Enhancement of novel Endo-polygalacturonase expression in Rhodotorula mucilaginosa PY18: insights from mutagenesis and molecular docking.

Pectinase is a particular type of enzyme that can break down pectin compounds and is extensively utilised in the agricultural field. In this study, twenty yeast isolates were isolated and assayed for pectinase activity. Molecular identification by PCR amplification and sequencing of internal transcribed spacer (ITS) regions of isolate no. 18 had the highest pectinase activity of 46.35 U/mg, was identified as Rhodotorula mucilaginosa PY18, and was submitted under accession no. (OM275426) in NCBI. Rhodotorula mucilaginosa PY18 was further enhanced through sequential mutagenesis, resulting in a mutant designated as Rhodotorula mucilaginosa E54 with a specific activity of 114.2 U/mg. Using Response Surface Methodology (RSM), the best culture conditions for the pectinase-producing yeast mutant Rhodotorula mucilaginosa E54 were pH 5, 72-h incubation, 2.5% xylose, and 2.5% malt extract, with a pectinase-specific activity of 156.55 U/mg. Then, the obtained sequences of the endo-polygalacturonase PGI gene from Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were isolated for the first time, sequenced, and submitted to NCBI accession numbers OQ283005 and OQ283006, respectively. The modelled 3D structure of the endo-PGI enzyme (485 residues) was validated using Ramachandran's plot, which showed 87.71, 85.56, and 91.57% in the most favourable region for template Rhodotorula mucilaginosa KR, strain Rhodotorula mucilaginosa PY18, and mutant Rhodotorula mucilaginosa E54, respectively. In molecular docking studies, the results of template Rhodotorula mucilaginosa KR endo-PG1 showed an interaction with an affinity score of - 6.0, - 5.9, and - 5.6 kcal/mol for active sites 1, 2, and 3, respectively. Rhodotorula mucilaginosa PY18 endo-PG1 showed an interaction affinity with a score of - 5.8, - 6.0, and - 5.0 kcal/mol for active sites 1, 2, and 3, respectively. Mutant Rhodotorula mucilaginosa E54 endo-PG1 showed an interaction affinity of - 5.6, - 5.5, - 5.5 and - 5.4 kcal/mol for active sites 1, 2, and 3, respectively. The endo-PGI genes of both the yeast strain Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were successfully cloned and expressed in E. coli DH5α, showing significantly higher endo-PG1 activity, which recorded 94.57 and 153.10 U/mg for recombinant Rhodotorula mucilaginosa pGEM-PGI-PY18 and recombinant mutant Rhotorula pGEM-PGI-E54, respectively.

Modeling and in-vivo evaluation of fibrinolytic enzyme produced by Bacillus subtilis Egy under solid state fermentation.

Blood clot formation increases cases of myocardial infarction (AMI) and stroke, thus urges directing much research works for treatment and prevention of the causes. One of these directions is the microbial production of fibrinolytic enzymes as thrombolytic agents. In the current work, Bacillus subtilis Egy has been used for enzyme production under solid state fermentation. Among twelve nutrient meals in addition to wheat bran as a control fodder yeast yielded the highest enzyme activity reaching 114U/g. Applying statistical model for optimization of enzyme production revealed that 3.6%, fodder yeast; 40%, moisture content; 6 days, incubation period and 2%, inoculum size were the optimum conditions for maximum fibrinolytic enzyme production (141.02 U/g) by Bacillus subtilis Egy under solid-state fermentation The model was significant and data were experimentally validated. The produced fibrinolytic enzyme was evaluated for in vitro and in vivo cytotoxicity. In-vivo examination of the enzyme resulted in no mortality during the first 24 h after treatment. After 14 days, the results revealed no significant changes detected in hematological parameters (RBCs, MCV, hemoglobin except WBCs which showed an increase for both sexes. Histopathological examination of liver and kidney of rats received oral and subcutaneous treatments showed normal architecture. The data showed the applicability of the produced enzyme for the treatment of blood clot with no significant effect on living cells or on physiological functions.

Optimization of Bacillus subtilis growth parameters for biosynthesis of silver nanoparticles by using response surface methodology.

