In the present investigation, we aimed to study biological petroleum wastewater treatment. A bioprocess based on the use of a mixed bacterial consortium along with the addition of Biosurfactant (BioS) and bioaugmentation with BioS producing strains were evaluated. The consortium was the combination of two bacterial strains with the capacity to metabolize hydrocarbons, Baci and B201. Two other strains BH1 and BSS2 selected for their ability to degrade hydrocarbons as well as to produce BioS were also exploited. The effect of ex-situ and in-situ produced BioS on hydrocarbon biodegradation were compared. Obtained results demonstrated a moderate capacity to remove crude oil from contaminated water of about 47% for the consortium. The best improvement of hydrocarbon biodegradation of about 84% was obtained when adding BH1 derived BioS solely. However, ex-situ produced BioS exhibited better results. Regarding the wastewater characteristics, we note a decrease of the COD/BOD5 Ratio to reach 2.64. Thus, the wastewater became easily biodegradable. The described bioprocess is of great interest for the bioremediation of petroleum effluents. BioS addition as well as bioaugmentation enhanced crude oil removal.

The study of amino acid interactions in the active site of scorpion venom phospholipases A2 could help to gain insights into the structure–function relationship and the biological activities of the enzyme. In the secreted phospholipase A2 of Scorpio maurus venom glands, Glutamate 63 and Tyrosine 122 amino acids play critical roles in the catalytic mechanism through interactions with residues around the calcium-binding loop. We constructed mutants at these positions by overexpression in Escherichia coli cells. After refolding and purification of recombinant enzymes, we studied their kinetic properties using pH-stat and monolayer techniques. The mutant Glutamate 63–Aspartate (E63D) exhibited a reduced activity, while the second mutant Tyrosine 122–Arginine (Y122R) retained some activity with a 14-fold reduction in catalytic efficiency. However, both mutants remained stable in pH values ranging from 2 to 12 whereas the double mutant D63–R122 was catalytically inactive. Comparative analysis of wild-type and mutant 3-D models showed various modifications of the hydrogen-binding network linking residues Glutamate 63 and Tyrosine 122. These modifications of interactions could explain the reduction in enzymatic activity. The kinetic behavior on phosphatidylcholine and phosphatidylethanolamine monolayers of three mutants was evaluated using a baro-stat system to assess the potential association between the hydrolysis of erythrocyte membrane phospholipids and the enzyme’s capability to penetrate phospholipid monolayers at high surface pressure. Mutants’ kinetic behaviors were similar to the wild-type form with slightly modified specific activities at high surface pressure. All mutants were more active on phosphatidylethanolamine than phosphatidylcholine films at high surface pressure. This study provided new information to further elucidate structure–function relationships of scorpion venom-secreted phospholipases A2 and the design of novel potent drug molecules.

Traditional wastewater treatments involve expensive mechanical and physiochemical methods, so researchers have been developing cost-effective, sustainable technologies that use enzymes to produce higher quality effluents and recover more energy and nutrients from wastewater. A thermostable, alkaline, and solvent-tolerant lipase was partially purified from thermophilic Bacillus stearothermophilus. The lipase displayed maximum activity at 50 °C and pH 11.0 and catalyzed both short- and long-chain triacylglycerols at similar rates. B. stearothermophilus lipase also exhibited high stability when incubated at 40 °C for 1 h with anionic and non-ionic surfactants. Studies show that thermostable enzymes can be improved through immobilization and modification of other reaction conditions. Therefore, B. stearothermophilus lipase was immobilized through adsorption on CaCO3, Celite 545, and silica gel with the CaCO3 support producing the best adsorption rate (89.33%). The optimal initial lipase activity was approximately 4500 U.g−1 after 60 min. Interestingly, 93% of the initial lipase activity was retained after six cycles, and almost 50% of the initial activity remained after 12 cycles. Furthermore, immobilization improved storage stability with 98.85% of the initial lipase activity retained after 60 days of storage at 4 °C. The biochemical characteristics of immobilized lipase shifted toward a slightly alkaline region, reaching maximum activity at pH 12. The optimal temperature of immobilized lipase was 60 °C. Immobilization also improved enzymatic stability by widening the pH range from 5–9 (for free lipase) to 4–11, and thermostability by reaching 65 °C. The application of immobilized lipase in wastewater treatment was observed through oil layer biodegradation. Notably, treating wastewater for 10 days with immobilized lipase almost removed the chemical oxygen demand (COD) from 1950.1 down to 4.04 mg.L−1. Similarly, lipid content was almost removed from 15,500 ± 546 mg.L−1 down to 12 mg.L−1. All results highlight the potential value of CaCO3-immobilized lipase as an effective biocatalyst for hydrolyzing wastewater.

