Characterization of inulolytic enzymes from the Jerusalem artichoke-derived Glutamicibacter mishrai NJAU-1.

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of ß (2-1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU's protein structure is then suggested. KEY POINTS: • A Glutamicibacter mishrai strain harbored exo-inulinase activity. • Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C. • Two exo-inulinases with differential substrate specificity were characterized.

Development and characterization of vancomycin-loaded levan-based microparticular system for drug delivery.

Encapsulation of vancomycin (VANCO) into biodegradable levan microparticles was achieved using a simple preparation technique. Microparticles were prepared by using levan polysaccharide produced by a halophilic bacterium Halomonas smyrnensis AAD6T. To optimize efficiency of encapsulation process by precipitation method, three parameters were studied: drug and polymer concentrations and preparation rotating speed. The particles were characterized in vitro. The size of levan microparticles was changed between 0.404 µm and 1.276 µm. The surface charge was detected between +4.1 mV and +6.5 mV. The highest drug encapsulation capacity of the system was 74.7% and was depending on the polymer concentration. In dissolution studies, initial burst effect around 10-20% from all the formulations was observed and then the release was slowed down and continued at a constant level. In vitro antibiotic release from the microparticles was controlled with the drug carrier system and release fit to Higuchi kinetic model. All the released samples collected at different time intervals during dissolution studies have exhibited intrinsic bactericidal activity against Bacillus subtilis ATCC 6633. WST-1 cell proliferation and viability studies showed that VANCO-loaded levan microparticles at concentrations between 100 µg/mL and 1000 µg/mL were nontoxic to L929 cells. As conclusion, levan microparticulate system could be a potential carrier of antibiotic drugs such as VANCO.

Functionally stable plasminogen activator inhibitor-1 in a family with cardiovascular disease and vitiligo.

Vitiligo is a common skin condition with a complex pathophysiology characterized by the lack of pigmentation due to melanocyte degeneration. In this study, we investigated PAI-1 antigen (Ag) and activity levels in a 34 year old male with extensive vascular disease, alopecia areata and vitiligo. Fasting PAI-1 Ag and activity levels were measured at 9 a.m. in the subject and family members. Both PAI-1 Ag (67 ± 38 vs. 18.6 ± 6.5 ng/ml, P < 0.001) and specific activity (15.8 ± 10.0 vs. 7.6 ± 6.0 IU/pmol, P < 0.04) levels of PAI-1 were moderately elevated in subjects compared to the controls. PAI-1 kinetic studies demonstrated a markedly enhanced stability of plasma PAI-1 activity in the family members. Specific activity at 16 h was significantly higher than expected activity levels (0.078 ± 0.072 vs. 0.001 ± 0.001 IU/ng/ml, P < 0.001). While the exact mechanism of increased stability of PAI-1 activity in vitiligo is not known, it is likely due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of vascular disease and associated melanocyte degeneration. Systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative to the near orphan status for vitiligo drug development.

Understanding the effects of chitosan, chia mucilage, levan based composite coatings on the shelf life of sweet cherry.

Sweet cherry (Prunus avium L.) fruits are prone to quality and quantity loss in shelf-life conditions and cold storage due to their short post-harvest life. Until now efforts have been made to extend the shelf life of the sweet cherry. However, an efficient and commercially scalable process remains elusive. To contribute to this challenge, here in this study, biobased composite coatings consisting of chitosan, mucilage, and levan, were applied on sweet cherry fruits and tested for postharvest parameters in both market and cold storage conditions. Results demonstrated that the shelf life of sweet cherries can be extended until the 30th day while retaining important post-harvest properties like decreased weight loss, fungal deterioration, increased stem removal force, total flavonoid, l-ascorbic acid, and oxalic acid. Given the cost-effectiveness of the polymers used, the findings of this study indicate the feasibility of extending the shelf-life of sweet cherries on a larger scale.

Draft genome sequence of Halomonas smyrnensis AAD6T.

Halomonas smyrnensis AAD6(T) is a Gram-negative, aerobic, exopolysaccharide-producing, and moderately halophilic bacterium that produces levan, a fructose homopolymer with many potential uses in various industries. We report the draft genome sequence of H. smyrnensis AAD6(T), which will accelerate research on the rational design and optimization of microbial levan production.

Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation.

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.

Levan nanostructured thin films by MAPLE assembling.

