmospheric and room temperature plasma and gamma rays. Two-round screening was adopted for obtaining positive mutants, and the best mutant B. amyloliquefaciens H-5 produced acetoin up to 68.2 g/L in shake flask. Then, culture conditions were optimized in 5-L fermentor to enhance acetoin production. Finally, 85.2 g/L acetoin was produced by B. amyloliquefaciens H-5, which was increased by 26.8% compared with that of the original strain B. amyloliquefaciens FMME088. These results indicated that the high-producing strain can be obtained efficiently by compound mutagenesis, which has a promising prospect for commercial scale process.

Highly efficient enzymatic preparation of isomalto-oligosaccharides from starch using an enzyme cocktail

Background: Current commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields. Results: To improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran ß-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced. Conclusions: Our method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.

Production, characterization and biological activities of acidic exopolysaccharide from marine Bacillus amyloliquefaciens 3MS 2017

Objective To evaluate in-vitro antioxidant, anti-inflammatory and antitumor abilities against human breast adenocarcinoma (MCF7) and human prostate cancer (PC3) as well as the suppressor effect of bacterial exopolysaccharide (BAEPS) on Ehrlich ascites carcinoma (EAC). Methods In-vitro antioxidants characters of BAEPS were determined using various methods, while anti-inflammatory activity was estimated against cyclooxygenase (COX-1 and COX-2). In-vitro study, anticancer against MCF7 and PC3 were assessed by the mitochondrial dependent reduction of yellow MTT. In in-vivo study against EAC progression, mice were inoculated with EAC cells and then were orally administered BAEPS at 200 mg/kg after 24 h (equals to 0.10 of determined LD

Production, characterization and biological activities of acidic exopolysaccharide from marine Bacillus amyloliquefaciens 3MS 2017

OBJECTIVE@#To evaluate in-vitro antioxidant, anti-inflammatory and antitumor abilities against human breast adenocarcinoma (MCF7) and human prostate cancer (PC3) as well as the suppressor effect of bacterial exopolysaccharide (BAEPS) on Ehrlich ascites carcinoma (EAC).@*METHODS@#In-vitro antioxidants characters of BAEPS were determined using various methods, while anti-inflammatory activity was estimated against cyclooxygenase (COX-1 and COX-2). In-vitro study, anticancer against MCF7 and PC3 were assessed by the mitochondrial dependent reduction of yellow MTT. In in-vivo study against EAC progression, mice were inoculated with EAC cells and then were orally administered BAEPS at 200 mg/kg after 24 h (equals to 0.10 of determined LD)/10 d.@*RESULTS@#BAEPS was acidic exopolysaccharide contained uronic acid (12.3%) and sulfate (22.8%) with constitution of glucose, galactose and glucuronic acid in a molar ratio 1.6:1.0:0.9, respectively, with a molecular mass of 3.76 × 10 g/mol. BAEPS appeared potent antioxidant characters as free radical scavenging, oxygen reactive species scavenging and metal chelation, while its reducing power was low. BAEPS showed selective anti-inflammatory activity against COX-2 than COX-1, COX-2 selective. BAEPS exhibited potent and selective effect to breast cell cancer MCF7, the death percentage was 65.20% with IC = 70 μg/mL and IC = 127.40 μg/mL. BAEPS decreased counted viable EAC cells and induced non-viable cells. BAEPS improved all assessed hematological parameters. These improvements were reflected in the increasing median survival time and significant increment (P < 0.05) in life span.@*CONCLUSIONS@#BAEPS has anti-tumor activity with a good margin of safety. The anti-tumor activity of BAEPS may be due to its content from sulfated groups and uronic acids and they have antioxidant and anti-inflammatory properties.