Secretory group V phospholipase A2 (PLA2-V) is known to be involved in inflammatory processes in cellular studies, nevertheless, the biochemical and the enzymatic characteristics of this important enzyme have been unclear yet. We reported, as a first step towards understanding the biochemical properties, catalytic characteristics, antimicrobial and cytotoxic effects of this PLA2, the production of PLA2-V from dromedary. The obtained DrPLA2-V has an absolute requirement for Ca2+ and NaTDC for enzymatic activity with an optimum pH of 9 and temperature of 45 °C with phosphatidylethanolamine as a substrate. Kinetic parameters showed that Kcat/Kmapp is 2.6 ± 0.02 mM−1 s−1. The enzyme was found to display potent Gram-positive bactericidal activity (with IC50 values of about 5 µg/mL) and antifungal activity (with IC50 values of about 25 µg/mL)in vitro. However, the purified enzyme did not display a cytotoxic effect against cancer cells.

Industrial processes have expanded with the ability to clone and express recombinant immobilized enzymes in microorganisms such as Pichia pastoris that have commercially attractive amounts of the appropriate genes. This report describes the overexpression in Pichia pastoris, immobilization, and functional characterization of a secreted phospholipase A2 from scorpion venom Scorpio maurus: rPLA2(-5). After 48 h of culture, the recombinant rPLA2(-5) was secreted into the culture medium and expressed at about 9 mg/L. Comparative analyses of the kinetics and hydrolysis of rPLA2(-5) monolayers at various surface pressures were conducted with the same form produced in Escherichia coli. As a second part of the study, rPLA2(-5) overexpressed in Pichia pastoris was immobilized by adsorption on CaCO3, with about 78 percent of the activity. In comparison to the free enzyme, rPLA2(-5) was studied for stability. Immobilization improved the thermal stability of rPLA2(-5) and even the stability at acidic pH. Moreover, we found that the immobilization improved the stability of rPLA2(-5) towards bile salts, Tween 80, Triton X-100, and SDS, as well as its stability towards many organic solvents. Until now, this is the first study to describe the overexpression and immobilization of a scorpion venom phospholipase A2 that possesses an interesting stability characteristic that makes it useful for a wide range of biotechnological applications.

The main objective of the current study was the extraction, purification, and enzymatic characterization of a potent proteinaceous amylase inhibitor from Moringa oleifera. The antimicrobial potential and insecticide effects against C. maculates insect larvae were also studied. The α-amylase inhibitor was extracted in methanol (with an inhibitory activity of 65.6% ± 4.93). Afterwards, the inhibitor αAI.Mol was purified after a heat treatment at 70 °C for 15 min followed by one chromatographic step of Sephadex G-50. An apparent molecular weight of 25 kDa was analyzed, and the N-terminal sequence showed the highest identity level (89%) with the monomeric α-amylase inhibitor from Triticum dicoccoides. αAI.Mol was found to tolerate pH values ranging from 5.0 to 11.0 and showed maximal activity at pH 9.0. Thermal stability was remarkably important, since the inhibitory activity was maintained at 55% after 1 h of incubation at 70 °C and at 53% after an incubation of 45 min at 80 °C. The potency of the current purified inhibitor against amylases from different origins indicates that αAI.Mol seems to possess the highest affinity toward human salivary α-amylase (90% inhibitory activity), followed by the α-amylase of insects Callosobruchus maculatus and Tribolium confusum (71% and 61%, respectively). The kinetic parameters were also calculated, and the Kmax and Vmax of the digestive amylase were estimated at 185 (mmol/min/mg) and 0.13 mM, respectively. The inhibitor possesses a strong bactericidal effect against Gram+ and Gram- strains, and the MIC values were >1 against B. cereus but >6 against E. coli. Interestingly, the rates of survival and pupation of C. maculates insect larvae were remarkably affected by the purified αAI.Mol from Moringa oleifera.

