In silico approaches to investigate enzyme immobilization: a comprehensive systematic review.

Enzymes are popular catalysts with many applications, especially in industry. Biocatalyst usage on a large scale is facing some limitations, such as low operational stability, low recyclability, and high enzyme cost. Enzyme immobilization is a beneficial strategy to solve these problems. Bioinformatics tools can often correctly predict immobilization outcomes, resulting in a cost-effective experimental phase with the least time consumed. This study provides an overview of in silico methods predicting immobilization processes via a comprehensive systematic review of published articles till 11 December 2022. It also mentions the strengths and weaknesses of the processes and explains the computational analyses in each method that are required for immobilization assessment. In this regard, Web of Science and Scopus databases were screened to gain relevant publications. After screening the gathered documents (n = 3873), 60 articles were selected for the review. The selected papers have applied in silico procedures including only molecular dynamics (MD) simulations (n = 20), parallel tempering Monte Carlo (PTMC) and MD simulations (n = 3), MD and docking (n = 1), density functional theory (DFT) and MD (n = 1), only docking (n = 11), metal ion binding site prediction (MIB) server and docking (n = 2), docking and DFT (n = 1), docking and analysis of enzyme surfaces (n = 1), only DFT (n = 1), only MIB server (n = 2), analysis of an enzyme structure and surface (n = 12), rational design of immobilized derivatives (RDID) software (n = 3), and dissipative particle dynamics (DPD; n = 2). In most included studies (n = 51), enzyme immobilization was investigated experimentally in addition to in silico evaluation.

Isolation, functional evaluation of probiotic properties and molecular identification of strains isolated from Iranian poultry's gut.

Background and Objectives: The use of probiotics as an alternative to antibiotics in poultry diets has attracted a lot of interest recently. In this context, the determination of probiotic characteristics was evaluated on several isolates obtained from Iranian poultry's gut. Materials and Methods: Probiotic characteristics such as hemolysis activity, acid, bile, gastric juice tolerate, in vitro adhesion assay, cell surface properties (hydrophobicity, auto-aggregation and co-aggregation) and antibiotic susceptibility test were evaluated. Finally, selected isolates identified molecularly after temperature-salt tolerance and extracellular enzyme activity (amylase, protease and cellulose). Results: Out of 362 strains isolated from native poultry in three geographical regions of Iran, nine strains (belong to Bacillus sp., Enterococcus sp., Pediococcus sp., Lactobacillus sp., Kluyveromyces sp.) showed resistance against gastrointestinal physiological conditions, desirable surface properties, ability to adhere to epithelial intestine cell line and antibiotic susceptibility. Also, these strains were discovered to be temperature-salt tolerant but, only a small number of them were able to produce hydrolase enzymes. Conclusion: According to the results, the selected strains can be introduced as native probiotic candidates for utilization in novel poultry feeds.

Antiviral activity of biosynthesized copper nanoparticle by Juglans regia green husk aqueous extract and Iron nanoparticle: molecular docking and in-vitro studies.

Background and Objectives: The interaction between nanoparticles (NPs) and viruses is attracting interest because of the antiviral potential of NPs. This study aims to investigate the antiviral potential of NPs against Herpes simplex virus types 1 (HSV-1). Materials and Methods: Molecular docking studies were conducted by Molegro virtual docker software. An extract of Juglans regia green husk was utilized to biosynthesize copper-oxide nanoparticles (CuNPs). The cytotoxicity of NPs was evaluated by MTT assay. Different treatment assays were conducted. Another assay was designed to employ the concentration of 300 µg/ml of CuNPs, which is the highest concentration that did not precipitate. Finally, chemically synthesized Iron oxide nanoparticles (FeNPs) were utilized to adsorb CuNPs. The antiviral effect of FeNPs was investigated, separately. Results: Docking results confirmed that NPs could interact with the HSV-1 glycoproteins and prevent viral entry. MTT assay results illustrated that the minimum non-toxic concentration (MNTD) of CuNPs is 100 µg/ml which did not exhibit antiviral properties. Employing a noncytotoxic concentration of FeNPs (300 mg/ml) in combination with cytotoxic concentration of CuNPs (300 µg / ml), eliminated the cytotoxicity effects of CuNPs. Exposure of the virus with the combination of CuNPs and FeNPs resulted in 4.5 log10 TCID50 reductions in HSV-1. While treating HSV-1 with only FeNPs reduced the titer of virus by 3.25 log10 TCID50. Conclusion: The results highlight that combination of CuNPs and FeNPs have antiviral activity against HSV-1. Moreover, FeNPs demonstrated antiviral properties against HSV-1 separately.