Synthesis of nanostructured thin films of pure and oxidized levan exopolysaccharide by matrix-assisted pulsed laser evaporation is reported. Solutions of pure exopolysaccharides in dimethyl sulfoxide were frozen in liquid nitrogen to obtain solid cryogenic pellets that have been used as targets in pulsed laser evaporation experiments with a KrF* excimer source. The expulsed material was collected and assembled onto glass slides and Si wafers. The contact angle studies evidenced a higher hydrophilic behavior in the case of oxidized levan structures because of the presence of acidic aldehyde-hydrogen bonds of the coating formed after oxidation. The obtained films preserved the base material composition as confirmed by Fourier transform infrared spectroscopy. They were compact with high specific surface areas, as demonstrated by scanning electron and atomic force microscopy investigations. In vitro colorimetric assays revealed a high potential for cell proliferation for all coatings with certain predominance for oxidized levan.

Molasses as fermentation substrate for levan production by Halomonas sp.

Levan is a homopolymer of fructose with many outstanding properties like high solubility in oil and water, strong adhesiveness, good biocompatibility, and film-forming ability. However, its industrial use has long been hampered by costly production processes which rely on mesophilic bacteria and plants. Recently, Halomonas sp. AAD6 halophilic bacteria were found to be the only extremophilic species producing levan at high titers in semi-chemical medium containing sucrose, and in this study, pretreated sugar beet molasses and starch molasses were both found to be feasible substitutes for sucrose. Five different pretreatment methods and their combinations were applied to both molasses types. Biomass and levan concentrations reached by the Halomonas sp. AAD6 cells cultivated on 30 g/L of pretreated beet molasses were 6.09 g dry cells/L and 12.4 g/L, respectively. When compared with literature, Halomonas sp. was found to stand out with its exceptionally high levan production yields on available fructose. Molecular characterization and monosaccharide composition studies confirmed levan-type fructan structure of the biopolymers. Rheological properties under different conditions pointed to the typical characteristics of low viscosity and pseudoplastic behaviors of the levan polymers. Moreover, levan polymer produced from molasses showed high biocompatibility and affinity with both cancerous and non-cancerous cell lines.

Optimization of ethanol production using newly isolated ethanologenic yeasts.

Yeasts are important microorganisms used for ethanol production; however, they are not equally efficient in the amount of ethanol production under different environmental conditions. It is, therefore, necessary to screen for elite strains to utilize them for commercial production of these commodities. In this study, yeasts were isolated from different Ethiopian traditional fermented alcoholic beverages (teji, tella, shamiata and areqe tinisis), milk and ergo, teff and maize dough, soil and compost, flowers, and fruits to evaluate their potential use for ethanol fermentation process. Isolates were screened for efficient ethanol production and the selected ones were identified using phenotypic and genetic characters using D1/D2 region of LSU rDNA sequence analysis. The yeast isolates were evaluated based on their growth and fermentation of different carbon sources. Response surface methodology (RSM) was applied to optimize temperature, pH and incubation time using central composite design (CCD) in Design-Expert 7.0.0. A total of 211 yeasts colonies were isolated of which 60% were ethanologenic yeasts (ethanol producers) and 40% were non-ethanol producers. The yeast population detected from various sources was in the range of 10 5 CFU from traditional foods and beverages to that of 10 3 CFU from fruits and soil samples. The data also showed that the number of colony types (diversity) did not correlate with population density. The highly fermentative isolates were taxonomically characterized into four genera, of which 65% of the isolates (ETP37, ETP50; ETP53, ETP89, ETP94) were categorized under Saccharomyces cerevisiae, and the remaining were Pichia fermentans ETP22, Kluyveromyces marxianus ETP87, and Candida humilis ETP122. The S. cerevisiae isolates produced ethanol (7.6-9.0 g/L) similar with K. marxianus ETP87 producing 7.97 g/L; comparable to the ethanol produced from commercial baker's yeast (8.43 g/L) from 20 g/L dextrose; whereas C. humilis ETP122 and P. fermentans ETP22 produced 5.37 g/L and 6.43 g/L ethanol, respectively. S. cerevisiae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122 tolerated 10% extraneous ethanol but the percentage of ethanol tolerance considerably decreased upon 15%. S. cerevisiae ETP53 produced ethanol optimally at pH 5.0, 60 h, and 34 o C. pH 4.8, temperature 36 o C, and 65 h of time were optimal growth conditions of ethanol fermentation by K. marxianus ETP87. The ethanol fermentation conditions of P. fermentans ETP22 was similar to S. cerevisiae ETP53 though the ethanol titer of S. cerevisiae ETP53 was higher than P. fermentans ETP22. Therefore, S. cerevisiae ETP53, K. marxianus and P. fermentans ETP22 are good candidates for ethanol production.

