A Combined Phyto- and Photodynamic Delivery Nanoplatform Enhances Antimicrobial Therapy: Design, Preparation, In Vitro Evaluation, and Molecular Docking.

Microbial combating is one of the hot research topics, and finding an alternative strategy is considerably required nowadays. Here, we report on a developed combined chemo- and photodynamic delivery system with a core of zinc oxide nanoparticles (ZnO NPs), porphyrin photosensitizer (POR) connected to alginate polymer (ALG), and berberine (alkaloid natural agent, BER) with favorable antimicrobial effects. According to the achieved main designs, the results demonstrated that the loading capacity and entrapment efficiency reached 22.2 wt % and 95.2%, respectively, for ZnO@ALG-POR/BER nanoformulation (second design) compared to 5.88 wt % and 45.1% for ZnOBER@ALG-POR design (first design). Importantly, when the intended nanoformulations were combined with laser irradiation for 10 min, they showed effective antifungal and antibacterial action against Candida albicans, Escherichia coli, and Staphylococcus aureus. Comparing these treatments to ZnO NPs and free BER, a complete (100%) suppression of bacterial and fungal growth was observed by ZnO@ALG-POR/BER nanoformulation treated E. coli, and by ZnOBER treated C. albicans. Also, after laser treatments, most data showed that E. coli was more sensitive to treatments using nanoformulations than S. aureus. The nanoformulations like ZnOBER@ALG-POR were highly comparable to traditional antibiotics against C. albicans and E. coli before laser application. The results of the cytotoxicity assessment demonstrated that the nanoformulations exhibited moderate biocompatibility on normal human immortalized retinal epithelial (RPE1) cells. Notably, the most biocompatible nanoformulation was ZnOBER@ALG-POR, which possessed ∼9% inhibition of RPE1 cells compared to others. High binding affinities were found between all three microbial strains' receptor proteins and ligands in the molecular docking interaction between the receptor proteins and the ligand molecules (mostly BER and POR). In conclusion, our findings point to the possible use of hybrid nanoplatform delivery systems that combine natural agents and photodynamic therapy into a single therapeutic agent, effectively combating microbial infections. Therapeutic efficiency correlates with nanoformulation design and microorganisms, demonstrating possible optimization for further development.

Co-administration of xylo-oligosaccharides produced by immobilized Aspergillus terreus xylanase with carbimazole to mitigate its adverse effects on the adrenal gland.

Carbimazole has disadvantages on different body organs, especially the thyroid gland and, rarely, the adrenal glands. Most studies have not suggested any solution or medication for ameliorating the noxious effects of drugs on the glands. Our study focused on the production of xylooligosaccharide (XOS), which, when coadministered with carbimazole, relieves the toxic effects of the drug on the adrenal glands. In addition to accelerating the regeneration of adrenal gland cells, XOS significantly decreases the oxidative stress caused by obesity. This XOS produced by Aspergillus terreus xylanase was covalently immobilized using microbial Scleroglucan gel beads, which improved the immobilization yield, efficiency, and operational stability. Over a wide pH range (6-7.5), the covalent immobilization of xylanase on scleroglucan increased xylanase activity compared to that of its free form. Additionally, the reaction temperature was increased to 65 °C. However, the immobilized enzyme demonstrated superior thermal stability, sustaining 80.22% of its original activity at 60 °C for 120 min. Additionally, the full activity of the immobilized enzyme was sustained after 12 consecutive cycles, and the activity reached 78.33% after 18 cycles. After 41 days of storage at 4 °C, the immobilized enzyme was still active at approximately 98%. The immobilized enzyme has the capability to produce xylo-oligosaccharides (XOSs). Subsequently, these XOSs can be coadministered alongside carbimazole to mitigate the adverse effects of the drug on the adrenal glands. In addition to accelerating the regeneration of adrenal gland cells, XOS significantly decreases the oxidative stress caused by obesity.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Nematicidal activity of sweet annie and garden cress nano-formulations and their impact on the vegetative growth and fruit quality of tomato plants.

