Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Production and statistical optimization of cholesterol-oxidase generated by Streptomyces sp. AN strain.

BACKGROUND: Cholesterol oxidases (CHOs) have attracted enormous attention because of their wide biotechnological potential. The present study explores the production of CHOs by Streptomyces sp. AN. Evaluation of culture conditions affecting enzyme production, medium optimization and released metabolite characteristics were also investigated. RESULTS: The current work reports the isolation of 37 colonies (bacteria/actinobacteria) with different morphotypes from different soil/water samples. The isolate-coded AN was selected for its high potency for CHO production. Morphological characteristics and the obtained partial sequence of 16srRNA of AN showed 99.38% identity to Streptomyces sp. strain P12-37. Factors affecting CHO production were evaluated using Plackett-Burman (PB) and Box-Behnken (BB) statistical designs to find out the optimum level of the most effective variables, namely, pH, starch, NH4NO3 and FeSO4.7H2O with a predicted activity of 6.56 U/mL. According to this optimization, the following medium composition was considered to be optimum (g/L): cholesterol 1, starch 6, MgSO4.7H2O 0.1, CaCl2 0.01, FeSO4.7H2O 0.1, NH4NO3 23.97, yeast extract (YE) 0.2, K2HPO4 0.01, KH2PO4 0.1, NaCl 0.01, Tween 20 0.01, pH 6.36 and incubation temperature (30 °C) for 9 days. Spectophotometric analysis for released metabolites against cholesterol (standard) via Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) was carried out. FTIR spectrum showed the appearance of new absorption peaks at 1644 and 1725cm-1; this confirmed the presence of the Keto group (C=O) stretch bond. Besides, fermentation caused changes in thermal properties such as melting temperature peak (99.26; 148.77 °C), heat flow (- 8; - 3.6 Mw/mg), capacity (- 924.69; - 209.77 mJ) and heat enthalpy (- 385.29; 69.83 J/g) by comparison to the standard cholesterol as recognized through DSC thermogram. These changes are attributed to the action of the CHO enzyme and the release of keto derivatives of cholesterol with different properties. CONCLUSION: Streptomyces sp. AN was endowed with the capability to produce CHO. Enzyme maximization was followed using a statistical experimental approach, leading to a 2.6-fold increase in the overall activity compared to the basal condition. CHO catalyzed the oxidation of cholesterol; this was verified by the appearance of a new keto group (C=O) peak at 1644 and 1725 cm-1 observed by FTIR spectroscopic analysis. Also, DSC thermogram demonstrates the alteration of cholesterol triggered by CHO.

Fabrication of biodegradable chicken feathers into ecofriendly-functionalized biomaterials: characterization and bio-assessment study.

This study aims to investigate novel applications for chicken feather waste hydrolysate through a green, sustainable process. Accordingly, an enzymatically degraded chicken feather (EDCFs) product was used as a dual carbon and nitrogen source in the production medium of bacterial cellulose (BC). The yield maximization was attained through applying experimental designs where the optimal level of each significant variable was recorded and the yield rose 2 times. The produced BC was successfully characterized by FT-IR, XRD and SEM. On the other hand, sludge from EDCFs was used as a paper coating agent. The mechanical features of the coated papers were evaluated by bulk densities, maximum load, breaking length, tensile index, Young's modulus, work to break and coating layer. The results showed a decrease in tensile index and an increase in elongation at break. These indicate more flexibility of the coated paper. The coated paper exhibits higher resistance to water vapor permeability and remarkable oil resistance compared to the uncoated one. Furthermore, the effectiveness of sludge residue in removing heavy metals was evaluated, and the sorption capacities were ordered as Cu ++ > Fe ++ > Cr ++ > Co ++ with high affinity (3.29 mg/g) toward Cu ++ and low (0.42 mg/g) towards Co ++ in the tested metal solution.

Anticandidal Potential of Two Cyanobacteria-Synthesized Silver Nanoparticles: Effects on Growth, Cell Morphology, and Key Virulence Attributes of Candida albicans.