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/β hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

Amylases are enzymes required for starch degradation and are naturally produced by many microorganisms. These enzymes are used in several fields such as food processing, beverage, and medicine as well as in the formulation of enzymatic detergents proving their significance in modern biotechnology. In this study, a three-stage growth mode was applied to enhance starch production and amylase detection from Chlorella vulgaris. Stress conditions applied in the second stage of cultivation led to an accumulation of proteins (75% DW) and starch (21% DW) and a decrease in biomass. Amylase activities were detected and they showed high production levels especially on day 3 (35 U/ml) and day 5 (22.5 U/ml) of the second and third stages, respectively. The bioinformatic tools used to seek amylase protein sequences from TSA database of C. vulgaris revealed 7 putative genes encoding for 4 α-amylases, 2 β-amylases, and 1 isoamylase. An in silico investigation showed that these proteins are different in their lengths as well as in their cellular localizations and oligomeric states though they share common features like CSRs of GH13 family or active site of GH14 family. In brief, this study allowed for the production and in silico characterization of amylases from C. vulgaris.

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

We aimed in the current study, the identification of a marine bacterial amylase produced by Bacillus pacificus, which was associated with Turbinaria ornata. Cultural conditions were optimized for the highest amylase production on Tryptic soy broth media supplemented with starch 1% at initial pH 9, 55 °C for 24 h. The newly purified amylase was characterized for a possible biotechnological application. Data indicated that the obtained amylase with a molecular weight of 40 kD and the N-terminal sequence of the first 30 amino acids of amBp showed a high degree of homology with known alpha amylase, and was stable at 60 °C of pH 11. Among the tested substrate analogs, amBp was almost fully active on Alylose and Alylopectine (97%), but moderately hydrolyzed glycogen < sucrose < maltose < lactose. Therefore, the current amylase mainly generated maltohexaose from starch. Mg2+ and Zn2+ improved amylase activity up to 170%. While ethylenediamine tetraacetic acid (EDTA) similarly induced the greatest activity with purified amylase, PCMB had the least effect. Regarding all these characteristics, amylase from marine bacterial symbionts amBp has a new promising feature for probable therapeutic, industrial, and nutritional applications.

Avian infectious bronchitis, associated coronavirus causes inflammation and damage to the lungs. To evaluate the molecular mechanisms behind this event, we investigated variations in the expression level of the proinflammatory factors, cyclo-oxygenase 1 (COX-1) and cyclo-oxygenases 2 (COX-2). In mammals, the increased generation of prostaglandins during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of COX-2. The recent sequencing of chicken genome and the identification of genes coding COX-1 and COX-2 allowed us to analyze their expression in lung inflammatory disease. The purpose of this study was to examine the relative expression and distribution of COX-1 and COX-2 in organs of normal hens and in hens with avian infection bronchitis. The expression levels of cyclo-oxygenase genes in several organs from uninfected chickens and infected ones were measured using a quantitative reverse transcription-PCR technique. Our results show that the expression level of COX-1 was highly up regulated in spleen, small intestine, lungs, and liver, but modestly up regulated in the pancreas. However, no significant change was observed in the expression levels of COX-2, in the small intestine, liver and spleen; suggesting a constitutive expression of this enzyme. Whereas, COX-2 mRNA expression was significantly decreased in the heart and lungs of infected chickens, ranging from 9-fold to 4-fold, respectively (p<0.05). Contrary to mammals, COX-2 is present constitutively in bird’s cells, while COX-1 is expressed primarily after inflammation.

