Cloning, Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium, Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS polyacrylamide gel electrophoresis (SDS-PAGE). Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results: Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS-PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion: In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity.

A Comparative Taxonomic Profile of Microbial Polyethylene and Hydrocarbon-Degrading Communities in Diverse Environments.

BACKGROUND: Polyethylene (PE) is one of the most abundant plastic wastes which accumulates in marine and terrestrial environments. As microbial degradation has been a promising approach for the bioremediation of polluted environments, identification of the microbial community profile where these pollutants accumulate, has recently been in focus. OBJECTIVE: We have investigated the taxonomic and functional characteristics of polyethylene- degrading microorganisms in a plastic waste recycling site in Tehran, Iran. MATERIALS AND METHODS: We have analyzed and compared a 16S rRNA dataset from this study with 15 datasets from 4 diverse plastic and oil polluted habitats to identify and evaluate bacterial communities involved in bioremediation. RESULTS: Our findings reveal that Proteobacteria, Actinobacteria, Acidobacteria and Cloroflexi were the dominant phyla and Actinobacteria, Alphaproteobacteria, Gammaproteobacteria and Acidimicrobia were dominant classes in these samples. The most dominant Kegg Orthology associated with PE bioremediation in these samples are related to peroxidases, alcohol dehydrogenases, monooxygenases and dioxygenases. CONCLUSIONS: Long-term presence of contaminants in soil could lead to changes in bacterial phyla abundance, resulting in metabolic adaptations to optimize biological activity and waste management in a diverse group of bacteria.

Considerable increase in Poly(3-hydroxybutyrate) production via phbC gene overexpression in Ralstonia eutropha PTCC 1615.

Introduction: Poly(3-hydroxybutyrate) (PHB) is a well-known biodegradable polymer produced by some microorganisms and can be a suitable alternative for petrochemical plastics. PHB synthase encoded by phb C gene is the main enzyme in PHB biosynthesis pathway in Ralstonia eutropha. The aim of current study was the transformation of R. eutropha PTCC 1615 with its own phb C gene and evaluation of the overexpression effect on PHB accumulation. Methods: DNA fragment including phbC gene and its promoter and terminator regions, was isolated from R. eutropha PTCC 1615, inserted into pET28a(+) vector, and transferred to the competent bacteria using calcium chloride and heat shock method. The effect of the cloned gene expression on PHB production was investigated with absorption of crotonic acid produced through PHB dehydration. Statistical analyses were carried out by SPSS software. Results: PHB content of cells of the engineered strain was 1.4 times more than that of the native bacteria. This significant difference can be an important finding for improvement of biopolymer production. Conclusion: Overexpression of phb C, the critical gene in PHB biosynthesis pathway, in R. eutropha PTCC 1615 had considerable effect on PHB accumulation.

A survey of intact low-density polyethylene film biodegradation by terrestrial Actinobacterial species.

Low-density polyethylene (LDPE) polymer is mainly used in the production of plastic bags and food packaging making up the largest volume of plastic pollutions. These polymers are potential substrates for bacteria in the bioremediation process. In this study, soil samples were collected from different plastic landfills in Iran and subsequently enriched in specific media (polyethylene as carbon source) to increase the population of LDPE-degrading bacteria. Seventeen PE-degrading bacteria, some novel, were isolated from Iranian soil samples and identified using 16S rDNA gene sequencing. These isolates were capable of degrading PE in a limited incubation period without the need for physicochemical pretreatments and comprise mostly of Actinobacteria which include the three genera of Streptomyces, Nocardia, and Rhodococcus. The isolates belonged to 17 different species of gram-positive Actinobacteria. In all, 11 species of the genus Streptomyces, 3 species of the genus Rhodococcus, and 3 species of the genus Nocardia were identified. The isolates with less than 99% 16S rRNA gene similarity to previously known species were suspected to be new species. Various analyses (weight loss, SEM, FTIR, and tensile strength test) to determine polyethylene biodegradation rate were carried out after a 60-day incubation period. Analysis of polyethylene biodegradation elucidates that Actinobacteria have a high ability for biodegradation of polyethylene-based plastics. Streptomyces sp. IR-SGS-T10 showed the highest reduction in weight of the LDPE film (1.58 mg/g/day) after 60 days of incubation without any pretreatments. Rhodococcus sp. IR-SGS-T11 shows the best reduction in the tensile property of LDPE film, while results from FTIR study for Streptomyces sp. IR-SGS-Y1 indicated a significant change in structural analysis.

