Coculture of bacterial levans and evaluation of its anti-cancer activity against hepatocellular carcinoma cell lines.

This research represents a novel study to assess how coculture affects levan yield, structure, bioactivities, and molecular weight. Among the 16 honey isolates, four bacterial strains recorded the highest levan yield. The Plackett-Burman design showed that the coculture (M) of isolates G2 and K2 had the maximum levan yield (52 g/L) and the effective factors were sucrose, incubation time, and sugarcane bagasse. The CCD showed that the most proper concentrations for maximum levan yield (81 g/L): were 130 g/L of sucrose and 6 g/f of sugarcane bagasse. Levan's backbone was characterized, and the molecular weight was determined. G2 and K2 isolates were identified based on 16 sRNA as Bacillus megaterium strain YM1C10 and Rhizobium sp. G6-1. M levan had promising antioxidant activity (99.66%), slowed the migration activity to a great extent, and recorded 70.70% inhibition against the hepatoblastoma cell line (HepG2) at 1000 µg/mL. Gene expression analysis in liver cancer cell lines (HePG2) revealed that M levan decreased the expression of CCL20), 2GRB2, and CCR6) genes and was superior to Doxo. While increasing the expression of the IL4R and IL-10 genes. The DNA damage values were significantly increased (P < 0.01) in treated liver cancer cell lines with levan M and Doxo. The results referred to the importance of each of the hydroxyl and carboxyl groups and the molecular weight in levans bioactivities.

Optimization strategy of Bacillus subtilis MT453867 levansucrase and evaluation of levan role in pancreatic cancer treatment.

Pancreatic cancer is the fourth most lethal cancer type worldwide. Due to multiple levan applications including anticancer activities, studies related to levansucrase production are of interest. To our knowledge, levan effect on pancreatic cancer cells has not been tested previously. In this work, among eighteen bacterial honey isolates, Bacillus subtilis MT453867 showed the highest levan yield (33 g/L) and levansucrase production (8.31 U/mL). One-factor-at-a-time technique increased levansucrase activity by 60% when MgSO4 was eliminated. The addition of 60 g/L banana peels enhanced the enzyme activity (192 U/mL). Placket Burman design determined the media composition for maximum levan yield (54.8 g/L) and levansucrase production (505 U/mL). The identification of levan was confirmed by thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C-nuclear-magnetic resonance, and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. The LD50 of crude levan was 4833 mg/kg body weight. Levan had strong antioxidant activity and significantly reduced the expression of CXCR4 and MCM7 genes in pancreatic cancer cells with significant DNA fragmentation. In conclusion, Bacillus subtilis MT453867 levan is a promising adjunct to pancreatic-anticancer agents with both anti-cancer and chemoprotective effects.

Optimization, Purification and Antitumor Activity of Kodamaea ohmeri ANOMY L-Asparaginase Isolated from Banana Peel.

OBJECTIVE: L-Asparaginase is an important enzyme that converts L-asparagine to L-aspartate and ammonia. Microbial L-asparaginase has important applications as anticancer and food processing agents. METHODS: This study reported the isolation, screening of a local yeast isolate from banana peel for L-asparaginase production using submerged fermentation, optimization of the production, purification, and anticancer assay of L-asparaginase. The yeast isolate was identified as Kodamaea ohmeri ANOMY based on the analysis of nuclear large subunit (26S) rDNA partial sequences. It was a promising L-asparaginase producer with a specific activity of 3059±193 U/mg in a non-optimized medium. The classical one-variable-at-a-time method was used to optimize the production medium components, and it was found that the elimination of K2HPO4 from the medium increased L-asparaginase specific activity (3100.90±180 U/mg). RESULTS: Statistical optimization of L-asparaginase production was done using Plackett-Burman and Box-Behnken designs. The production medium for the maximum L-asparaginase specific activity (8500±578U/mg) was as follows (g/L): L-asparagine (7.50), NaNO3 (0.50), MgSO4.7H2O (0.80), KCl (0.80) associated with an incubation period of 5 days, inoculum size of 5.60 %, and pH (7.0). The optimization process increased L-asparaginase production by 2.78-fold compared to the non-optimized medium. L-Asparaginase was purified using ammonium sulphate precipitation followed by gel filtration on a Sephadex G-100 column. Its molecular weight was 66 KDa by SDS-PAGE analysis. CONCLUSION: The cell morphology technique was used to evaluate the anticancer activity of L-asparaginase against three different cell lines. L-Asparaginase inhibited the growth of HepG-2, MCF-7, and HCT-116 cells at a concentration of 20, 50, and 60 µL, respectively.