The selection of suitable natural raw materials in the cosmetic research and development is a key point, in order not only to obtain the expected results but also to avoid undesirable side effects. In this study, spirulina platensis, pomegranate (Punica granatum) peel, and moringa leaves alone were evaluated for anti-oxidant and antimicrobial properties. The chemical composition (moisture, dry matter, protein, lipid, and ash) and total polyphenols, flavonoids, and carotenoids content were evaluated in the three extracts. Total antioxidant capacity and ferric reducing activity power of extracts were also studied. Using agar diffusion method, the anti-Micrococcus luteus, Staphylococcus aureus, E. coli, Listeria monocytogenes, Salmonella typhimurium, and Enterococus faecalis activities were measured. Interestingly, after combinations, pomegranate peel/spirulina (A), and moringa/spirulina (B): 25%/75% and 50%/50%, we have found that pomegranate peel can be incorporated into cosmetic formulations as an excellent preservative due to its exceptionally amount of phenolic compounds, powerful antioxidant activity, and its antibacterial activity against pathogenic strains.

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyzadioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6–7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl2 and sorbitol, whereas the presence of Ca2+ and Zn2+ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.

The kinetic aspects of lipolysis by pulmonary phospholipase A2 (ChPLA2-V), chicken intestinal phospholipase A2 (ChPLA2-IIA) and chicken pancreatic phospholipase A2 (ChPLA2-IB), from chicken have been compared using the monomolecular films technique, on short-chain phospholipids (with three different head groups) and on long-chain phospholipids. The main conclusions from our experimental data indicate that the maximum catalytic activities of ChPLA2-V on 1,2 phosphatidylcholine and 1,2 phosphatidylethanolamine reached 15.26 and 36.12 moles/cm2.min.mM, respectively, at a pressure of 15 and 35 dynes/cm, respectively. Whereas, those of ChPLA2-IB were 3.58 (at the pressure of 20 dynes/cm) and 4.9 moles/cm2.min.mM. However, hydrolysis of phosphatidylglycerol monolayers (C12PG), were very much higher compared with all the substrates tested with 122 moles/cm2.min. Surprisingly, the hydrolysis rate of ChPLA2-V on long-chain phosphatidylglycerol (C18PG) was very low (1.45 moles/cm2.min) compared with all tested substrates, even with the use of p-cyclodextrin. And thus, the fatty acid preference of ChPLA2-V was 2-decanoyl > 2-oleoyl with a PG head group. In order to gain significant correlations between enzyme's structures and their relative functions, we tried to examine the surface electrostatic potentials of the various secreted phospholipase 2 (sPLA2) from chicken. In the present study, we detailed that the substrate affinity, specificity and the hydrolysis rates of sPLA2 at each interface is governed by the surface electrostatic potentials and hydrophobic interactions operative at this surface.

Aida karray*, Ameni ktata, Naima mounedi and Adel Sayari Author Affiliations Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Tunisia Received: July 17, 2020 | Published: July 28, 2020 Corresponding author: Aida Karray, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra, université de Sfax-Tunisia DOI: 10.26717/BJSTR.2020.29.004759

Lipases are hydrolytic enzymes owing much importance in industrial applications. These enzyme-based detergents are ecofriendly and produce a wastewater with low level of COD (chemical oxygen demand). In the present work, a novel halophilous, thermoalkaline, and detergent-tolerant lipase produced by a newly isolated Aeribacillus pallidus strain VP3 was studied. Considerable interest has been given to this lipase by the improvement of its catalytic activity through the optimization of the pH, the (C/N) ratio, and the inoculum size, using the response surface methodology based on the Box-Behnken design of experiments. A total of 16 experiments were conducted, and the optimized pH, (C/N) ratio, and inoculum size were 10, 1, and 0.3, respectively. The results of the analysis of variance (ANOVA) test indicated that the established model was significant (p value < 0.05). The optimization of the production conditions leads to 2.83-fold of increase in the catalytic activity calculated as the ratio of the activity obtained after optimization (68 U) and the initial activity before optimization (24 U). All in all, the lipase of Aeribacillus pallidus could be considered as a potential candidate to be incorporated in detergent formulations since it shows a good stability towards detergents and wash performance.