Evaluation of Helicobacter pylori OipA protein as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice.

OBJECTIVES: Outer inflammatory protein A (OipA) is an essential adhesin of Helicobacter pylori. We aimed to evaluate the effects of a recombinant OipA in the induction of crucial cytokines as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice. MATERIALS AND METHODS: C57BL/6 mice were divided into nine groups according to the disposition of antigen and adjuvant and route of administration: subcutaneous (sc) or gavage. The administrated recombinant purified OipA and propolis concentrations were 10 µg/ml and 40 µg/ml, respectively. After vaccination, we measured expression levels of IFN-γ and IL-4 cytokine genes in the spleen cells of mice by real-time PCR. RESULTS: All results were contrasted with the negative sample. By sc injection, the expression of INF-γ was increased 3.5 and 2.9-fold for OipA and OipA plus propolis, respectively. By gavage 4.4 and 11-fold increase was found for OipA and OipA plus propolis, respectively. The administration of propolis by gavage showed more increase than Sc injection concerning the production of INF-γ. The 11-fold increase for injection of OipA plus propolis by gavage was comparable OipA plus Freund's adjuvant injected subcutaneously. This result suggested an excellent immunological response toward OipA concerning the production of INF-γ in mice. In all cases there were no notable IL-4 production increases. CONCLUSION: The results confirm the efficiency of OipA in induction of IFN-γ production, and thereby the cellular immune response. Propolis could be a suitable adjuvant.

The Role of Helicobacter pylori Proinflammatory Outer Membrane Protein and Propolis in Immunomodulation on U937 Macrophage Cell Model.

BACKGROUND: Regarding the important role of proinflammatory outer membrane protein (OipA) in the pathogenesis of Helicobacter pylori infection and immunomodulatory activity of propolis, we aimed to evaluate the immunogenicity effect of a purified recombinant OipA protein and propolis in the induction of two cytokines, interferon-gamma (IFN-γ) and interleukin-4 (IL-4), in a macrophage cell model. MATERIALS AND METHODS: The recombinant protein used in the present study corresponding to the oipA expressing a 34-35 kDa protein. OipA protein was purified by Ni-NTA affinity chromatography. The purified OipA protein (2.5- 40 µg /mL) and the propolis ethanolic extract (5-40 µg/mL) were incubated with phorbol 12-myristate 13-acetate-treated human myelomonocytic cell line U937 cells. IL-4 and IFN-γ levels were measured after 48 hours of incubation using enzyme-linked immunosorbent assay. RESULTS: The amounts of IL-4 and IFN-γ were significantly increased. The optimum concentration of OipA for the secretion of IL-4 was 5 µg/ml (P<0.0001). At higher concentrations, the amount of IL-4 diminished until suppression at 40 µg/mL. The optimum concentration of propolis, resulting in the most significant increased secretion of both IL-4 and IFN-γ was 40 µg/mL (P=0.0001 and P=0.0004). CONCLUSION: We found that an OipA concentration of 10 µg/mL was more effective for IFN-γ production; however, it was not effective for the high production of IL-4. Therefore, it is postulated that the OipA could mainly induce a Th1 response through the production of IFN-γ. We also observed propolis's capability to induce IFN-γ production; however, the effective concentration for this was the same as for IL-4. Therefore, as an adjuvant, proper concentration of propolis is required for OipA to give the optimum response.

Ecological role of Acinetobacter calcoaceticus GSN3 in natural biofilm formation and its advantages in bioremediation.

This study assessed the role of a new Acinetobacter calcoaceticus strain, GSN3, with biofilm-forming and phenol-degrading abilities. Three biofilm reactors were spiked with activated sludge (R1), green fluorescent plasmid (GFP) tagged GSN3 (R2), and their combination (R3). More than 99% phenol removal was achieved during four weeks in R3 while this efficiency was reached after two and four further operational weeks in R2 and R1, respectively. Confocal scanning electron microscopy revealed that GSN3-gfp strains appeared mostly in the deeper layers of the biofilm in R3. After four weeks, almost 7.07 × 107 more attached sludge cells were counted per carrier in R3 in comparison to R1. Additionally, the higher numbers of GSN3-gfp in R2 were unable to increase the efficiency as much as measured in R3. The presence of GSN3-gfp in R3 conveyed advantages, including enhancement of cell immobilization, population diversity, metabolic cooperation and ultimately treatment efficiency.