Improving the Thermostability and Catalytic Activity of an Inulosucrase by Rational Engineering for the Biosynthesis of Microbial Inulin.

Thermostability and enzymatic activity are two vital indexes determining the application of an enzyme on an industrial scale. A truncated inulosucrase, Laga-ISΔ138-702, from Lactobacillus gasseri showed high catalysis activity. To further enhance its thermostability and activity, multiple sequence alignment (MSA) and rational design based on the modeled structure were performed. Variants A446E, S482A, I614M, and A627S were identified with an improved denaturation temperature (Tm) of more than 1 °C. A combinational mutation method was further carried out to explore the synergistic promotion effects of single-point mutants. Additionally, 33 residues at the N-terminus were truncated to construct mutant M4N-33. The half-life of M4N-33 at 55 °C increased by 120 times compared to that of Laga-ISΔ138-702, and the relative activity of M4N-33 increased up to 152% at the optimal pH and temperature (pH 5.5 and 60 °C). Molecular dynamics (MD) simulations illustrated the decreased b-factor of the surface loop of M4N-33.

Improving the catalytic behaviors of Lactobacillus-derived fructansucrases by truncation strategies.

Fructansucrases (FSs), including inulosucrase (IS) and levansucrase (LS), are the members of the Glycoside Hydrolase family 68 (GH68) enzymes. IS and LS catalyze the polymerization of the fructosyl moiety from sucrose to inulin- and levan-type fructans, respectively. Lactobacillus-derived FSs have relatively extended N- and C-terminal sequences. However, the functional roles of these sequences in their enzymatic properties and fructan biosynthesis remain largely unknown. Limosilactobacillus reuteri (basionym: Lactobacillus reuteri) 121 could produce both IS and LS, abbreviated as Lare121-IS and Lare121-LS, respectively. In this study, it was found that the terminal truncation displayed an obvious effect on their activities and the N-terminal truncated variants, Lare121-ISΔ177-701 and Lare121-LSΔ154-686, displayed the highest activities. Melting temperature (Tm) and the thermostability at 50 °C were measured to evaluate the stability of various truncated versions, revealing the different effects of N-terminal on the stability. The average molecular weight and polymerization degree of the fructans produced by different truncated variants did not change considerably, indicating that N-terminal truncation had low influence on fructan biosynthesis. In addition, it was found that N-terminal truncation could also improve the activity of other reported FSs from Lactobacillus species.

Levan enhanced the NF-κB suppression activity of an oral nano PLGA-curcumin formulation in breast cancer treatment.

Chemoresistance (CR) is one of the reasons why chemotherapy agents like Gemcitabine (GMC) remain insufficient in healing breast cancer. Activation of Nuclear Factor-kappa B (NF-κB) during chemotherapy is known as an important factor in the development of CR. The hydrophobic polyphenol curcumin is shown to inhibit NF-κB and hence CR. The aim of this work was to increase the poor bioavailability of curcumin by loading it into the nano-micelles made of Poly (Lactide-co-Glycolide) (PLGA) and levan, where levan as a natural fructose homopolymer makes the nano-micelle more stable and increases its uptake using the fructose moieties. In this study, a PLGA-levan-curcumin formulation (PLC) was designed and characterized. The size was measured as 154.16 ± 1.45 nm with a 67.68% encapsulation efficiency (EE%). The incorporation between the components was approved. Levan made the nano-micelles stable for at least three months, increased their uptake, and led to a 10,000-fold increase in the solubility of curcumin. The enhanced bioavailability of curcumin reduced the NF-κB levels elevated by GMC, both in vitro and in vivo. The PLC showed a complete tumor treatment, while GMC only showed a rate of 52%. These point to the great potential of the PLC to be used simultaneously with chemotherapy.

Exopolysaccharides from extremophiles: from fundamentals to biotechnology.