One step green synthesis of Cu nanoparticles by the aqueous extract of Juglans regia green husk: assessing its physicochemical, environmental and biological activities.

Juglans regia (J. regia) green husk is an abundant agricultural waste. In this study, an economical, rapid and green synthetic route was introduced for the biosynthesis of copper nanoparticles (CuNPs) by applying the aqueous extract of J. regia green husk at the ambient conditions. Ultra Violet-Visible (UV-Visible) analysis revealed that the Surface Plasmon Resonance (SPR) of the CuNP was 212 nm. The average hydrodynamic and metallic core diameters of the CuNPs were about 53-28 nm, respectively. X-ray Diffraction (XRD) analysis presented that the CuNPs were amorphous. The CuNPs exhibited the highest free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging efficiency. These nanoparticles (NPs) showed antibacterial, antifungal and antibiofilm properties. They presented photocatalytic activity against Methyl Orange (MO). Besides, the potential of these NPs for the fast and precise colorimetric detection of Hg2+ was remarkable. The biosynthesized CuNPs are introduced as a multifunctional nanomaterial with various applications in medicine and environmental cases. The CuNPs were produced through an environmentally green process by the aqueous extract of dried J. regia green husk at the ambient condition. The CuNPs confirmed that this type of nanomaterial is a multifunctional agent with significant antibacterial, antifungal, antibiofilm, antioxidant, photocatalytic activities. Besides, it is a promising colorimetric sensor for the detection of Hg2+ in an aqueous complex media.

Green synthesis of AgCl/Ag3PO4 nanoparticle using cyanobacteria and assessment of its antibacterial, colorimetric detection of heavy metals and antioxidant properties.

In this study, the extract of two strains of cyanobacteria was used for the synthesis of silver nanoparticles (NPs). UV-vis spectroscopy, X-ray diffraction, dynamic light scattering and field emission scanning electron microscopy (FESEM) analyses were carried out to characterise the NPs. The antioxidant activity and heavy metal detection properties were investigated; moreover, their minimum inhibitory concentration and minimum bactericidal concentration against the multi-drug resistant bacteria were determined. The most abundant materials in these extracts were carbohydrates, so the biosynthesis of NPs using exopolysaccharide (EPS) was also investigated. The surface plasmon resonance of NPs had a peak at 435 nm and EPS NPs at 350-450 nm. The NPs produced by Nostoc sp. IBRC-M5064 extract revealed the face-centred cubic (fcc) structure of AgCl, while NPs of N. pruniforme showed the fcc crystalline structure of Ag3PO4 and AgCl. The FESEM showed the spherical shape of these NPs. The AgCl/Ag3PO4 colloid, in comparison with AgCl, showed better antioxidant activity and antibacterial effect. The heavy metal detection analysis of NPs revealed that the NPs of both stains involved in Hg (NO3)2 detection.

Biochemical and computational study of an alginate lyase produced by Pseudomonas aeruginosa strain S21.