Characterization of gyrA and parC mutations in ciprofloxacin-resistant Pseudomonas aeruginosa isolates from Tehran hospitals in Iran.

BACKGROUND AND OBJECTIVES: Pseudomonas aeruginosa, a major cause of several infectious diseases, has become a hazardous resistant pathogen. One of the factors contributing to quinolone resistance in P. aeruginosa is mutations occurring in gyrA and parC genes encoding the A subunits of type II and IV topoisomerases, respectively, in quinolone resistance determining regions (QRDR) of the bacterial chromosome. MATERIALS AND METHODS: Thirty seven isolates from patients with burn wounds and 20 isolates from blood, urine and sputum specimen were collected. Minimum Inhibitory Concentrations (MICs) of ciprofloxacin were determined by agar diffusion assay. Subsequently, QRDRs regions of gyrA and parC were amplified from resistant isolates and were assessed for mutations involved in ciprofloxacin resistance after sequencing. RESULTS: Nine isolates with MIC≥8 µg/ml had a mutation in gyrA (Thr83→Ile). Amongst these, seven isolates also had a mutation in parC (Ser87→ Leu or Trp) indicating that the prevalent mutation in gyrA is Thr83Ile and Ser87Leu/Trp in parC. No single parC mutation was observed. CONCLUSION: It seems that mutations in gyrA are concomitant with mutations in parC which might lead to high-level ciprofloxacin resistance in P. aeruginosa isolates from patients with burn wounds and urinary tract infections.

Highly efficient phenol degradation in a batch moving bed biofilm reactor: benefiting from biofilm-enhancing bacteria.

In this study, the efficiency improvement of three moving bed biofilm reactors (MBBRs) was investigated by inoculation of activated sludge cells (R1), mixed culture of eight strong phenol-degrading bacteria consisted of Pseudomonas spp. and Acinetobacter spp. (R2) and the combination of both (R3). Biofilm formation ability of eight bacteria was assessed initially using different methods and media. Maximum degradation of phenol, COD, biomass growth and also changes in organic loading shock were used as parameters to measure the performance of reactors. According to the results, all eight strains were determined as enhanced biofilm forming bacteria (EBFB). Under optimum operating conditions, more than 90% of initial COD load of 2795 mg L-1 was reduced at 24 HRT in R3 while this reduction efficiency was observed in concentrations of 1290 mg L-1 and 1935 mg L-1, in R1 and R2, respectively. When encountering phenol loading shock-twice greater than optimum amount-R1, R2 and R3 managed to return to the steady-state condition within 32, 24 and 18 days, respectively. SEM microscopy and biomass growth measurements confirmed the contribution of more cells to biofilm formation in R3 followed by R2. Additionally, established biofilm in R3 was more resistant to phenol loading shock which can be attributed to the enhancer role of EBFB strains in this reactor. It has been demonstrated that the bacteria with both biofilm-forming and contaminant-degrading abilities are not only able to promote the immobilization of other favorable activated sludge cells in biofilm structure, but also cooperate in contaminant degradation which all consequently lead to improvement of treatment efficiency.