Exopolysaccharides (EPSs) make up a substantial component of the extracellular polymers surrounding most microbial cells in extreme environments like Antarctic ecosystems, saline lakes, geothermal springs or deep sea hydrothermal vents. The extremophiles have developed various adaptations, enabling them to compensate for the deleterious effects of extreme conditions, e.g. high temperatures, salt, low pH or temperature, high radiation. Among these adaptation strategies, EPS biosynthesis is one of the most common protective mechanisms. The unusual metabolic pathways revealed in some extremophiles raised interest in extremophilic microorganisms as potential producers of EPSs with novel and unusual characteristics and functional activities under extreme conditions. Even though the accumulated knowledge on the structural and theological properties of EPSs from extremophiles is still very limited, it reveals a variety in properties, which may not be found in more traditional polymers. Both extremophilic microorganisms and their EPSs suggest several biotechnological advantages, like short fermentation processes for thermophiles and easily formed and stable emulsions of EPSs from psychrophiles. Unlike mesophilic producers of EPSs, many of them being pathogenic, extremophilic microorganisms provide non-pathogenic products, appropriate for applications in the food, pharmaceutical and cosmetics industries as emulsifiers, stabilizers, gel agents, coagulants, thickeners and suspending agents. The commercial value of EPSs synthesized by microorganisms from extreme habitats has been established recently.

Improved Exopolymer Production by Chromohalobacter canadensis Cultures for Its Potential Cosmeceutical Applications.

Several exopolymers with different chemical composition and correspondingly variety in their physico-chemical properties from halophilic microorganisms have still been described, however, with a low production yield. Chromohalobacter canadensis 28 isolated from Pomorie saltern synthesized an unusual exopolymer (EP) containing 72% γ-polyglutamic acid (PGA), an essential cosmeceutical additive. Current work suggests a novel approach for effective EP synthesis by C. canadensis 28 using continuous cultures. Highest production was observed at low dilution rates reaching a level of 2.1 mg/mL at D = 0.035, similar to those in batch cultures (2.34 mg/mL), however avoiding all disadvantages of discontinuous fermentation processes. At steady state, the total quantities of the synthesized EP after 48 h cultivation for the given equipment volume in D = 0.035 h-1 and D = 0.075 h-1 were 8.67 and 12 g, correspondingly, while it was 2.9 g for batch culture. Process parameters did not change after a ten-day run at D = 0.35 h-1. A degree of purity of EP fraction received from continuous cultures was significantly increased up to 93-96%. A lack of cytotoxicity and high cell viability were observed for human dermal fibroblast cells after 24 h incubation with crude EP from C. canadensis 28 and purified PGA fraction that could suggest its high potential for cosmetic applications.

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28.

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2-L bioreactor at agitation speeds at interval 600-1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale-up of bioreactors mass transfer parameters were estimated and lowest values of KLa obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ-polyglutamic acid (PGA) showed higher viability than control group.

Preconcentrations and determinations of copper, nickel and lead in baby food samples employing Coprinus silvaticus immobilized multi-walled carbon nanotube as solid phase sorbent.

Preconcentrations of Cu(II), Ni(II) and Pb(II) ions by using Coprinus silvaticus immobilized multiwalled carbon nanotube (MWCNT) were investigated. Effects of important parameters on preconcentration procedure were examined. The best pH values of for Cu(II), Ni(II) and Pb(II) were found to be 6.0, 6.0 and 4.0, respectively. Flow rate of sample solution was 2.0 mL min-1, while desorption was achieved at 1.0 mL min-1 flow rate. Preconcentration factors were achieved as 60 for Cu(II), Ni(II) and 70 for Pb(II) (by dividing initial sample volume to final volume). LODs were calculated as 0.014, 0.016 and 0.093 ng mL-1, respectively for Cu(II), Ni(II) and Pb(II). Accuracy of the method was checked by applying to certified reference samples. Inductively coupled plasma optical emission spectrometer (ICP OES) was employed for measurements of Cu(II), Ni(II) and Pb(II) in digested baby food samples.

Adsorptive removal of bisphenol A from aqueous solutions using phosphonated levan.