OBJECTIVES: Alginates play a key role in mucoid Pseudomonas aeruginosa colonization, biofilm formation, and driving out of cationic antibiotics. P. aeruginosa alginate lyase (AlgL) is a periplasmic enzyme that is necessary for alginate synthesis and secretion. It also has a role in depolymerization of alginates. Using AlgLs in cystic fibrosis patients along with antibiotics enhances bacterial killing and host healing. In this study, we investigated the different biochemical properties of a newly isolated AlgL from P. aeruginosa S21 to complete the databank of AlgLs. MATERIALS AND METHODS: The enzyme was extracted from the periplasmic space of the bacteria by the heat shock method. Using the TBA method, the enzyme activity and biochemical properties were assessed. The mutability of P. aeruginosa S21 AlgL to increase its thermal stability was investigated. The most favorable mutations were studied computationally. The molecular dynamics simulation (MDS) package GROMACS was used for determining the effect of S34R mutation on enzyme's thermal stability. RESULTS: Data showed that this enzyme has the best activity at 37 °C and pH 7.5 and it can degrade mannuronate blocks, guluronate blocks, and sodium alginate. After 7 hr at 80 °C, 45% of the enzyme activity was retained. This enzyme needed 15 min to completely degrade accessible sodium alginate. Tris buffer, pH 8.5 and Britton-Robinson buffer, pH 7.0 were the preferable buffers for the enzyme activity. MDS of native and mutated enzymes showed desirable results. CONCLUSION: P. aeruginosa S21 AlgL can be used in medical and industrial applications to degrade alginates.

Protective effect of bleomycin on 5-azacitidine induced cytotoxicity and apoptosis in mice hematopoietic stem cells via Bcl-2/Bax and HMGB1 signaling pathway.

Antineoplastic drugs cause severe cytotoxicity for normal cells, especially hematopoietic stem cells (HSCs). However, bleomycin (BLM) is glycopeptide antibiotic that is effective on various cancers and has either low or no myelosuppression effects. The aim of the present study was to investigate the effect of BLM on 5-Azacitidine (5-AZA) induced cytotoxicity in bone marrow HSCs. 5-AZA reduced HSC cell viability in a time and dose-dependent manner with an IC50 value of 16 µM. However, pretreatment of the cells with BLM for 4 h induced an antagonistic cytotoxicity with an increased IC50 of 64 µM. 5-AZA decreased the colony formation ability of HSC cells in semi-solid agar culture and this effect was attenuated by BLM. 5-AZA significantly downregulated high mobility group Box1 (HMGB1) and Bcl-2 gene expression but upregulated Bax gene expression, while BLM impeded the action of 5-AZA. Pretreatment with BLM remarkably decreased HMGB1 release into culture media that was induced by 5-AZA. The cells were distribution at the sub/G1 phase. Annexin/PI staining of the cells, poly (ADP-ribose) polymerase (PARP) cleavage, and anion superoxide production indicated that BLM limited 5-AZA induced apoptotic cell death. In conclusion, BLM in combination with 5-AZA effectively reduces the adverse cytotoxic effects of 5-AZA on bone marrow hematopoietic stem cells, providing a new chemotherapeutic strategy.

Microencapsulation of Omeprazole by Lactobacillus acidophilus ATCC 4356 Surface Layer Protein and Evaluation of its Stability in Acidic Condition.

The present study introduces a novel method for encapsulation of the acid-labile drug called Omeprazole using Lactobacillus acidophilus (L. acidophilus) ATCC 4356 S-layer protein. Before preparing the Omeprazole suspension, a series of preliminary studies were performed on the Omeprazole powder. For this purpose, some parameters such as melting point, IR spectrum, UV spectrum, and the particle size of Omeprazole powder were investigated. The size reduction process was done in order to achieve an ideal formulation. Ultimately, the resulting powder had an average particle size of 35.516 µm and it was almost uniform. After calculating the amount of S-layer protein required for complete covering of drug particles, the effect of different factors on the drug coating process was investigated with one factor at a time method. Then stability of coated Omeprazole was evaluated in acetate buffer (pH 5). Finally, the maximum coat of drug particles was determined using S- layer protein of Lactobacillus acidophilus ATCC 4356 at 25 °C for 2 h, shaking rate of 100 rpm and ratio of 2:1 for S-layer protein amount/Omeprazole Surface in Tris hydrochloride buffer medium (50 mM, pH 8). The coating of Omeprazole by the S-layer protein decreased the drug decomposition rate up to 2.223.

Antifungal, antibacterial, antibiofilm and colorimetric sensing of toxic metals activities of eco friendly, economical synthesized Ag/AgCl nanoparticles using Malva Sylvestris leaf extracts.