Background : Tunisia has natural resources favorable to fishing with a coastline that extends for a total length of 1,300 kilometres. The large quantities processed show that tuna canneries generate an important amounts of discarded high quality species. These wastes are, of course, problems, but they also offer excellent opportunities for biotechnological exploitation. The disposal of these wastes has always been expansive and has often a harmful impact on the environment, but thanks to the evolution of techniques and the development of markets, it is now possible to transform this waste stream into useful and marketable products. Results : In the present work, we focused primary on the characterization of the red muscles of a tuna cannery for inclusion in the formulation of young dog’s wet food. The composition of these co-products indicates a high protein content (80%), and a moderate fat and mineral content (11% and 8%, respectively). Nevertheless, the carbohydrate content in the red muscles of this fish remains low (2%). To increase the carbohydrate content whish constitute the principal energy source and fiber proportion to improve product digestibility by the animals, cereals (maize flour and rice flour) were used. Four formulae were tested, using the same manufacturing process. thA physicochemical study of the finished products was carried out and the Limit Date of Consumption was determined and estimated to 3 years and 3 months. Microbiological analyses were carried out to ensure the safety of the finished product and the results showed the absence of pathogens and the compliance of the product with current standards. Conclusion : Thanks to their high protein content, tuna discarded products represent an important source for the development of a new animal feed product, including the young dog.

Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

Background : Alkaline, thermostable bacterial lipases are largely used in detergent applications, since they substitute the use of synthetic detergents which are known to cause substantial environmental problems. These enzymes based detergent are eco friendly and produce a waste water with low level of COD (Chemical Oxygen Demand). In the present study, we investigated a newly isolated Aeribacillus pallidus strain produces, without induction, a novel halophilous, thermo-alkaline and detergent- tolerant lipase.Results: Considerable interest has been given to this lipase by the improvement of its catalytic activity through the optimization of the pH, the (C/N) ratio and the inoculums size, using the response surface methodology based on the Box-Behnken Design of experiments. A total of 16 experiments were conducted, and the optimized pH, (C/N) ratio and inoculums size were 10, 1 and 0.3 respectively. The results of the analysis of variance (ANOVA) test indicated that the established model was significant (p value < 0.05).Conclusions: The optimization of the production conditions leads to 6.68-fold of increase in the catalytic activity with a maximum of 68 U/mL. All in all, the lipase of Aeribacillus pallidus could be considered as a potential candidate to be incorporated in detergent formulations since it shows a good stability towards detergents and wash performance.

The present study investigates the biochemical characterization of an extracellular (phospho)lipase from a wood fungus Peziza sp. (medium optimization, inducer concentration and substrate specificity measurements). The strain was identified on the basis of ITS1/ITS4 primers. A 604 bp fragment was amplified by PCR and the obtained nucleotide sequence, showed 99% identity with the ITS region of isolates named as Peziza sp. Interestingly, Peziza sp. has both lipase and phospholipase activities with the same level which require both the presence of Ca2+ and bile salts. Our result shows that the lipase hydrolyzes preferably the olive oil at 45 °C, pH 8. Whereas, the phospholipase activity was detected on pure PC at 45 °C, pH 9. Lipid extraction from dry biomass using chloroform/methanol (2/1) and quantitative measurement using electron microscope showed that intracellular triglycerides content was significantly high and reaches 20.88%. Gas chromatography analysis shows a majority of C18:1 (76, 98%) and C18:2 (9, 33%). Whereas, saturated fatty acids ranging C16–C20 represent only 11.5% of total lipids composition.