Response surface methodology based optimization for degradation of align in Laminaria japónica feedstuff via fermentation by Bacillus in Apostichopus japónicas farming

Background: The alga Laminaria japonica is the most economically important brown seaweed cultured in China, which is used as food and aquatic animal feedstuff. However, the use of L. japonica as a feedstuff in Apostichopus japonicasfarming is not ideal because A. japonicas does not produce enough enzyme activity for degrading the large amount of algin present in L. japonica. In this study, semi solid fermentation of the L. japonica feedstuff employing a Bacillus strain as the microbe was used to as a mean to degrade the algin content in L. japonica feedstuff. Results: The Bacillus strain, Bacillus amyloliquefaciens WB1, was isolated by virtue of its ability to utilize sodium alginate as the sole carbon source. Eight factors affecting growth and algin-degrading capacity of WB1 were investigated. The results of Plackett-Burman design indicated that fermentation time, beef extract, and solvent to solid ratio were the significant parameters. Furthermore, the mutual interaction between the solvent to solid ratio and beef extract concentration was more significant than the other pairs of parameters on algin degradation. Optimal values obtained from Central-Composite Design were 113.94 h for fermentation time, 0.3% (w/v) beef extract and 44.87 (v/w) ratio of solvent to feedstuff. Under optimal conditions, 56.88% of the algin was degraded when a 50-fold scale-up fermentation was carried out, using a 5-L fermenter. Conclusions: This study provides an alternative and economical way to reduce the algin content in L. japonicathrough degradation by WB1, making it a promising potential source of feed for cultured L japonica.

Efecto de las PGPB sobre el crecimiento Pennisetum clandestinum bajo condiciones de estrés salino / PGPB effect on the growth Pennisetum clandestinum under salt stress

Esta investigación tuvo como objetivo el aislamiento y caracterización de bacterias con potencial para promover el crecimiento del pasto Pennisetum clandestinum en suelos salinos simulados. De muestras de suelo rizosférico de P. clandestinum se aislaron 92 bacterias Gram positivas, diez aislados crecieron en agar nutritivo suplementado con NaCl (2,578 M). Los aislados se evaluaron bajo condiciones de invernadero; las cepas identificadas como KISA 34 y KISA 71 fueron seleccionadas como las mejores con base a la prueba estadística de Dunnet (p≤ 0,05) y fueron identificadas molecularmente como Bacillus amyloliquefaciens KISA 34 y Bacillus subtilis KISA 71. Estas cepas tienen la capacidad de producir; amonio, exopolisacáridos y celulosa, y tanto en presencia como ausencia de NaCl, produjeron índoles totales y solubilización de fósforo. La evaluación de las cepas en invernadero evidenció que el T6 - KISA 34 + KISA 71+ 25 % (Roca Fosfórica RF) con respecto al T2- testigo químico completo incrementaron de manera significativa la biomasa y el desarrollo de la planta (p≤ 0.05). Los resultados de esta investigación demostraron que las cepas aisladas tienen la capacidad de crecer en suelos salinos conservando sus características como promotoras de crecimiento vegetal con efectos positivos sobre P. clandestinum.

This research aims the isolation and characterization of bacteria with potential to promote the growth of grass Pennisetum clandestinum in simulated saline soils. 92 Gram positive bacterias were isolated from Rhizosphere soil samples of P. clandestinum. Ten isolated bacteria grew on nutrient agar supplemented with NaCl (2.578 M). Isolates were evaluated under greenhouse conditions; the strains identified as KISA 34 and KISA 71 were selected as the best based on the statistical test of Dunnet (p ≤ 0.05) and were identified Bacillus amyloliquefaciens KISA 34 and Bacillus subtilis KISA 71. These strains have the ability to produce ammonium, exopolysaccharides and cellulose, both in absence and presence of NaCl. The strains produced indoles and phosphorus solubilization. The evaluation of strains in greenhouse showed that the T6 - KISA 34 + KISA 71+ 25 % (phosphate rock RF) significantly increased biomass and plant development (p≤ 0.05) compared with T2 - full fertilization . The results of this research showed that isolates have the ability to grow in saline soils retaining its characteristics as promoting plant growth with positive effects on P. clandestinum.

Mosquitocidal Bacillus amyloliquefaciens: Dynamics of growth & production of novel pupicidal biosurfactant.