Screening, cloning and expression of a novel alginate lyase gene from P. aeruginosa TAG 48 and its antibiofilm effects on P. aeruginosa biofilm.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen and utilizes several virulence factors for pathogenesis. One of the most important factors is alginate, found in the biofilm which enables P. aeruginosa to establish chronic lung infections. MATERIALS AND METHODS: In this study, 25 clinical alginate-degrading isolates were selected. Biochemical and molecular approach were carried out to identify the isolates by 16S rDNA gene amplification. Growth conditions and enzyme production were the criteria for selection. Since the main objective of the project was the production and characterization of alginate lyase and its effect on biofilm elimination, the P. aeruginosa sp.TAG48 alginate lyase-encoding gene was isolated, cloned, sequenced and expressed in E.coli DH5α. The resultant enzyme was purified by affinity chromatography. Ciprofloxacin, tobramycin and cefixime were also used to test the effectiveness of these antibiotics on P. aeruginosa biofilm by minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC). The synergistic effects of these antibiotics and the recombinant alginate lyase on biofilm were evaluated. RESULTS: Results indicate that the addition of alginate (0.2%-0.8%) and NaCl (0.2-0.5 M) to the medium significantly increases cell growth followed by higher enzyme production (p≤ 0.05). Moreover, substrate specificity of alginate lyase produced by P. aeruginosa sp.TAG48 shows the enzyme is capable of degrading both polyM and polyG alginate and acts bifunctionally. Results from the antimicrobial characteristics of the antibiotics and the enzyme have shown MBIC for ciprofloxacin, tobramycin, cefixime and enzyme in the following concentrations 4, 32, 256 and 18.75 µg/ml, and MBEC: 32, 128, ≥ 512 and 37.5 µg/ml, respectively. The study of synergism between the antibiotics and the enzyme to prevent growth and eradication of P. aeruginosa sp.TAG48 biofilm shows that alginate lyase exhibits synergy with tobramycin and cefixime but not with ciprofloxacin. CONCLUSION: The results indicate that the use of purified novel alginate lyase with antibiotics could be a beneficial alternative for the treatment of P. aeruginosa infections. Elucidation of mechanisms involved in antibiotic resistance and the role of biofilm structure could assist physicians select optimum treatment regimen.

The role of probiotic Lactobacillus acidophilus ATCC 4356 bacteriocin on effect of HBsu on planktonic cells and biofilm formation of Bacillus subtilis.

Bacillus subtilis is a Gram positive, aerobic and motile bacterium. Biofilm formation is an important feature of this bacterium which confers resistance to antimicrobial agents. The use of new antimicrobial reagents which eliminate biofilms are important and necessary. In this study, the effect of secondary metabolites (bacteriocin) from Lactobacillus acidophilus ATCC 4356 on Bacillus subtilis BM19 in the presence and absence of HBsu which is involved in the growth of planktonic cells and biofilm formation, is reported. HBsu nucleoprotein plays several roles in different processes of Bacillus subtilis cells such as replication, transcription, cell division, recombination and repair. In this study, for the first time, the effect of HBsu on biofilm formation is presented. RESULTS: In the absence of HBsu, purified bacteriocin from L. acidophilus ATCC 4356 was more effective in inhibiting growth of B. subtilis BM19 planktonic cells as well as biofilm formation. The presence of HBsu on the other hand led to increased biofilm formation.

Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production.

BACKGROUND: Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. METHODS: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Resuls: The results showed the ability of Bacillus sp. TAG8 to utilize alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. CONCLUSION: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples.

Ex situ study of Enterococcus faecalis survival in the recreational waters of the southern coast of the Caspian Sea.

BACKGROUND AND OBJECTIVES: The US Environmental Protection Agency has suggested faecal enterococci as the primary bacterial indicators. Of more importance is their direct correlation with swimmer-associated gastroenteritis in recreation water quality monitoring. In contrast to other seawater bodies with 3.5% salinity, the recreational waters in the southern coast of the Caspian Sea possess its own salinity (about 1% w/v) and thus require further investigations to determine the capacity of Enterococcus faecalis as the sole primary microbial index in this unique aquatic environment. MATERIALS AND METHODS: The survey of the presence and survival of E. faecalis as a microbial index in the recreational waters of the southern Caspian Sea was carried out using a microcosm as an experimental model. The concentration of E. faecalis cells in samples of seawater were estimated by a standard membrane filtration method using m-Enterococcus agar as the selective culture medium. As the current standard culture-based methods are not reliable enough for the detection of non-growing, damaged and under-tension bacteria, PCR was used to identify the possible VBNC form of the bacterium after disappearance of the culturable cells. RESULTS AND CONCLUSION: A continuous decline in the number of culturable E. faecalis cells resulted in apparent elimination of the bacteria from seawater in a defined period. Detection of intact DNA was possible in the following 60 days. The salinity of about 1% and the self-purification properties of the Caspian Sea make the conditions feasible for the use of this microorganism as a measure of water quality throughout the region. The results confirmed the presence of damaged bacterial cells, namely VBNC forms, indicating the necessity of examining of the sea water samples by using molecular approaches or repair procedures.

