AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer.

A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents. KEY POINTS: • A novel thermostable esterase has been retrieved through functional metagenomics • The esterase is detergent-tolerant, which is attractive for some applications • The esterase can be expressed in a yeast mesophilic host to enhance its yield.

Bioprospecting the potential of metabolites from a Saharan saline soil strain Nocardiopsis dassonvillei GSBS4.

Saharan soil samples collected in El-Oued province have been investigated for actinobacteria as a valuable source for the production of bioactive metabolites. A total of 273 isolates were obtained and subjected to antagonistic activity tests against human pathogenic germs. A strain with a broad-spectrum antimicrobial activity was selected and identified as Nocardiopsis dassonvillei GSBS4, with high sequence similarities to N. dassonvillei subsp. dassonvilleiT X97886.1 (99%) based on polyphasic taxonomy approach and 16S ribosomal ribonucleic acid gene sequence analysis. The GSBS4 ethyl acetate crude extract showed strong antibacterial activity towards pathogenic bacteria and Candida albicans. It inhibited biofilm formation by Staphylococcus aureus and methicillin-resistant S. aureus with minimum inhibitory concentrations estimated at 0.144 and 1.15 mg·mL-1 , respectively. A 44% biofilm reduction was obtained for S. aureus and 61% for Pseudomonas aeruginosa. Furthermore, phenols composition of the crude extract showed a significant dose-dependent antioxidant activity by α-diphenyl-ß-picrylhydrazyl (57.21%) and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (64.29%) radicals scavenging assays. Although no inhibition was obtained on human coronavirus human coronavirus (HCoV) 229E and on model enterovirus (poliovirus 1) infection, a dose-dependent increase in cell viability of HCoV 229E-infected cells was noticed as the viability increased from 21% to 37%. Bioassay-guided fractionation of the crude extract gave a fraction showing antibacterial activity, which was analyzed by liquid chromatography-electrospray mass spectrometric technique, providing structural features on a major purple metabolite.

Statistical Optimization and Partial Characterization of Xylanases Produced by Streptomyces sp. S1M3I Using Olive Pomace as a Fermentation Substrate.

Xylanase production by Streptomyces sp. S1M3I was optimized by response surface methodology (RSM), followed by a partial characterization of these enzymes. Olive pomace was used as a substrate for growing Streptomyces sp. S1M3I in submerged fermentation. Effects of incubation time, pH, temperature, carbon source, nitrogen source, and inoculum size on xylanase production were studied, through the one-factor-at-a-time method. Then, a 33-factorial experimental design with RSM and the Box-Behnken design was investigated for the major influence factors. Maximum xylanase production (11.28 U/mL) was obtained when the strain was grown in mineral medium supplemented with 3% (w/v) of olive pomace powder and 0.3% (w/v) of ammonium sulfate, at a pH 7.4 and an incubation temperature of 40 °C. The xylanases in the supernatant degraded all tested substrates, with higher activity for the low-viscosity wheat arabinoxylan substrate. Two xylanases with close molecular masses were detected by zymogram analysis: Xyl-1 and Xyl-2 with molecular masses of 24.14 kDa and 27 kDa, respectively. The optimization of enzyme production parameters of Streptomyces sp. S1M3I and the characterization of these enzymes are prerequisites to enhancing xylanase production yield, which is crucial for further biotechnological processes.

Integrated Metabolomic, Molecular Networking, and Genome Mining Analyses Uncover Novel Angucyclines From Streptomyces sp. RO-S4 Strain Isolated From Bejaia Bay, Algeria.

Multi-omic approaches have recently made big strides toward the effective exploration of microorganisms, accelerating the discovery of new bioactive compounds. We combined metabolomic, molecular networking, and genomic-based approaches to investigate the metabolic potential of the Streptomyces sp. RO-S4 strain isolated from the polluted waters of Bejaia Bay in Algeria. Antagonistic assays against methicillin-resistant Staphylococcus aureus with RO-S4 organic extracts showed an inhibition zone of 20 mm by using the agar diffusion method, and its minimum inhibitory concentration was 16 µg/ml. A molecular network was created using GNPS and annotated through the comparison of MS/MS spectra against several databases. The predominant compounds in the RO-S4 extract belonged to the angucycline family. Three compounds were annotated as known metabolites, while all the others were putatively new to Science. Notably, all compounds had fridamycin-like aglycones, and several of them had a lactonized D ring analogous to that of urdamycin L. The whole genome of Streptomyces RO-S4 was sequenced to identify the biosynthetic gene cluster (BGC) linked to these angucyclines, which yielded a draft genome of 7,497,846 bp with 72.4% G+C content. Subsequently, a genome mining analysis revealed 19 putative biosynthetic gene clusters, including a grincamycin-like BGC with high similarity to that of Streptomyces sp. CZN-748, that was previously reported to also produce mostly open fridamycin-like aglycones. As the ring-opening process leading to these compounds is still not defined, we performed a comparative analysis with other angucycline BGCs and advanced some hypotheses to explain the ring-opening and lactonization, possibly linked to the uncoupling between the activity of GcnE and GcnM homologs in the RO-S4 strain. The combination of metabolomic and genomic approaches greatly improved the interpretation of the metabolic potential of the RO-S4 strain.