Silver nanoparticles (AgNPs) are among the most widely biosynthesized and used nanomaterials. They have different unique properties and a wide range of applications. This study is concerned with optimization of the growth conditions of Bacillus subtilis NRC1 for the biosynthesis of AgNPs using two designs of response surface methodology (RSM) statistical analysis. The data obtained from Plackett-Burman design (PBD) followed by central composite design (CCD), showed a good agreement between the experimental and predicted values of AgNPs biosynthesis. The optimum conditions were 0.7% (w/v) casein hydrolysate, 5% dextrin (w/v), pH 7.5 and 57 × 106 CFU/ml inoculum size. The model was highly valid and could be applied with a confidence factor of 99.47%. Minimal inhibitory concentration (MIC) of these AgNPs synthesized using the extracellular filtrate after growth of Bacillus subtilis NRC1 in the optimized medium was found to be 41-43µg/ml for all tested microorganisms with exception of Pseudomonas aeruginosa where MIC was 169 µg/ml.

Optimization of Bacillus subtilis NRC1 growth conditions using response surface methodology for sustainable biosynthesis of gold nanoparticles.

Gold nanoparticles (AuNPs) have different unique properties and a wide range of applications in different fields. Thereby, there is a growing urgency for the production of AuNPs using a safe and an economic method. In this study, optimization of fermentation conditions by Bacillus subtilis NRC1 for extracellular AuNPs synthesis using response surface methodology was achieved. The data obtained from Plackett-Burman design followed by Box-Behnken design indicated the accuracy and reliability of the model and it could be used to navigate the design space with a reasonable accuracy. Numerical optimization of Bacillus subtilis NRC1 active extracellular filtrate production, showed the optimum conditions of 0.74% (w/v) casein hydrolysate, 3.99% (w/v) dextrin, 47 × 106 CFU/ml inoculum size at pH 7.76 and 25 [Formula: see text]C to give the maximum AuNPs biosynthesis. The model was highly valid and the obtained data had a confidence factor of 98.48%. Statistical optimization resulted in a 2.6-fold increase in AuNPs production compared with that of the non-optimized medium.

Potential of silver nanoparticles synthesized using low active mosquitocidal Lysinibacillus sphaericus as novel antimicrobial agents.

Silver nanoparticles (AgNPs) were synthesized using extracellular filtrates of some Lysinibacillus sphaericus (Ls) strains under simple conditions. Ls synthesized AgNPs showed the optical absorption peaks at 388-412 nm as detected by UV-visible spectrophotometer. Transmission electron micrographs of bacterial synthesized AgNPs revealed that they were polycrystalline with spherical, hexagonal, cuboidal, rod and irregular shapes. The average diameter of the tested AgNPs were ranged from 14-21 nm and they were negatively charged as detected by DLS (-18.2 to -28.9). FTIR spectra showed the presence of nitrogenous biomolecules capping the synthesized AgNPs. The filtrates of tested Ls strains showed nitrate reductase activity (1.45-2.56 µmol/ml/min). Tested AgNPs showed bactericidal activity against Gram positive and Gram negative bacteria, fungicidal activity against yeast and filamentous fungi, and virucidal activity against rotavirus. In addition, it showed synergistic antimicrobial effect to cephradine and nizoarm against all tested microorganisms. Cytotoxicity test revealed the safety of the tested nanoparticles at tested concentrations.Finally, Ls strains represent microbial sources for ecofriendly, simple and economic biosynthesis of antimicrobial AgNPs. Also, this research may contribute to the medicinal chemistry and pharmaceutical industry for the development of new products used for the public health.

Biosynthesis of silver nanoparticles using isolated Bacillus subtilis: characterization, antimicrobial activity, cytotoxicity, and their performance as antimicrobial agent for textile materials.

