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A Bacillus licheniformis isolate with high l-asparaginase productivity was recovered upon screening two hundred soil samples. This isolate produces the two types of bacterial l-asparaginases, the intracellular type I and the extracellular type II. The catalytic activity of type II enzyme was much higher than that of type I and reached about 5.5 IU/ml/h. Bioinformatics analysis revealed that l-asparaginases of Bacillus licheniformis is clustered with those of Bacillus subtilis, Bacillus haloterans, Bacillus mojavensis and Bacillus tequilensis while it exhibits distant relatedness to l-asparaginases of other Bacillus subtilis species as well as to those of Bacillus amyloliquefaciens and Bacillus velezensis species. Upon comparison of Bacillus licheniformis l-asparaginase to those of the two FDA approved l-asparaginases of E. coli (marketed as Elspar) and Erwinia chrysanthemi (marketed as Erwinaze), it observed in a cluster distinct from- and with validly predicted antigenic regions number comparable to those of the two mentioned reference strains. It exhibited maximum activity at 40 °C, pH 8.6, 40 mM asparagine, 10 mM zinc sulphate and could withstand 500 mM NaCl and retain 70% of its activity at 70 °C for 30 min exposure time. Isolate enzyme productivity was improved by gamma irradiation and optimized by RSM experimental design (Box–Behnken central composite design). The optimum conditions for maximum l-asparaginase production by the improved mutant were 39.57 °C, 7.39 pH, 20.74 h, 196.40 rpm, 0.5% glucose, 0.1% ammonium chloride, and 10 mM magnesium sulphate. Taken together, Bacillus licheniformis l-asparaginase can be considered as a promising candidate for clinical application as antileukemic agent.Electronic supplementary materialThe online version of this article (10.1186/s13568-019-0751-3) contains supplementary material, which is available to authorized users.

Ammi majus L.; Family Apiaceae; is a plant indigenous to Egypt. Its fruits contain bioactive compounds such as furanocoumarins and flavonoids of important biological activities. An endophytic fungus was isolated from the fruits and identified as Aspergillus amstelodami (MK215708) by morphology, microscopical characterization, and molecular identification. To our knowledge this is the first time an endophytic fungus has been isolated from the fruits. The antimicrobial activity of the Ammi majus ethanol fruits extract (AME) and fungal ethyl acetate extract (FEA) were investigated, where the FEA showed higher antimicrobial activity, against all the tested standard strains. Phytochemical investigation of the FEA extract yielded five prenylated benzaldehyde derivative compounds isolated for the first time from this species: Dihydroauroglaucin (1), tetrahydroauroglaucin (2), 2-(3,6-dihydroxyhepta-1,4-dien-1-yl)-3,6-dihydroxy-5-(dimethylallyl)benzaldehyde (3), isotetrahydroauroglaucin )4), and flavoglaucin (5). Structure elucidation was carried out using (1H- and 13C-NMR). Fractions and the major isolated compound 1 were evaluated for their antimicrobial and antibiofilm activity. Compound 1 showed high antimicrobial activity against Escherichia coli with minimum inhibitory concentration (MIC) = 1.95 µg/mL, Streptococcus mutans (MIC = 1.95 µg/mL), and Staphylococcus aureus (MIC = 3.9 µg/mL). It exhibited high antibiofilm activity with minimum biofilm inhibitory concentration (MBIC) = 7.81 µg/mL against Staphylococcus aureus and Escherichia coli biofilms and MBIC = 15.63 µg/mL against Streptococcus mutans and Candida albicans and moderate activity (MBIC = 31.25 µg/mL) against Pseudomonas aeruginosa biofilm. This reveals that dihydroauroglaucin, a prenylated benzaldehyde derivative, has a broad spectrum antimicrobial activity. In conclusion, it was observed that the MICs of the FEA are much lower than that of the AME against all susceptible strains, confirming that the antimicrobial activity of Ammi majus may be due to the ability of its endophytic fungi to produce effective secondary metabolites.

An exhaustive screening program was applied for scoring a promising l-asparaginase producing-isolate. The recovered isolate was identified biochemically and molecularly and its l-asparaginase productivity was optimized experimentally and by Response Surface Methodology. The produced enzyme was characterized experimentally for its catalytic properties and by bioinformatics analysis for its immunogenicity. The promising l-asparaginase producing-isolate was selected from 722 recovered isolates and identified as Stenotrophomonas maltophilia and deposited at Microbiological Resources Centre (Cairo Mircen) under the code EMCC2297. This isolate produces both intracellular (type I) and extracellular (type II) l-asparaginases with about 4.7 fold higher extracellular l-asparaginase productivity. Bioinformatics analysis revealed clustering of Stenotrophomonas maltophilia l-asparaginase with those of Pseudomonas species and considerable closeness to the two commercially available l-asparaginases of E. coli and Erwinia chrysanthemi. Fourteen antigenic regions are predicted for Stenotrophomonas maltophilia l-asparaginase versus 16 and 18 antigenic regions for the Erwinia chrysanthemi and E. coli l-asparaginases. Type II l-asparaginase productivity of the test isolate reached 4.7 IU/ml/h and exhibited maximum activity with no metal ion requirement at 37 °C, pH 8.6, 40 mM asparagine concentration and could tolerate NaCl concentration up to 500 mM and retain residual activity of 55% at 70 °C after half an hour treatment period. Application both of random mutation by gamma irradiation and Response Surface Methodology that determined 38.11 °C, 6.89 pH, 19.85 h and 179.15 rpm as optimum process parameters could improve the isolate l-asparaginase productivity. Maximum production of about 8 IU/ml/h was obtained with 0.4% dextrose, 0.1% yeast extract and 10 mM magnesium sulphate. In conclusion l-asparaginase of the recovered Stenotrophomonas maltophilia EMCC2297 isolate has characters enabling it to be used for medical therapeutic application.

Streptococcus mutans has been considered as the major etiological agent of dental caries, mostly due to its arsenal of virulence factors, including strong biofilm formation, exopolysaccharides production, and high acid production. Here, we present the antivirulence activity of fatty acids derived from the endophytic fungus Arthrographis kalrae isolated from Coriandrum sativum against Streptococcus mutans. The chemical composition of the fatty acids was analyzed by gas chromatography–mass spectrometry GC-MS and revealed nine compounds representing 99.6% of fatty acids, where unsaturated and saturated fatty acids formed 93.8% and 5.8 % respectively. Oleic and linoleic acids were the major unsaturated fatty acids. Noteworthy, the fatty acids at the concentration of 31.3 mg L–1 completely inhibited Streptococcus mutans biofilm, and water insoluble extracellular polysaccharide production in both polystyrene plates, and tooth model assay using saliva-coated hydroxyapatite discs. Inhibition of biofilm correlated significantly and positively with the inhibition of water insoluble extracellular polysaccharide (R = 1, p < 0.0001). Furthermore, Arthrographis kalrae fatty acids at a concentration of 7.8 mg L–1 exhibited acidogenesis-mitigation activity. They did not show bactericidal activity against Streptococcus mutans and cytotoxic activity against human oral fibroblast cells at the concentration used. On the other hand, saliva-coated hydroxyapatite discs treated with sub-minimum biofilm inhibitory concentration of fatty acids showed disturbed biofilm architecture with a few unequally distributed clumped matrices using fluorescence microscopy. Our findings revealed that the intracellular fatty acid arrays derived from endophytic Arthrographis kalrae could contribute to the biofilm-preventing alternatives, specifically Streptococcus mutans biofilms.