Background & objectives: A strain of Bacillus amyloliquefaciens (VCRC B483) producing mosquito larvicidal and pupicidal biosurfactant was isolated from mangrove forest soil. The present study was aimed at studying the kinetics of growth and production of the mosquitocidal biosurfactant by this bacterium. Methods: Dynamics of growth, sporulation and production of mosquitocidal biosurfactant were studied by standard microbiological methods. The mosquitocidal biosurfactant was precipitated from the culture supernatant and bioassayed against immature stages of   mosquito vectors to determine lethal dose and lethal time. The activity, biological and biochemical properties of the biosurfactant have also been studied. Results: The pupal stages of mosquitoes were found to be more vulnerable to the biosurfactant produced by this bacterium with Anopheles stephensi being the most vulnerable species. The median lethal time (LT50) was found to be 1.23 h when the pupal stages of the above species were exposed to lethal concentration LC90 (9 μg/ml) dosage of the biosurfactant. Production of biosurfactant was found to increase with incubation time and maximum biomass, maximum quantity of biosurfactant (7.9 mg/ml), maximum biosurfactant activity (6 kBS unit/mg) and maximum mosquitocidal activity (5 μg/ml) were attained by 72 h of growth. The lipopeptide nature of the biosurfactant was confirmed by β-haemolysis, lipase activity, biofilm forming capacity, thermostability and biochemical analysis. Interpretation & conclusions: The mosquitocidal biosurfactant produced by B. amyloliquefaciens (VCRC B483) may be a prospective alternative molecule for use in mosquito control programmes involving bacterial biopesticides.

Biocontrol Activity of Bacillus amyloliquefaciens CNU114001 against Fungal Plant Diseases

A total of 62 bacterial isolates were obtained from Gomsohang mud flat, Mohang mud flat, and Jeju Island, Republic of Korea. Among them, the isolate CNU114001 showed significant antagonistic activity against pathogenic fungi by dual culture method. The isolate CNU114001 was identified as Bacillus amyloliquefaciens by morphological observation and molecular data analysis, including 16SrDNA and gyraseA (gyrA) gene sequences. Antifungal substances of the isolate were extracted and purified by silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. The heat and UV ray stable compound was identified as iturin, a lipopeptide (LP). The isolate CNU114001 showed broad spectrum activity against 12 phytopathogenic fungi by dual culture method. The semi purified compound significantly inhibits the mycelial growth of pathogenic fungi (Alternaria panax, Botrytis cinera, Colletotrichum orbiculare, Penicillium digitatum, Pyricularia grisea and Sclerotinia sclerotiorum) at 200 ppm concentration. Spore germ tube elongation of Botrytis cinerea was inhibited by culture filtrate of the isolate. Crude antifungal substance showed antagonistic activity against cucumber scleotiorum rot in laboratory, and showed antagonistic activity against tomato gray mold, cucumber, and pumpkin powdery mildew in greenhouse condition.

Potential pathogenicity of Bacillus amyloliquefaciens to infants / 临床儿科杂志

Bacillus amyloliquefaciensin can cause infections and the building related ill-health syndrome among newborns, infants and other immunocompromised populations by polluting hospital disinfectants, such as alcohol 75%, moisture housing and food. In this article, we reviewed the prevalence of Bacillus amyloliquefaciens in environment, food and intrahospital infection, the toxicology by describing its toxicity in mammalian cells, the physicoehemical characteristics by analyzing its special structure of bacterial spores. Its pathogenicity through animal model of disease was discussed as well.(J Glin Pediatr,2010,28(2) :190-192)

Exploring the Antifungal Activity of Bacillus amyloliquefaciens NK10.BAhjaWT / 微生物学通报

Bacillus are well known antibiotic producers. In this study,dozens of Bacillus strains from different sources were screened. Among them,a strain with strong antifungal activity was found. With 16S rDNA test and Biolog assay,this strain was identified to be Bacillus amyloliquefaciens. The fermentation conditions were optimized in small conical flasks. After ammonium sulfate salting out,dialysis,freezing vacuum dehydration,the crude protein extracts were obtained. The thermal stability,pH stability,protease stability,ion stability and antifungal spectrum of this protein were studied further. Scanning electronic microscope was also used to explore the antifungal mechanism.