Silver nanoparticles (AgNPs) have unique properties and a large range of applications. Biosynthesis of stable AgNPs using the extracellular filtrate of Bacillus subtilis was proved by the characteristic surface plasmon resonance at about 420-430 nm. They were polycrystalline with spherical, hexagonal, and irregular shapes and they were negatively charged (-40 mV) with an average diameter of 20 nm. FTIR spectrum confirmed the presence of protein molecules coating AgNPs. The optimum conditions for the synthesis of tested AgNPs were 1:6 filtrate dilution, 1 mM AgNO3, pH 7, 30 °C , 48 h contact time under static and illuminating conditions. The synthesized AgNPs showed antibacterial activities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, and Salmonella typhi, antifungal activity against Candida albicans and antiviral activity against rotavirus. Also, they showed potent cytotoxic effects on lung and hepatic carcinoma human cell lines. Meanwhile, the acute toxicity study against mice showed no significant changes in hematological, biochemical, and histological parameters of AgNPs treated mice. They also showed mild hepatoprotective effect in thioacetamide (TAA) - induced hepatic fibrosis in rats. AgNPs treated textiles fabrics showed high antimicrobial activities against different pathogenic microorganisms as well as UV protection adequacy.

Semi-pilot scale production of biodiesel from waste frying oil by genetically improved fungal lipases.

This paper addresses the issue of combining the usage of waste frying oil (WFO), as a feedstock, and a lipase produced in solid-state fermentation (SSF), as a biocatalyst, for semi-pilot scale production of biodiesel as fatty acid methyl esters (FAME). Two fungal mutants namely; Rhizopus stolonifer 1aNRC11 mutant F (1F) and Aspergillus tamarii NDA03a mutant G (3G) were used as a cocatalyst. The two mutants were cultivated separately by SSF in a tray bioreactor. The dried fermented solid of 1F and 3G mutants were used in a ratio of 3:1, respectively, for WFO transesterification. Optimization of several semi-pilot process stages including SSF and WFO transesterification reaction conditions resulted in 92.3% conversion of WFO to FAME. This FAME yield was obtained after 48 h using 10% cocatalyst (w/w of WFO), 10% water (w/w of WFO) and 3:1 methanol/ WFO molar ratio at 30 °C and 250 rpm. A preliminary economic evaluation of produced biodiesel price (190 $/Ton) is less than half the price of petroleum diesel in Egypt (401$/Ton) and is about 40.3% the price of biodiesel produced using a pure enzyme, which is a promising result. This strategy makes the biodiesel synthesis process greener, economical and sustainable.

Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization.

Different bacterial isolates with amylolytic activity were insulated from various honey samples. The most active isolate was identified by the molecular 16SrRNA sequence technique as Bacillus atrophaeus NRC1. The bacterium showed maximum amylase production under optimum culture conditions at pH 6.0, 40 °C and after 24 h incubation. Two amylase isoenzymes (AmyI and AmyII) from Bacillus atrophaeus NRC1 have been purified to homogeneity by using ammonium sulfate precipitation, Sephacryl S-200 and DEAE-Sepharose chromatography. The major isoenzyme, AmyI, had a specific activity 4635 U/mg proteins with molecular weight of 61 kDa using SDS-PAGE electrophoresis. The maximum activity of AmyI against starch was determined at pH 6.0 and 50 °C. AmyI was stable up to 50 °C after incubation for 30 min, retained 65 and 23% of its activity at 60 and 70 °C, respectively. Pre-incubation with Ca2+, Mg2+ and Ba2+ cations for 30 min enhanced the enzyme activity; while it was completely inhibited by Hg2+. Varied inhibition degree of the enzyme activity was determined with K+, Ni2+, Zn2+, Na2+ and Cu2+ ions. AmyI was inhibited by EDTA, PMSF and SDS, while it was activated by l-Cysteine-HCl and DTT. AmyI had the ability to degrade starch, amylopectin, glycogen, amylose and lacked the affinity towards ß-1,4-linked xyloses.

Multi-bioactive silver nanoparticles synthesized using mosquitocidal Bacilli and their characterization.