Root-knot nematode is one of the major problems that face the agricultural production of several vegetable crops. Chemical nematicides have been banned because of their healthy and environmental undesirable attributes. So, this study aimed to evaluate the potential use of sweet annie (Artimisia annua) and garden cress (Lepidium sativum) as green routes for the development of effective and eco-friendly alternative nematicides. Nematicidal activity of sweet annie and garden cress aqueous extracts (500 g/L) in the original and nano-forms were evaluated against Meloidogyne incognita in tomato planted in infected soil under greenhouse conditions. Nineteen phenolic compounds were identified in A. annua extract, which was dominated by chlorogenic acid (5059 µg/100 mL), while 11 compounds were identified in L. sativum extract, that dominated by p-hydroxybenzoic acid (3206 µg/100 mL). Nano-particles were characterized with smooth surface, spherical shape and small size (50-100 nm). Under laboratory, the nano-formulations showed mortality percentage of M. incognita J2 greater than the original extract from. Vegetative growth parameters of tomato plants treated with A. annua and L. sativum extracts significantly improved compared to the control plants. Also, biochemical analysis revealed that the extracts were able to induce tomato plants towards the accumulation of phenolic compounds and increasing the activity of defensive enzymes (protease, polyphenol oxidase and chitinase) resulting in systemic resistance. Regarding tomato fruits yield and quality, the studied treatments significantly improved the yield and physicochemical parameters of tomato fruits in terms of fruit weight, diameter, TSS, pH, lycopene content and color attributes gaining higher sensorial acceptance by the panelist. Generally, both extracts represent promising nematicide alternatives and have potential use in crop management. The nano-form of A. annua extract outperformed the nematicidal activity of other studied treatments.

Safe production of Aspergillus terreus xylanase from Ricinus communis: gene identification, molecular docking, characterization, production of xylooligosaccharides, and its biological activities.

BACKGROUND: The production of industrial enzymes such as xylanase using sufficient cost-effective substrates from potent microorganisms is considered economically feasible. Studies have reported castor cake (Ricinus communis) as the most potent and inexpensive alternative carbon source for production of xylanase C by using Aspergillus terreus (A. terreus). RESULTS: A. terreus strain RGS Eg-NRC, a local isolate from agro-wastes, was first identified by sequencing the internal transcribed spacer region of a nuclear DNA encoding gene cluster deposited in GenBank (accession number MW282328). Before optimization of xylanase production, A. terreus produced 20.23 U/g of xylanase after 7 days using castor cake as a substrate in a solid-state fermentation (SSF) system that was employed to achieve ricin detoxification and stimulate xylanase production. Physicochemical parameters for the production of xylanase were optimized by using a one-variable-at-a-time approach and two statistical methods (two-level Plackett-Burman design and central composite design, CCD). The maximum xylanase yield after optimization was increased by 12.1-fold (245 U/g). A 60-70% saturation of ammonium sulfate resulted in partially purified xylanase with a specific activity of 3.9 IU/mg protein. At 60 °C and pH 6, the partially purified xylanase had the highest activity, and the activation energy (Ea) was 23.919 kJmol. Subsequently, antioxidant capacity and cytotoxicity tests in normal Ehrlich ascites carcinoma human cells demonstrated xylooligosaccharides produced by the xylanase degradation of xylan as a potent antioxidant and moderate antitumor agent. Further investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis then determined the molecular weight of partially purified xylanase C to be 36 kDa. Based on the conserved regions, observations revealed that xylanase C belonged to the glycosyl hydrolase family 10. Next, the xylanase-encoding gene (xynC), which has an open reading frame of 981 bp and encodes a protein with 326 amino acids, was isolated, sequenced, and submitted to the NCBI GenBank database (accession number LC595779.1). Molecular docking analysis finally revealed that Glu156, Glu262, and Lys75 residues were involved in the substrate-binding and protein-ligand interaction site of modeled xylanase, with a binding affinity of -8.7 kcal. mol-1. CONCLUSION: The high production of safe and efficient xylanase could be achieved using economical materials such as Ricinus communis.