Assessment of Biofilm Formation and Resistance to Imipenem and Ciprofloxacin among Clinical Isolates of Acinetobacter baumannii in Tehran.

BACKGROUND: Biofilms are communities of bacteria attached to the surfaces in an extracellular polymeric matrix which are associated with many chronic infections in humans. Acinetobacter spp. are emerging as a major cause of nosocomial infections and Acinetobacter baumannii is the predominant species associated with this kind of infections. OBJECTIVES: In the present study, the potential of biofilm formation of clinical isolates, A. baumannii, was assessed by using crystal violet method. Furthermore, susceptibility pattern of these strains to ciprofloxacin and imipenem was determined. METHODS AND MATERIALS: Biofilm formation by 75 A. baumannii isolates was evaluated by using microtiter plate and tube methods and crystal violet staining. Tube method was carried out under static and shaking conditions. Then, the susceptibility of isolates to ciprofloxacin and imipenem was determined. RESULTS: Results showed that in tube method under shaking, 22% of clinical isolates were strong biofilm producers while 23% of them were not able to form biofilms. In this experiment, 18% and 42% of isolates were considered as moderate and weak biofilm-forming strains, respectively. In microtiter plate tests, 18% of strains were strong-biofilm producers and 25% of them were notable biofilm producers. In this assessment, 10% and 47% were considered as moderate and weak biofilm-forming isolates, respectively. The susceptibility tests, using microdilution method, confirmed that 92% of these isolates were resistant and 6.6% were susceptible to ciprofloxacin, although these results for imipenem were 68% and 24%, respectively. CONCLUSIONS: It can be concluded that most of A. baumannii isolates can form biofilm in microtiter plate and tube. The results also verified that most of these isolates were resistant to ciprofloxacin and imipenem.

Study of antagonistic effects of Lactobacillus strains as probiotics on multi drug resistant (MDR) bacteria isolated from urinary tract infections (UTIs).

OBJECTIVE(S): Urinary tract infection (UTI) caused by bacteria is one of the most frequent infections in human population. Inappropriate use of antibiotics, often leads to appearance of drug resistance in bacteria. However, use of probiotic bacteria has been suggested as a partial replacement. This study was aimed to assess the antagonistic effects of Lactobacillus standard strains against bacteria isolated from UTI infections. MATERIALS AND METHODS: Among 600 samples; those with ≥10,000 cfu/ml were selected as UTI positive samples. Enterococcus sp., Klebsiella pneumoniae, Enterobacter sp., and Escherichia coli were found the most prevalent UTI causative agents. All isolates were screened for multi drug resistance and subjected to the antimicrobial effects of three Lactobacillus strains by using microplate technique and the MICs amounts were determined. In order to verify the origin of antibiotic resistance of isolates, plasmid curing using ethidium bromide and acridine orange was carried out. RESULTS: No antagonistic activity in Lactobacilli suspension was detected against test on Enterococcus and Enterobacter strains and K. pneumoniae, which were resistant to most antibiotics. However, an inhibitory effect was observed for E. coli which were resistant to 8-9 antibiotics. In addition, L. casei was determined to be the most effective probiotic. RESULTS from replica plating suggested one of the plasmids could be related to the gene responsible for ampicillin resistance. CONCLUSION: Treatment of E. coli with probiotic suspension was not effective on inhibition of the plasmid carrying hypothetical ampicillin resistant gene. Moreover, the plasmid profiles obtained from probiotic-treated isolates were identical to untreated isolates.

Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity.

BACKGROUND AND OBJECTIVES: Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS-degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. MATERIALS AND METHODS: Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS). RESULTS AND CONCLUSION: Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation.

Cane molasses as a source of precursors in the bioproduction of tryptophan by Bacillus subtilis.

BACKGROUND AND OBJECTIVES: The essential amino acid L-tryptophan can be produced by a condensation reaction between indole and L-serine, catalyzed by B. subtilis with tryptophan synthase activity. Application of the tryptophan is widespread in the biotechnology domain and is sometimes added to feed products as a food fortifier. MATERIALS AND METHODS: The optimum concentration of the Iranian cane molasses was determined by measuring the amount of biomass after growth in 1 to 30 g/mL of molasses. The maximum amount of biomass was obtained in 10 g/mL molasses. Chromatographic methods, TLC and HPLC, were used to assay the amount of tryptophan produced in the presence of precursors of tryptophan production (indole and serine) and/or molasses. RESULTS: Our results indicate the importance of the Iranian cane molasses not only as carbon source, but also as a source of precursors for tryptophan production. CONCLUSION: This report evaluates the potential of cane molasses as an economical source for tryptophan production by B. subtilis, hence eliminating the requirement for additional serine and indole as precursors.