Silver nanoparticles (AgNPs) were synthesized using cell-free filtrates of some mosquitocidal Bacilli. They showed the optical absorption peaks at 386-412 nm. They were polycrystalline spherical, hexagonal, cuboidal, rod and anisotropic shapes as detected by TEM. These nanoparticles were negatively charged with sizes ranging from 15 to 21 nm average diameter as detected by DLS. FTIR spectra showed that the main absorption bands of biomolecules capping AgNPs appeared at average wave numbers of 3435 cm-1 [ν(N-H) of amide A overlapped by ν(O-H)], 1631 cm-1 [(ν(C=O) of amide I], 1396 cm-1 [ν(C-N) of amide I], 2929 cm-1 (aliphatic C-H) and 1040 cm-1 (C-C-O). FTIR spectra confirmed the presence of protein biomolecules in the bacterial filtrate-formed coat covering AgNPs through free amide groups resulting in their stabilization in the aqueous medium. Nitrate reductase activity was found in all tested bacterial filtrates and ranged from 1.66 to 2.51 µmol/ml/min. These findings point to the probable role of nitrate reductase in reducing silver ions to silver nanoparticles and their stabilization. Tested AgNPs were multi-bioactive nanometals and showed mosquitocidal, bactericidal, fungicidal and virucidal activities. In addition, they exhibited highly synergistic mosquitocidal effect to spore toxin complex of mosquitocidal Bacilli at a very low concentration. AgNPs exhibited activities that were not or slightly cytotoxic to MA 104 cell line at tested concentrations. Therefore, they can be applied in the medical field. Finally, this study offered a simple, highly efficient, eco-friendly, economic method for biosynthesis of multi-bioactive AgNPs by some mosquitocidal Bacilli.

Optimization of biosurfactant production by Bacillus brevis using response surface methodology.

The present study aims to evaluate and validate a statistical model for maximizing biosurfactant productivity by Bacillus brevis using response surface methodology. In this respect, twenty bacterial isolates were screened for biosurfactant production using hemolytic activity, oil spreading technique, and emulsification index (E24). The most potent biosurfactant-producing bacterium (B. brevis) was used for construction of the statistical response surface model. The optimum conditions for biosurfactant production by B. brevis were: 33 °C incubation temperature at pH 8 for 10 days incubation period and 8.5 g/L glucose concentration as a sole carbon source. The produced biosurfactant (BS) (73%) exhibited foaming activity, thermal stability in the range 30-80 °C for 30 min., pH stability, from 4 to 9 and antimicrobial activity against (Escherichia coli). The BS gave a good potential application as an emulsifier.

UPLC/Tandem mass and antimicrobial activity of Khaya senegalensis (A. Juss.) / UPLC/Tándem masa y la actividad antimicrobiana de Khaya senegalensis

Characterization of flavonoids and limonoids in the defatted acetone extract of Khaya senegalensis flowers (A. Juss.) contents was performed using ultra performance liquid chromatography (UPLC) coupled with ultraviolet (UV) and electrospray ionization (ESI) mass spectrometry, furthermore, tandem mass spectrometry (MS/MS) was performed to assist in the structural elucidation. The antimicrobial effect was tested against representative gram positive and negative bacteria and candida. Cytotoxicity of extract was evaluated using the mitochondrial- dependent reduction of MTT. The method used enabled identification of five flavonoid glycosides (di and mono- sugar) and twelve limonoids of different types viz: mexicanolides, phragmalins and angolensate were tentatively identified. The extract was effective against tested microorganism revealing potent growth inhibitory effect on Salmonella typhimurium ATCC 25566, Escherichia coli NRRN 3008 , Pseudomonas aeruginosa ATCC 10145 and fungus Candida albicans EMCC105, MIC ≤ 25µg/µl while MIC ≤ 50 µg/µl for Bacillus cereus, staphylococcus aureus ATCC 6538. Extract showed cytotoxicity against MCF7 (Breast carcinoma cell line) compared to doxorubicin, IC50=88.1(µg/mL) but no activity on HCT 116 (Colon carcinoma cell line) and HepG2 (liver cell carcinoma) was observed. Bioactive compounds in K senegalensis flowers acetone extract possesses promising antimicrobial activity with low cytotoxic effect warrants further investigation for their therapeutic and prophylactic roles

