Using Ceriporiopsis subvermispora CZ-3 laccase for indigo carmine decolourization and denim bleaching

In this study, the production of laccase by Ceriporiopsis subvermispora CZ-3 has been studied under semi solid-state conditions using natural waste as solid support materials. Different concentrations of xylidine derivatives were also investigated as inducer affecting laccase production. Melon peel having the lowest C/N ratio in comparison to other supports led to the highest activity levels, reaching maximum values of about 3000 UL⁻¹ for C. subvermispora CZ-3 in the presence of 2,4-xylidine. Laccase produced by this fungus was partially purified by ammonium sulphate precipitation and Sephacryl S-100 HR size exclusion chromatography. Several kinetic parameters of enzyme were also determined with 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) as a substrate. On the other hand, several compounds were investigated with respect to mediator effect for decolourization of indigo carmine and denim bleaching by this enzyme. It was observed that most of the compounds have mediator effect for decolourization of indigo carmine whereas 1-hydroxybenzotriazol is an appropriate compound for indigo dyed denim bleaching.     

Decolorization and biodegradation of Indigo carmine by a textile soil isolate Paenibacillus larvae

The potential of recently isolated bacteria Paenibacillus larvae for the effective decolorization of Indigo carmine was evaluated. The effects of operational parameters (temperature, pH, dye concentration, shaking/non shaking) were tested. Maximum extent of decolorization was observed when the medium was incorporated with 10 g/l of yeast extract and peptone. Decolorization was strongly inhibited at non-shaken conditions as well as incorporation of inorganic sources (sodium nitrite and ammonium chloride) in the medium. Maximum decolorization was observed at 30°C (100%) and 40°C (92%) at 8 h of incubation. The LC-MS and NMR analysis confirms the oxidation of Indigo carmine .The primary degradation products were found to be Isatin sulfonic acid and anthranilicacid.     

Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores

A strain of B. subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium. Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 °C. The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.     

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10⁷ spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10⁴ spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10⁵ spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed.     

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf=0.60, as a result of dye degradation.     

Permeability of gentamicin into the inside of Bacillus subtilis spores

Abstract The penetration of gentamicin into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were located mainly in the coat regions of spores and were not observed in the cortex or core regions. This result suggests the existence of an outer membrane inside the coat region as the primary permeability barrier to gentamicin.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

Injury and repair in biocide-treated spores of Bacillus subtilis

Abstract Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37°C but not in broth at 22°C, sterile filtered water at 4, 22 or 37°C or germination medium at 37°C. Repair appeared to occur primarily during outgrowth and was initiated soonest for iodine-treated spores and latest for glutaraldehyde-treated ones.     

New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 μM (3-5 μg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 μM (2-4 μg mL(-1)) and >150 μM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: Purification, characterization and indigo carmine degradation

γ-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu²⁺-centre. It worked optimally at 55 °C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 even after 60 days at 4 °C. K_m and V_max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 °C) to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT.     

Spore-displayed streptavidin: a live diagnostic tool in biotechnology

Streptavidin, which is one of the most widely used proteins in biotechnological application field and is active only in tetrameric form, was surface expressed on the surface of Bacillus subtilis spore. Spore coat protein of B. subtilis, CotG, was used as an anchoring motif to display streptavidin. FACS using anti-streptavidin antibody was used for the verification of surface localization of expressed CotG-streptavidin fusion protein. FACS and dot-blot were used for the verification of biological activity of displayed streptavidin with FITC-labeled biotin.     

Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55 degrees C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.     

枯草杆菌的芽胞在肉鸡肠道中的生活状态和分布

目的探讨枯草芽胞杆菌的芽胞在肉鸡肠道中的生活状态和分布。方法以20日龄AA肉鸡为研究对象,饲喂枯草芽胞菌剂,分别测定鸡粪中芽胞数量和鸡肠道不同部位的活菌数量。结果饲喂3h后,鸡粪中开始检测到芽胞的存在,24h达到最高值,直至饲喂120h后,肠道内的芽胞基本排除。排出芽胞总量为饲喂芽胞总数的3．0倍左右,同时研究还表明：芽胞在实验肉鸡的十二指肠2内开始萌发,并进行了繁殖,在小肠的后端,即小肠3和小肠4,活菌数量达到高峰。结论部分芽胞进入小肠后即可开始萌发,并进行生长繁殖,而且在肠道内有短暂滞留。     

Activation of subtilin precursors by Bacillus subtilis extracellular serine proteases subtilisin (AprE), WprA,and Vpr

The maturation of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 includes posttranslational modifications of the propeptide and proteolytic cleavage of the leader peptide. To identify subtilin processing activities, we used antimicrobial inactive subtilin precursors consisting of the leader peptide which was still attached to the fully matured propeptide. Two extracellular B. subtilis proteases were able to activate subtilin precursors, the commercially available serine protease prototype subtilisin (AprE) and WprA. The latter was isolated from B. subtilis WB600, a strain deficient in six extracellular proteases. Surprisingly, the aprE wprA double mutant of the ATCC 6633 strain was still able to produce active subtilin, however, with a reduced production rate. No subtilin processing was found within the culture supernatant of the WB800 strain, which is deficient in eight extracellular proteases. Vpr was identified as the third protease capable to process subtilin.     

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis

We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B. subtilis protoplast, which expressed PrsA-CelA was 15 times higher compared to control strain. More than 85% of total cellulase activity was observed in surface displayed format and less than 15% of total cellulase activity was found in supernatant. Flow cytometric analysis of protoplast of PrsA-CelA fusion expressing bacteria provided another proof of uniform expression of fusion protein onto cytoplasmic membrane of B. subtilis. Without lysozyme treatment, only part of cellulase activity (10%) was observed in whole cell fraction.     

PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.