Selective isolation, antimicrobial screening and phylogenetic diversity of marine actinomycetes derived from the Coast of Bejaia City (Algeria), a polluted and microbiologically unexplored environment.

AIMS: The current study aimed to evaluate the occurrence of actinomycetes in the Coast of Bejaia City using selective isolation, as well as their bioactivity and phylogenitic diversity. METHODS AND RESULTS: Different selective media and methods were used, leading to the isolation of 103 actinomycete strains. The number of strains was influenced by isolation procedures and their interactions based on a three-way ANOVA and a post hoc Tukey test, which revealed that using M2 medium, dilution of samples followed by moderate heat treatment, and sampling at 10-20 m yielded the highest numbers of actinomycetes. The isolates were screened for their antimicrobial activity against human pathogenic microorganisms using agar and well diffusion methods. Of all the isolates, ten displayed activity against at least one Gram-positive bacterium, of which P21 showed the highest activity against Staphylococcus aureus, Methicillin-resistant S. aureus and Bacillus subtilis, with a diameter of 32, 28 and 25 mm respectively. Subsequently, active isolates were assigned to Streptomyces spp. and Nocardiopsis spp. based on 16S rRNA gene sequencing, including a putative new Streptomyces species (S3). The phenotypic characteristics of the P21 strain were determined, and interesting enzymatic capacities were shown. CONCLUSION: The recovery of actinomycetes along the Coast of Bejaia City was influenced by the isolation procedure. Ten strains displayed interesting antibacterial activity against Gram-positive bacteria, of which the P21 strain was selected as the most active strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a new insight into the occurrence of actinobacteria in the Coast of Bejaia. It suggests also that polluted environments such as Bejaia Bay could provide access to interesting actinomycetes as sources of antibiotic leads.

Epidemiological investigation of subclinical bovine mastitis in Algeria and molecular characterization of biofilm-forming Staphylococcus aureus.

Thirty dairy farms were selected for this study; the first objective of our study was to investigate the prevalence of subclinical bovine mastitis (SCM) in Eastern Algeria, from 600 lactating cows, and to identify potential risk factors associated with the occurrence of bovine mastitis and bacteria isolation using logistic regression. The second objective was to evaluate biofilm formation capacity and detect biofilm-associated genes of S. aureus, isolated from SCM cases. Molecular typing was investigated by spa typing. The prevalence of mastitis at cow and quarter level was 37.66% (226/600) and 27.17% (555/2042), respectively. Stage of lactation, cow breed, milk production, and the study area were factors associated with SCM. In addition, the most frequent pathogens isolated from mastitic milk were coagulase-negative staphylococci (CNS), E. coli, and S. aureus. The study area was highly associated with SCM caused by S. aureus; cows from Setif province were 18 times more affected with SCM caused by S. aureus compared to cows from Batna province (OR = 18.6, 95%CI 2.038-171.2), but were less affected with SCM caused by CNS isolates (OR = 0.17, 95%CI 0.033-0.868). Moreover, cows with milk production less than 10 L per day increased (p < 0.05) the prevalence of mastitis caused by E. coli. All the S. aureus isolates had biofilm-forming ability, and 41.66% of isolates were positive for adhesion genes (icaA, icaD, fbnA, and clfA). This study, therefore, warrants the need for improving sanitary measures and strict hygienic measures, and presents the first insight into biofilm-forming ability of S. aureus strains causing mastitis in dairy herds in Algeria, which will help in tracking the evolution of epidemic strains responsible for causing bovine mastitis.

Actinobacteria Derived from Algerian Ecosystems as a Prominent Source of Antimicrobial Molecules.