Se ha procedido a la caracterización de los flavonoides y limonoides del extracto acetónico de las flores de la especie Khaya senegalensis (A. Juss.) El análisis de sus componentes se realizó mediante cromatografía líquida de ultra resolución (UPLC) con detección ultravioleta (UV) y de espectrometría de masas de ionización por electrospray (ESI), así como espectrometría de masas (MS / MS) para ayudar en la elucidación estructural de los compuestos. Se comprobó el efecto antimicrobiano en bacterias gram positivas y negativas y levaduras como el género Candida. La citotoxicidad del extracto se evaluó mediante la reducción mitocondrial dependiente de MTT. El método de análisis permitió la identificación de cinco glucósidos flavonoides (di y mono-azúcar) y doce limonoides de diferentes tipos:se identificaron tentativamente mexicanolidos, phragmalinas y angolensato. El extracto fue efectivo contra microorganismos (≤ MIC 25µg / l ), revelando potente efecto inhibidor del crecimiento de Salmonella typhimurium (ATCC 25566), Escherichia coli (NRRN 3008), Pseudomonas aeruginosa (ATCC 10145) y el hongo Candida albicans (EMCC105). Igualmente fue eficaz a MIC ≤ 50 mg / l sobre Bacillus cereus, Staphylococcus aureus (ATCC 6538). Igualmente, el extracto mostró citotoxicidad contra línea celular de carcinoma de mama (MCF7) en comparación con la doxorrubicina, (IC50 = 88.1 (µg/ mL), pero no modificó la actividad en una línea celular de carcinoma colon (HCT 116) y en células de carcinoma del hígado (HepG2). Los compuestos bioactivos en extracto acetónico de flores de K. senegalensis poseen una actividad antimicrobiana prometedora con bajos efectos citotóxicos que garantizan la necesidad de una mayor investigación para conocer completamente sus papeles terapéuticos y profilácticos

Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710.

Two inulinases (Inu2 and Inu3) were purified from Rhizopus oligosporus NRRL 2710 by chromatography on DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of Inu2 and Inu3 were determined to be 76 and 30 kDa, respectively. Inu2 and Inu3 had the same pH optimum at 5.0, temperature optimum at 50 and 60 °C, and thermal stability up to 60 and 70 °C for 1 h, respectively. Inu2 and Inu3 had low km values (0.93 and 0.70 mM, respectively) indicating the high affinity toward inulin. Mg2+, Ca2+, Zn2+ and EDTA did not significantly influence the enzyme activity. Ni2+, Cu2+, Fe2+ and Co2+ showed a partial inhibitory effect, and Hg2+ had a strong inhibitory effect. p-Chloromercuribenzoate had a partial inhibitory effect on Inu2. From these findings, R. oligosporus inulinases can be beneficial enzymes for industrial enzymatic production of high fructose syrup.

Efficient mosquitocidal toxin production by Bacillus sphaericus using cheese whey permeate under both submerged and solid state fermentations.

Whey permeate (WP) was used efficiently for production of mosquitocidal toxin by Bacillus sphaericus 2362 (B. sphaericus 2362) and the Egyptian isolate, B. sphaericus 14N1 (B. sphaericus 14N1) under both submerged and solid state fermentation conditions. Under submerged fermentation, high mosquitocidal activity was produced by B. sphaericus 2362 and B. sphaericus 14N1 at 50-100% and 25-70% WP, respectively. Initial pH of WP was a critical factor for toxin production by both tested organisms. The highest toxicity was obtained at initial pH 7. Egyptian isolate, B. sphaericus 14N1 was tested for growth and toxin production under solid state fermentation conditions (SSF) by using WP as moistening agent instead of distilled water. The optimum conditions for production of B. sphaericus 14N1 on wheat bran-WP medium were 10 g wheat bran/250 ml flask moistened with 10-70% WP at 50% moisture content, inoculum size ranged between 17.2x10(7) and 34.4x10(7) and 6 days incubation under static conditions at 30 degrees C. Preliminary pilot-scale production of B. sphaericus 14N1 under SSF conditions in trays proved that wheat bran-WP medium was efficient and economic for industrial production of mosquitocidal toxin by B. sphaericus.