Silver nanoparticles, one of the most popular nanomaterials, are used extensively in medicine and industries. The present study biosynthesized spherical Ag/AgCl nanoparticles with a size range of 10-50 nm in less than 5 min. The synthesis was performed in a single step, in a low-cost and eco-friendly manner, from the aqueous extract of Malva Sylvestris leaves. The aqueous extract had a large number of phenolic compounds and carbohydrates as reducing and capping agents. The nanoparticles also showed significant antibacterial and anti-biofilm activities against some multi drug resistant bacteria. They additionally showed antifungal activities on several Candida species. The highest concentration of Ag/AgCl-NPs (62.5 µg/ml) was required in order to inhibit P. aeruginosa B 52, C. glabrata and C. parapsilosis growth. The lowest concentration of Ag/AgCl-NPs (7.8125 µg/ml) inhibited the growth of C. orthopsilosis, P. aeruginosa ATCC 27853 and B. subtilis ATCC 6633. A total of 125 µg/ml of Ag/AgCl-NPs was used to prevent P. aeruginosa B 52 biofilm growth. The concentration of 62.5 µg/ml Ag/AgCl-NPs also eradicated both P. aeruginosa 48 and P. aeruginosa B 52 biofilms. The results showed that Hg2+ and Pb2+ contaminants in water could be colorimetrically detected by these nanoparticles.

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.

Biosynthesised AgCl NPs using Bacillus sp. 1/11 and evaluation of their cytotoxic activity and antibacterial and antibiofilm effects on multi-drug resistant bacteria.

Silver nanoparticles (AgNPs) have attracted the attention of researchers due to their properties. Biological synthesis of AgNPs is eco-friendly and cost-effective preferred to physical and chemical methods, which utilize environmentally harmful agents and large amounts of energy. Microorganisms have been explored as potential biofactories to synthesize AgNPs. Bacterial NP synthesis is affected by Ag salt concentration, pH, temperature and bacterial species. In this study, Bacillus spp., isolated from soil, were screened for AgNP synthesis at pH 12 with 5 mM Ag nitrate (AgNO3) final concentration at room temperature. The isolate with fastest color change and the best ultraviolet-visible spectrum in width and height were chosen as premier one. AgNO3 and citrate salts were compared in terms of their influence on NP synthesis. Spherical Ag chloride (AgCl) NPs with a size range of 35-40 nm were synthesized in 1.5 mM Ag citrate solution. Fourier transform infrared analysis demonstrated that protein and carbohydrates were capping agents for NPs. In this study, antimicrobial and antitumor properties of the AgNP were investigated. The resulting AgCl NPs had bacteriostatic activity against four standard spp. And multi-drug resistant strain of Pseudomonas aeruginosa. These NPs are also cytotoxic to cancer cell lines MCF-7, U87MG and T293.

Lithium chloride attenuates mitomycin C induced necrotic cell death in MDA-MB-231 breast cancer cells via HMGB1 and Bax signaling.

The clinical use of potent anticancer drug mitomycin C (MMC) has limited due to side effects and resistance of cancer cells. The aim of this study was to investigate whether lithium chloride (LiCl), as a mood stabilizer, can affect the sensitivity of MDA-MB-231 breast cancer cells to mitomycin C. The cells were exposed to various concentrations of mitomycin C alone and combined with LiCl and the viability determined by trypan blue and MTT assays. Proteins were analyzed by western blot and mRNA expression of HMGB1 MMP9 and Bcl-2 were analyzed by RT-PCR. Flow cytometry was used to determine the cell cycle arrest and percent of apoptotic and necrotic cells. Concentration of Bax assessed by ELISA. Exposure of the cells to mitomycin C revealed IC50 value of 20 µM, whereas pretreatment of the cells with LiCl induced synergistic cytotoxicity and IC50 value declined to 5 µM. LiCl combined with mitomycin C significantly down-regulated HMGB1, MMP9 and Bcl-2 gene expression but significantly increased the level of Bax protein. In addition, the content of HMGB1 in the nuclei decreased and pretreatment with LiCl reduced the content of HMGB1 release induced by MMC. LiCl increased mitomycin C-induced cell shrinkage and PARP fragmentation suggesting induction of apoptosis in these cells. LiCl prevented mitomycin C-induced necrosis and changed the cell death arrest at G2/M-phase. Taking all together, it is suggested that LiCl efficiently enhances mitomycin C-induced apoptosis and HMGB1, Bax and Bcl-2 expression may play a major role in this process, the findings that provide a new therapeutic strategy for LiCl in combination with mitomycin C.