Degradation of a textile reactive azo dye by a combined biological-photocatalytic process: Candida tropicalis Jks2 -Tio2/Uv.

In the present study, the decolorization and degradation of Reactive Black 5 (RB5) azo dye was investigated by biological, photocatalytic (UV/TiO2) and combined processes. Application of Candida tropicalis JKS2 in treatment of the synthetic medium containing RB5 indicated complete decolorization of the dye with 200 mg/L in less than 24 h. Degradation of the aromatic rings, resulting from the destruction of the dye, did not occur during the biological treatment. Mineralization of 50 mg/L RB5 solution was obtained after 80 min by photocatalytic process (in presence of 0.2 g/L TiO2). COD (chemical oxygen demand) was not detectable after complete decolorization of 50 mg/L RB5 solution. However, photocatalytic process was not effective in the removal of the dye at high concentrations (≥200 mg/L). With 200 mg/L concentration, 74.9% of decolorization was achieved after 4 h illumination under photocatalytic process and the absorbance peak in UV region (attributed to aromatic rings) was not completely removed. A two-step treatment process, namely, biological treatment by yeast followed by photocatalytic degradation, was also assessed. In the combined process (with 200 mg/L RB5), absorbance peak in UV region significantly disappeared after 2 h illumination and about 60% COD removal was achieved in the biological step. It is suggested that the combined process is more effective than the biological and photocatalytic treatments in the remediation of aromatic rings.

Bioprocessing of seleno-oxyanions and tellurite in a novel Bacillus sp. strain STG-83: a solution to removal of toxic oxyanions in presence of nitrate.

Bioremediation of toxic nonmetal and metalloid oxyanions is of great interest. In this study, among 148 bacterial isolates from two types of polluted water, strain STG-83 showed maximum oxyanion reduction and resistance ability. Sequencing of the 16S rDNA gene of STG-83 showed that the strain is closely related to Bacillus pumilus and morphological and biochemical tests confirmed the result. The strain was nitrate negative, but it could reduce half of tellurite in solution containing 1-mM concentration and completely reduced selenite and selenate in solutions containing 1-mM concentrations. Both reduction to elemental form and volatilization occurred in case of all oxyanions tested, according to hydride generation atomic absorption spectroscopy and proton induced X-ray emission analytical methods. The strain was able to tolerate remarkably high concentrations of selenite (640 mM), selenate (320 mM), and tellurite (1250 microM); and tolerance to tellurite increased in presence of selenite and selenate. Biochemical tests and zymogram of extracted culture solutions on gel electrophoresis showed that the strain was nitrate negative and therefore nitrate did not interfere with reduction of other oxyanions. Thus, the strain opens up good opportunities for the bioremediation of polluted waters in natural environment, since nitrate usually inhibits or decelerates reduction of the mentioned toxic oxyanions.

A study of varicella zoster virus glycoprotein C regulatory region response to viral activators in vitro.

A study was conducted to analyze the response of varicella zoster virus (VZV) glycoprotein C gene (ORF14) regulatory sequences downstream as well as upstream of the transcription site to VZV transactivators, IE4 and IE62 and p29, the single-stranded DNA binding protein, in vitro by transiently transfecting a permissive human melanoma cell line (Mewo). This glycoprotein has been shown to be an important factor in VZV pathogenesis and therefore the regulation of its expression has been of much interest. In this study, the promoter region of gC as well as another VZV glycoprotein, gI (as a positive control), was amplified and cloned into a promoter less plasmid expressing the luciferase gene as a reporter. The activities of the regulatory regions from both glycoproteins were assessed by quantifying the luciferase activity. The results show that the luciferase assay is a powerful means of measuring promoter activity; nevertheless, the promoter region and cognate downstream and upstream sequences of the true late gC gene were not responsive to these viral proteins, indicating that other viral/cellular factors and/or viral replication could be involved in gC synthesis during the VZV infection cycle.