In this study, the potential use of phosphonated Halomonas Levan (PhHL) as a natural and cost effective adsorbent for Bisphenol A (BPA), was systematically investigated via the study of the adsorption equilibrium, kinetics, and reuse potential as well as the interpretation of adsorption mechanism. The effects of pH and temperature on the adsorption were also evaluated. The maximum amount of BPA adsorbed on the unit weight of PhHL was determined as 104.8 (∓5.02) mg/g (at 298 K) and the maximum adsorption capacity was calculated as 126.6 mg/g by Sips model. FTIR and XPS studies were conducted to elucidate the adsorption mechanism. Based on the obtained results OH-pi and CH-pi interactions were found to be effective in the adsorption mechanism. The reuse ability was studied with three cycles of adsorption-desorption, and the results showed that the BPA adsorbed per gram of the PhHL decreased 28.6% after the third cycle. This study has shown that PhHL can be used as an effective adsorbent for the removal of BPA from aqueous solutions. The obtained results may be useful in the development of PhHL based adsorption systems for the removal of EDCs with similar chemical properties to BPA.

Development and optimization of a novel PLGA-Levan based drug delivery system for curcumin, using a quality-by-design approach.

This study aimed to develop a PLGA, Levan-based drug delivery system (DDS) of Curcumin using a quality-by-design (QbD) approach to reveal how formulation parameters affect the critical quality attributes (CQAs) of this DDS and to present an optimal design. First, a risk assessment was conducted to determine the impact of various process parameters on the CQAs of the DDS (i.e., average particle size, ZP, encapsulation efficiency and polydispersity index). Plackett-Burman design revealed that potential risk factors were Levan molecular weight, PLGA amount and acetone amount. Then, the optimization of the DDS was achieved through a Box-Behnken Design. The optimum formulation was prepared using low molecular weight Levan (134 kDa), 51.51 mg PLGA and 10 ml acetone. The model was validated and the optimized formulation was further characterized using different physic-chemical methods. The study resulted in the most stable NP with a spherical and uniform shape and physical stability tests indicated its stability for at least 60 days at room temperature. In conclusion, this study was an effort for developing a DDS which solubilizes Curcumin in clinically applicable concentrations.

Encapsulated melatonin in polycaprolactone (PCL) microparticles as a promising graft material.

Electrospraying assures many advantages with taking less time and costing less relatively to the other conventional particle production methods. In this research, we investigated the encapsulation of melatonin (MEL) hormone in polycaprolactone (PCL) microparticles by using electrospraying method. Morphology analysis of the produced particles completed with Scanning Electron Microscopy (SEM). SEM images demonstrated that micro-particles of 3 wt% PCL solution has the most suitable particle diameter size (2.3 ±â€¯0.64 µm) for melatonin encapsulation. According to the characterization of the particles, electrospraying parameters like optimal collecting distance, the flow rate of the solution and voltage of the system detected as 8 cm, 0.5 ml/h, and 10 kV respectively. For determining the chemical bonds of scaffold Fourier-Transform Infrared Spectroscopy (FTIR) were used and FTIR results showed that melatonin successfully loaded into PCL micro-particles. Drug release kinetics of the melatonin loaded particles indicated that melatonin released with a burst at the beginning and release behavior became sustainable over a period of 8 h with the encapsulation efficiency of about 73%. In addition, both in-vitro and in-vivo studies of the graft materials also completed. Primary human osteoblasts (HOB) cells and female Sprague Dawley rats were used in in-vitro and in-vivo studies. Test results demonstrate cell population, and bone volume of the rats grafted with composites has remarkably increased, this caused remodelling in bone structure. Overall, these findings indicate that encapsulation of melatonin in the PCL particles with electrospray method is optimum for new synthetic graft material.

From healing wounds to resorbable electronics, levan can fill bioadhesive roles in scores of markets.

Levan is a fructose homopolysaccharide which gained attention recently for its unusual combination of properties distinguishing it from other natural biodegradable polysaccharides like chitosan, cellulose or starch. Among the strongest bioadhesives, film-forming levan is garnering interest for its role in some simple solutions to difficult problems. One of these is illustrated by the elegant research using laser-based techniques to construct levan films for healing wounds and burned tissue. Another is the development of bioresorbable electronic implants. Levan has been found in habitats as diverse as salterns and thermal waters to tropical plants and sugar factories. This review of the low viscosity, levan adhesive describes the mechanisms by which it forms bonds and the reasons behind some of its practical and industrial applications. Here we present descriptions from the literature for feasible approaches ready to transition from the laboratory to those searching for answers in fields as varied as medicine, packaging and furniture assembly.