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.

One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm.

OBJECTIVES: Pseudomonas aeruginosa is a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl) is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. MATERIALS AND METHODS: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. RESULTS: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. CONCLUSION: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

Production of Xanthanases by Paenibacillus spp.: Complete Xanthan Degradation and Possible Applications.

Background: A number of microorganisms and their enzymes have been reported as xanthan depolymerizers. Paenibacillus species are well-known polysaccharide hydrolyzing bacteria. However, Paenibacillus alginolyticus and Paenibacillus sp. XD are the only species in the genus which are now known to degrade xanthan. Objectives: Complete biodegradation of the xanthan exopolysaccharide is a rarely found capability among microorganisms. The aim of this study is to survey xanthanase producing bacteria with an appropriate bioactivity for the biopolymer degradation under different environmental conditions. Materials and Methods: The bacteria were isolated based on viscosity reduction of the xanthan solution. Bacterial isolates were identified using rep-PCR (repetitive element-based genomic fingerprinting) and 16S rDNA sequencing. Xanthanases were identified using rep-PCR (repetitive element-based genomic fingerprinting) and 16S rDNA sequencing. Xanthanases were characterized by measuring their activity at different temperatures, pH values, and NaCl concentrations. Degradation of other polysaccharides and xanthan degradation products were investigated based on the screening plate method and TLC (thin-layer chromatography), respectively. Results:Six isolates from different Paenibacillus species with a complete xanthan degrading capability were isolated from Urmia Lake. Phylogenetic analysis placed these strains within the genus Paenibacillus with the closest relatives that were found to be P. nanensis, P. phyllosphaerae, P. agaridevorans, P. agarexedens, and P. taohuashanense. These isolates displayed different levels of the xanthan biodegradation activity in temperatures ranging from 15 to 55°C and pH values from 4 to 11. Xanthanolytic activity was generally prevented in presence of NaCl (> 0.1 mol.L-1). Furthermore, the isolated Paenibacillus spp. could degrade several other polysaccharides including xylan, CMC (carboxymethyl cellulose), starch, alginate, and pectin. Conclusion: Novel strains of the six different Paenibacillus species that were introduced in the present study are able to produce xanthanases with interesting characteristics. In light of the results from this study, special applications, particularly in healthcare, medicine, and the environment is hereby proposed for these enzymes.

Antagonistic activities of some probiotic lactobacilli culture supernatant on Serratia marcescens swarming motility and antibiotic resistance.

BACKGROUND AND OBJECTIVES: Serratia marcescens, a potentially pathogenic bacterium, benefits from its swarming motility and resistance to antibiotic as two important virulence factors. Inappropriate use of antibiotics often results in drug resistance phenomenon in bacterial population. Use of probiotic bacteria has been recommended as partial replacement. In this study, we investigated the effects of some lactobacilli culture supernatant on swarming, motility and antibiotic resistance of S. marcescens. MATERIALS AND METHODS: Antimicrobial activity of lactobacilli supernatant and susceptibility testing carried out on S. marcescens isolates. Pretreatment effect of lactobacilli culture supernatant on antibiotic - resistance pattern in S. marcescens was determined by comparison of the MIC of bacteria before and after the treatment. RESULTS: Our results showed that pretreatment with L. acidophilus ATCC 4356 supernatant can affect the resistance of Serratia strains against ceftriaxone, but it had no effect on the resistance to other antibiotics. Furthermore, culture supernatant of lactobacilli with concentrations greater than 2%, had an effect on the swarming ability of S. marcescens ATCC 13880 and inhibited it. CONCLUSION: Probiotic bacteria and their metabolites have the ability to inhibit virulence factors such as antibiotic resistance and swarming motility and can be used as alternatives to antibiotics.