Candida albicans is an opportunistic human fungal pathogen responsible for 90-100% of mucosal and nosocomial infections worldwide. The emergence of drug-resistant strains has resulted in adverse consequences for human health, including numerous deaths. Consequently, there is an urgent need to identify and develop new antimicrobial drugs to counter these effects. Antimicrobial nanoagents have shown potent inhibitory activity against a number of pathogens through targeting their defense systems, such as biofilm formation. Here, we investigated the anticandidal activity of silver nanoparticles biosynthesized by the cyanobacterial strains Desertifilum sp. IPPAS B-1220 and Nostoc Bahar_M (D-SNPs and N-SNPs, respectively), along with that of silver nitrate (AgNO3), and examined the mechanisms underlying their lethal effects. For this, we performed agar well diffusion and enzyme activity assays (lactate dehydrogenase, adenosine triphosphatase, glutathione peroxidase, and catalase) and undertook morphological examinations using transmission electron microscopy. The effects of the three treatments on Hwp1 and CDR1 gene expression and protein patterns were assessed using qRT-PCR and SDS-PAGE assays, respectively. All of the three treatments inhibited C. albicans growth; disrupted membrane integrity, metabolic function, and antioxidant activity; induced ultrastructural changes in the cell envelope; and disrupted cytoplasmic and nuclear contents. Of the three agents, D-SNPs showed the greatest biocidal activity against C. albicans. Additionally, the D-SNP treatment significantly reduced the gene expression of Hwp1 and CDR1, suggestive of negative effects on biofilm formation ability and resistance potential of C. albicans, and promoted protein degradation. The mechanism involved in the biocidal effects of both D-SNPs and N-SNPs against C. albicans could be attributed to their ability to interfere with fungal cell structures and/or stimulate oxidative stress, enabling them to be used as a robust antimycotic agent.

Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization.

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine-metallo enzyme type.

Effective multi-functional biotechnological applications of protease/keratinase enzyme produced by new Egyptian isolate (Laceyella sacchari YNDH).

BACKGROUND: Due to a multitude of industrial applications of keratinolytic proteases, their demands are increasing. The present investigation studied the production and monitoring of the most possible multi-functional applications of YNDH thermoalkaline keratin-degrading enzyme. RESULTS: This work is considered the first that reported YNDH strain closely related to Laceyella sacchari strain; YNDH is a producer of protease/keratinase enzyme and able to degrade natural keratin such as feathers, wool, human hairs, and nails. Experimental design Plackett-Burman (PBD) was applied to evaluate culture conditions affecting the production of thermoalkaline protease/keratinase. Afterwards, Box-Behnken design (BBD) was applied to find out the optimum level of significant variables namely, NH4Cl, yeast extract, and NaNO3 with a predicted activity of 1324.7 U/ml. Accordingly, the following medium composition and parameters were calculated to be optimum (%w/v): NH4Cl, 0.08; feather, 1; yeast extract, 0.04; MgSO4.7H2O, 0.02; NaNO3, 0.016; KH2PO4, 0.01; K2HPO4, 0.01; pH, 8; inoculum size; 5%, cultivation temperature (Temp.) 45 °C and incubation time 48 h. The studied enzyme can degrade keratin-azure, remove proteinaceous materials, and is able to remove hairs from goat hides. These interesting characteristics make this enzyme a good candidate in many applications especially in detergent (Det.), in leather industries, and in pharmaceuticals particularly in nail treatment. CONCLUSION: The promising properties of the newly keratin-degrading protease enzyme from Laceyella sacchari strain YNDH would underpin its efficient exploitation in several industries to cope with the demands of worldwide enzyme markets.

Novel Biogenic Silver Nanoparticle-Induced Reactive Oxygen Species Inhibit the Biofilm Formation and Virulence Activities of Methicillin-Resistant Staphylococcus aureus (MRSA) Strain.

Emerging antibiotic-resistant bacteria result in increased mortality and have negative economic impacts. It is necessary to discover new strategies to create alternative antibacterial agents that suppress the bacterial resistance mechanism and limit the spread of serious infectious bacterial diseases. Silver nanoparticles may represent a new medicinal agents as alternative antibiotics affect different bacterial mechanisms such as virulence and resistance. In addition to that of silver nitrate (AgNO3) and ampicillin, for the first time, the inhibitory effect of silver nanoparticles synthesized using Desertifilum sp. (D-SNPs) was evaluated against five pathogenic bacteria using the agar well diffusion method. Also, the influence of D-SNPs and AgNO3 on bacterial antioxidant and metabolic activities was studied. The antibacterial activity of D-SNPs and AgNO3 against methicillin-resistant Staphylococcus aureus (MRSA) strains was studied at the morphological and molecular level. D-SNPs and AgNO3 have the ability to inhibit the growth of the five bacterial strains and resulted in an imbalance in the CAT, GSH, GPx and ATPase levels. MRSA treated with D-SNPs and AgNO3 showed different morphological changes such as apoptotic bodies formation and cell wall damage. Moreover, both caused genotoxicity and denaturation of MRSA cellular proteins. Additionally, TEM micrographs showed the distribution of SNPs synthesized by MRSA. This result shows the ability of MRSA to reduce silver nitrate into silver nanoparticles. These data indicate that D-SNPs may be a significant alternative antibacterial agent against different bacteria, especially MDR bacteria, by targeting the virulence mechanism and biofilm formation, leading to bacterial death.

Cytotoxic effect of green silver nanoparticles against ampicillin-resistant Klebsiella pneumoniae.