Actinobacteria, in particular "rare actinobacteria" isolated from extreme ecosystems, remain the most inexhaustible source of novel antimicrobials, offering a chance to discover new bioactive metabolites. This is the first overview on actinobacteria isolated in Algeria since 2002 to date with the aim to present their potential in producing bioactive secondary metabolites. Twenty-nine new species and one novel genus have been isolated, mainly from the Saharan soil and palm groves, where 37.93% of the most abundant genera belong to Saccharothrix and Actinopolyspora. Several of these strains were found to produce antibiotics and antifungal metabolites, including 17 new molecules among the 50 structures reported, and some of these antibacterial metabolites have shown interesting antitumor activities. A series of approaches used to enhance the production of bioactive compounds is also presented as the manipulation of culture media by both classical methods and modeling designs through statistical strategies and the associations with diverse organisms and strains. Focusing on the Algerian natural sources of antimicrobial metabolites, this work is a representative example of the potential of a closely combined study on biology and chemistry of natural products.

Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel Streptomyces sp. strain from a Saharan soil.

The novel bioactive actinobacterial strain GSBNT10 obtained from a Saharan soil, was taxonomically characterized using a polyphasic approach. 16S rRNA gene sequence analysis supported the classification of the isolate within the genus Streptomyces indicating it as a novel species. The major metabolite responsible of the bioactivity was purified and structurally characterized as actinomycin D (act-D) by mass spectrometric and nuclear magnetic resonance analyses Plackett-Burman design (PBD) and response surface methodology (RSM) were applied in order to optimize the medium formulation for the production of this bioactive metabolite. By PBD experiments, NaNO3, K2HPO4 and initial pH value were selected as significant variables affecting the metabolite production. Central Composite Design (CCD) showed that adjustment of the fermentative medium at pH 8.25, K2HPO4 at 0.2 gL-1 and NaNO3 at 3.76 gL-1 were the values suiting the production of act-D. Moreover, the results obtained by the statistical approach were confirmed by act-D detection using the HPLC equipped with a diode array detector and coupled online with electrospray-mass spectrometry (ESIMS) technique. act-D production was highly stimulated, obtaining a good yield (656.46 mgL-1) which corresponds to a 58.56% increase compared with the non-optimized conditions and data from LC-ESIMS technique efficiently confirmed the forecast from RSM.

Characterization and optimization of extracellular L-Asparaginase production by selected Actinomycete strain isolated from an algerian wheat bran.

L-Asparaginase is an enzyme that hydrolyses the amino acid L-Asparagine into aspartic acid and ammonia. As a medication, L-Asparaginase is used in chemotherapy to treat acute lymphoblastic leukaemia by depleting circulating Asparagine and depriving tumor cells. Interest in Actinomycetes as potential producers of antibiotics and enzymes encouraged us to investigate an isolated strain (CA01) from soft wheat bran.The Actinomycete strain was characterized based on its morphological and biochemical characteristics and selected due to a proved promising ability to produce L-Asparaginase optimized in both solid and liquid media cultures.The conditions of enzyme production were standardized according to a one-factor-at-a-time (OFAT) experimental design.To obtain optimal medium combination, a Box-Behnken Response Surface Methodology (RSM) has been adopted by choosing the most influential factors. The optimal conditions for the enzyme production were (g/l): L-Asparagine 10.7; Glucose 2.7; starch 7, in based medium containing (g/l): K2HPO4 0.5; MgSO4, 7H2O 0.1, corresponding to an optimal enzymatic activity of 8.03 IU/ml at 27.83°C. The maximum production of enzyme was reached on the sixth day of experiment. The ANOVA test (P value ˂ 0.05) and adjusted R2 values close to the experimental R2 show that the obtained model of the active L-Asparaginase of CA01 strain production is significant with the following linear terms: temperature, substrate concentration, Glucose concentration and there squared.

Streptomyces thermoviolaceus SRC3 strain as a novel source of the antibiotic adjuvant streptazolin: A statistical approach toward the optimized production.