Characteristics of surface layer proteins from two new and native strains of Lactobacillus brevis.

In this work, some important characteristics of surface layer (S-layer) proteins extracted from two new and native Lactobacillus strains, L.brevis KM3 and L.brevis KM7, were investigated. The presence of S-layer on the external surface of L.brevis KM3 was displayed by thin sectioning and negative staining. SDS-PAGE analysis were shown same dominant protein bands approximately around 48kDa for both S-layer proteins. Moreover, the S-layer reappeared when LiCl treated cells were allowed to grow again. Protein secondary structure and thermal behavior were evaluated by using circular dichroism (CD) and differential scanning calorimetry (DSC), respectively. Both S-layer proteins had high content of ß-sheet and low amount of α-helix. The thermograms of lyophilized S-layer proteins of L.brevis KM3 and L.brevis KM7 showed one transition peak at 67.9°C and 59.14°C, respectively. To determine monodispersity of extracted S-layer proteins, dynamic light scattering (DLS) was used. The results indicated that the main population of S-layer molecules in two tested lactobacillus strains were composed of monomer with an expected diameter close to 10nm. Furthermore, Zeta potential measurements were showed positive potential for both S-layer proteins, as expected. Our results could be used as the basis for biotechnological applications of these two new S-layer proteins.

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.

The inhibitory effect of bacteriocin produced by Lactobacillus acidophilusATCC 4356 and Lactobacillus plantarum ATCC 8014 on planktonic cells and biofilms of Serratia marcescens.

BACKGROUND/AIM: The spread of antibiotic-resistant pathogens has resulted in the need for new treatments. The aim of the present study is to investigate the effect of bacteriocin from Lactobacillus acidophilus ATCC 4356 and Lactobacillus plantarum ATCC 8014 on planktonic and biofilm forms of Serratia marcescens strains. MATERIALS AND METHODS: The direct antagonism of the L. plantarum and L. acidophilus cell-free supernatant on S. marcescens cultures was determined using an optical density assay. The bacteriocin was partial purified by ammonium sulfate precipitation. Its molecular weight was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The effect of bacteriocins on the biofilm of S. marcescens strains was then determined with 2,3,5-triphenyl tetrazolium chloride. RESULTS: The purified bacteriocin from L. plantarum ATCC 8014 and partially purified bacteriocin from L. acidophilus ATCC 4356 displayed noticeable inhibitory activity against planktonic and biofilm forms of S. marcescens strains. SDS-PAGE analysis revealed that the apparent molecular weight of bacteriocin from L. planetarium was 63 kDa, and that of bacteriocin from L. acidophilus was 68 or 48 kDa. CONCLUSION: The bacteriocins could be effective compounds to control surface-attached pathogenic bacteria and can be used as therapeutic agents after acceptable in vivo experimentation.

Binding of topotecan to chromatin: Insights into cooperative binding and comparison with DNA.

Topotecan (TPT) is an anticancer drug widely used in cancer therapy. Although the interaction of TPT with DNA is a subject of few reports, no work has been reported on the binding affinity of TPT to DNA-histone complex in chromatin structure. In the present study we have focused on the effect of TPT on chromatin employing various types of spectroscopy and equilibrium dialysis techniques. The results showed that TPT quenched with chromatin chromophores and decreased fluorescence emission intensity corresponding to aromatic residues of histone proteins. The UV absorbance at 260 and 210 nm in decreased in a dose dependent manner. Upon binding of the drug, ellipticity at 222 nm in the circular dichroism profile became more positive implying reduction of α-helix content of histones. The binding is positive cooperative with association constant (Ka) of 2.65×10(2) M(-1) and 1.11×10(2) M(-1) for chromatin and DNA respectively indicating higher affinity of TPT to chromatin compared to DNA. From the results it is concluded that in the cell nucleus, TPT, as a potent anticancer drug, exerts its biological action through binding to chromatin and in this process not only DNA but also histone proteins play a fundamental role.