Role of levan extracted from bacterial honey isolates in curing peptic ulcer: In vivo.

Peptic ulcer is one of the worldwide diseases where 10% of adults are affected by peptic ulcers at least once in their lifetime. The goal of this study was to evaluate the effect of levan in treating peptic ulcer. The bacterial honey isolates called Bacillus sp. levan was utilized. Levan was chemically characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), 1H and 13C NMR analysis. Levan was used to treat gastric ulcers induced in rats by oral administration of 5 mL/kg ethanol. Microscopic examination of stomach sections indicated that treatment with 200 mg/kg levan effectively healed the ulcers. Levan had no antimicrobial activity against a common cause of ulcers such as Helicobacter pylori bacteria. Rather, we proposed that the high adhesion (manifested as a protective coating) and prebiotic activity of levan may account for the observed beneficial effects. The immunohistochemical examination showed that levan led to a noticeable Bacillus sp. levan reduction in NF-κB in the upper gastric mucosa. The results concluded that the role of levan was more protective rather than preventive and suggested that levan could play a fundamental role in solving the peptic ulcer problems.

Optimization of Bacillus licheniformis MAL tyrosinase: in vitro anticancer activity for brown and black eumelanin.

The influence of tyrosinase in catalyzes/stimulates the eumelanin production was studied. Accordingly, bacterial sp. was isolated and identified as Bacillus licheniformis based on 16S rRNA. It could grow and gave monophenolase and diphenolase productivity in medium contained tyrosin and Cu2+ only. The tyrosinase enzymes were optimized by studying different environmental and nutritional factors. The maximum monophenolase and diphenolase productivity were obtained at 60 °C, pH9, Cu2+(0.01g), liver extract (1 g/L) and the oxygen level fixed at 20%. Also, the mannose as a carbon source increased the monophenolase production 6.2 times. For the first time, two types of eumelanin were extracted by hydrochloric acid treatment. The black and brown eumelanin weighed (0.1 g/100 mL and 0.7 g/100 mL respectively) and characterized by using FTIR and UV/Vis spectroscopy techniques. Their morphological structure and its elemental composition were characterized by SEM and EDAX respectively. The black melanin showed promising anticancer activity towards HEPG-2 and HCT-116 cell lines with IC50 values (6.15, 5.54 µg) compared to Doxorubicin (4.05, 4.45 µg) respectively.

Scaling up of levan yield in Bacillus subtilis M and cytotoxicity study on levan and its derivatives.