Considering the harmful effects and high spread of drug-resistant Klebsiella pneumoniae, many researchers have been trying to produce new antibacterial agents to combat the emergence of multidrug-resistant (MDR) strains of this bacterium. Recent progress in the nanomedicine field has provided opportunities for synthesizing unique nanoagents to battle MDR bacteria by targeting virulence and resistance signalling. The biocidal effects of 14.9 nm silver nanoparticles fabricated using Nostoc sp. Bahar M (N-SNPs) and AgNO3 were examined against drug-resistant K. pneumoniae using the agar well diffusion method. Transmission electron microscopy (TEM) was used to detect the ultrastructural changes caused by N-SNPs and AgNO3. To address the mode of action of N-SNPs and AgNO3, CAT, GPx, LDH and ATPase levels were assessed. The toxicity of N-SNPs and AgNO3 was evaluated against the mfD, flu, hly, 23S, hns, hcp-1, VgrG-1 and VgrG-3 genes as well as cellular proteins. N-SNPs showed the greatest inhibitory activity against K. pneumoniae, with MIC and MBC values of 0.9 and 1.2 mg mL-1, respectively. Furthermore, N-SNPs and AgNO3 induced apoptotic features, including cell shrinkage and cell atrophy. N-SNPs were more potent bactericidal compounds than AgNO3, causing increased leakage of LDH and GPx activities and depletion of ATPase and CAT activities, resulting in induced oxidative stress and metabolic toxicity. Compared to AgNO3, N-SNPs exhibited the highest toxicity towards the selected genes and the greatest damage to bacterial proteins. N-SNPs were the most potent agents that induced bacterial membrane damage, oxidative stress and disruption of biomolecules such as DNA and proteins. N-SNPs may be used as effective nanodrugs against MDR bacteria.

Lethal Mechanisms of Nostoc-Synthesized Silver Nanoparticles Against Different Pathogenic Bacteria.

BACKGROUND: Increasing antibiotic resistance and the emergence of multidrug-resistant (MDR) pathogens have led to the need to develop new therapeutic agents to tackle microbial infections. Nano-antibiotics are a novel generation of nanomaterials with significant antimicrobial activities that target bacterial defense systems including biofilm formation, membrane permeability, and virulence activity. PURPOSE: In addition to AgNO3, the current study aimed to explore for first time the antibacterial potential of silver nanoparticles synthesized by Nostoc sp. Bahar_M (N-SNPs) and their killing mechanisms against Streptococcus mutans, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium. METHODS: Potential mechanisms of action of both silver species against bacteria were systematically explored using agar well diffusion, enzyme (lactate dehydrogenase (LDH) and ATPase) and antioxidant (glutathione peroxidase and catalase)   assays, and morphological examinations. qRT-PCR and SDS-PAGE were employed to investigate the effect of both treatments on mfD, flu, and hly gene expression and protein patterns, respectively. RESULTS: N-SNPs exhibited greater biocidal activity than AgNO3 against the four tested bacteria. E. coli treated with N-SNPs showed significant surges in LDH levels, imbalances in other antioxidant and enzyme activities, and marked morphological changes, including cell membrane disruption and cytoplasmic dissolution. N-SNPs caused more significant upregulation of mfD expression and downregulation of both flu and hly expression and increased protein denaturation compared with AgNO3. CONCLUSION: N-SNPs exhibited significant inhibitory potential against E. coli by direct interfering with bacterial cellular structures and/or enhancing oxidative stress, indicating their potential for use as an alternative antimicrobial agent. However, the potential of N-SNPs to be usable and biocompatible antibacterial drug will evaluate by their toxicity against normal cells.

Bacterial Diversity and Bioremediation Potential of the Highly Contaminated Marine Sediments at El-Max District (Egypt, Mediterranean Sea).

Coastal environments worldwide are threatened by the effects of pollution, a risk particularly high in semienclosed basins like the Mediterranean Sea that is poorly studied from bioremediation potential perspective especially in the Southern coast. Here, we investigated the physical, chemical, and microbiological features of hydrocarbon and heavy metals contaminated sediments collected at El-Max bay (Egypt). Molecular and statistical approaches assessing the structure of the sediment-dwelling bacterial communities showed correlations between the composition of bacterial assemblages and the associated environmental parameters. Fifty strains were isolated on mineral media supplemented by 1% crude oil and identified as a diverse range of hydrocarbon-degrading bacteria involved in different successional stages of biodegradation. We screened the collection for biotechnological potential studying biosurfactant production, biofilm formation, and the capability to utilize different hydrocarbons. Some strains were able to grow on multiple hydrocarbons as unique carbon source and presented biosurfactant-like activities and/or capacity to form biofilm and owned genes involved in different detoxification/degradation processes. El-Max sediments represent a promising reservoir of novel bacterial strains adapted to high hydrocarbon contamination loads. The potential of the strains for exploitation for in situ intervention to combat pollution in coastal areas is discussed.