This study focused on kinetics of levan yield by Bacillus subtilis M, in a 150 L stirred tank bioreactor under controlled pH conditions. The optimized production medium was composed of (g/L): commercial sucrose 100.0, yeast extract 2.0, K2HPO4 3.0 and MgSO4⋅7H2O 0.2; an increase in both carbohydrates consumption and cell growth depended on increasing the size of the stirred tank bioreactor from 16 L to 150 L. The highest levansucrase production (63.4 U/mL) and levan yield of 47 g/L was obtained after 24 h. Also, the specific levan yield (Yp/x) which reflects the cell productivity increased with the size increase of the stirred tank bioreactor and reached its maximum value of about 29.4 g/g cells. These results suggested that B. subtilis M could play an important role in levan yield on a large scale in the future. Chemical modifications of B. subtilis M crude levan (CL) into sulfated (SL), phosphorylated (PL), and carboxymethylated levans (CML) were done. The difference in CL structure and its derivatives was detected by FT-IR transmission spectrum. The cytotoxicity of CL and its derivatives were evaluated by HepGII, Mcf-7 and CaCo-2. In general most tested levans forms had no significant cytotoxicity effect. In fact, the carboxymethylated and phosphrylated forms had a lower anti-cancer effect than CL. On the other hand, SL had the highest cytotoxicity showing SL had a significant anti-cancer effect. The results of cytotoxicity and cell viability were statistically analyzed using three-way ANOVA.

Levansucrase optimization using solid state fermentation and levan biological activities studies.

Bacillus subtilis NRC1aza produced levansucrase under solid state fermentation using starch as support. A sequential optimization strategy, based on statistical experimental designs is employed to enhance enzyme productivity. First, a 2-level Plackett-Burman design was applied for bioprocess parameters screen that significantly increase levansucrase production. Second optimization step was performed using fractional factorial design in order to optimize the amounts of highest positive variables that had significant effect on levansucrase productivity. Maximal enzyme productivity of 170 U/gds was achieved in presence of glucose, yeast extract, and pH 8. In vitro, experiments confirmed that LevCR and LevQT had an antitumor activity against different animal and human cancer cell lines by demonstrating inhibitory effects on growth of Ehrlich ascites carcinoma cell line, human MCF-7 breast and liver HepG2 cancer cell lines, in particular LevQT was found to be efficacious compared to anticancer drug, cisplatin. Result focused in LevCR as strong fibrinolytic agent.

Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage'.

Honey isolate Bacillus subtilis M was cultivated in shake flasks and in 16-l bioreactor cultures to investigate cell growth, bio-metabolites production kinetics and bioprocess scalability. The respective maximal levan and levansucrase productions of 59.5 g/l and 74.1 U/ml were achieved in bioreactor cultures under pH controlled condition (pH=7.0) after only 24 h. Crude levan (levE) was isolated, characterized and fractionated into F1, F2, and F3 with different molecular weight (21.8, 13.118, 9.53 kDa). (1)H NMR and (13)C NMR spectroscopy proved that LevE and their fractions were mainly ß-(2, 6)-linked levan-type polysaccharide. The cancer chemo-preventive activity indicated that the levE and its fraction 3 were promising inhibitors of cytochrome P-450 1A activity, inducers of glutathione-S-transferase activity in Murine hepatomaHepa1c1c7cells and possessed highest radical scavenging affinity to both ROO and OH. They inhibited the induced-DNA fragmentation. None of the tested samples triggered apoptosis or necrosis in splenocytes, except F2.

Antitumor and antioxidant activities of levan and its derivative from the isolate Bacillus subtilis NRC1aza.

The novel levansucrase produced from Bacillus subtilis NRC1aza yielded two types of levan L1 and L2 with different molecular weights 85.23 kDa and 31.95 kDa, respectively. The levan identification was detected by paper chromatography and high-pressure liquid chromatography. The antioxidant activity of levan and their derivatives (SL1 and SL2) exhibited a strong free radical scavenging activity with DPPH. The antitumor activity of SL1 was tested against different human cancer cell lines. The cell death was explored mechanistically through evaluation of Apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase activity, mitochondrial transmembrane potential (Δψm), cytochrome C release, total caspases, caspase-3, and caspase-9, and cell cycle. SL1 showed high selective cytotoxicity against HepG2 cells. SL1 led to DNA damaging and fragmentation that was associated with induced apoptosis via mitochondrial pathway, which initiated by the impairment of Δψm and then increased mitochondria, released cytochrome c, that in turn activated caspase-9 and caspase-3 and induced apoptosis.