Streptomyces thermoviolaceus SRC3, a newly isolated actinobacterial strain from Algerian river sediments, exhibited a broad activity against various bacterial and yeast human pathogens (Salmonella Typhi ATCC 14028, Vibrio cholerae ATCC 14035, MRSA ATCC 43300 and Candida albicans ATCC 10231). The strain SRC3 was selected from thirty nine actinobacterial isolates and identified as S. thermoviolaceus based on morphology, cultural properties, physiological analyses and 16S rRNA gene sequencing. Culture parameters for the antibiotic production were optimized by sequential statistical strategy including Plackett-Burman design (PBD) and Response Surface Methodology (RSM). In PBD experiments, KCl, K2HPO4, MgSO4·7H2O, pH value and incubation time emerged as the most significant in affecting the output of antimicrobial activities. These factors were further optimized using Central Composite Design (CCD). The best achieved conditions were: KCl (0.01%), K2HPO4 (0.1%), MgSO4·7H2O (0.02%) and 9 days incubation for anti-S. Typhi compounds, KCl (0.051%), MgSO4·7H2O (0.05%) and 5 days incubation for C. albicans inhibitors. The metabolite responsible for the bioactivities was purified, structurally characterized (by NMR, MS, UV and IR analyses) and identified as streptazolin, recently reported as a promising antibiotic adjuvant.

Selective Isolation and Screening of Actinobacteria Strains Producing Lignocellulolytic Enzymes Using Olive Pomace as Substrate.

BACKGROUND: Olive pomace, as the main by-product of the olive oil industry, is recently recycled as fermentation substrate for enzyme production. OBJECTIVES: Actinobacteria isolates were separated from an Algerian soil under olive pomace cultivation and were evaluated for their lignocellulolytic enzymes production. MATERIALS AND METHODS: Isolates of Actinobacteria were separated from soils around oil mills using four isolation media, among them three were enriched by olive pomace. The isolates were screened for their cellulolytic, xylanolytic and ligninolytic activities. Isolates with potential of producing lignocellulose-degrading enzymes were selected under submerged fermentation based olive pomace. RESULTS: Ninety isolates of Actinobacteria were separated from soil samples. M3 medium (raw pomace autoclaved alone) was the best isolation medium (68 strains), whereas, the soil from oil mill with continuous system (S1) led to separation of 52 strains. Among the 90 isolates, 82 were shown promising enzyme activity, 19 isolates were presented the largest zone diameter (<30 mm). S1M3I and S1M3II isolates were exhibited the highest values. CONCLUSIONS: Olive pomace with medium low cost and high titers of enzymes can be valorized by culture of Actinobacteria to produce lignocellulolytic enzymes for industrial applications.

Partial characterization of xylanase produced by Caldicoprobacter algeriensis, a new thermophilic anaerobic bacterium isolated from an Algerian hot spring.

To date, xylanases have expanded their use in many processing industries, such as pulp, paper, food, and textile. This study aimed the production and partial characterization of a thermostable xylanase from a novel thermophilic anaerobic bacterium Caldicoprobacter algeriensis strain TH7C1(T) isolated from a northeast hot spring in Algeria. The obtained results showed that C. algeriensis xylanase seems not to be correlated with the biomass growth profile whereas the maximum enzyme production (140.0 U/ml) was recorded in stationary phase (18 h). The temperature and pH for optimal activities were 70 °C and 11.0, respectively. The enzyme was found to be stable at 50, 60, 70, and 80 °C, with a half-life of 10, 9, 8, and 4 h, respectively. Influence of metal ions on enzyme activity revealed that Ca(+2) enhances greatly the relative activity to 151.3 %; whereas Hg(2+) inhibited significantly the enzyme. At the best of our knowledge, this is the first report on the production of xylanase by the thermophilic bacterium C. algeriensis. This thermo- and alkaline-tolerant xylanase could be used in pulp bleaching process.

Antibacterial polyketides from the marine alga-derived endophitic Streptomyces sundarbansensis: a study on hydroxypyrone tautomerism.

Polyketide 13 [=2-hydroxy-5-((6-hydroxy-4-oxo-4H-pyran-2-yl)methyl)-2- propylchroman-4-one] and three related known compounds 7, 9 and 11 were obtained and structurally characterized from Streptomyces sundarbansensis strain, an endophytic actinomycete isolated from the Algerian marine brown algae Fucus sp. Compound 13 was obtained as the major metabolite from optimized culture conditions, by using Agar state fermentation. Due to tautomeric equilibrium, 13 in CD(3)OD solution was able to incorporate five deuterium atoms, as deduced by NMR and ESI-MS/MS analysis. The 2-hydroxy-γ-pyrone form was established for these metabolites based on the comparison of their experimental IR spectra with the DFT calculated ones, for both the corresponding 4-hydroxy-α-pyrone and 2-hydroxy-γ-pyrone forms. During antibacterial evaluation, compound 13 stood out as the most active of the series, showing a selective activity against the gram positive pathogenic methicillin-resistant S. aureus (MRSA, MIC = 6 µΜ), with a bacteriostatic effect.

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus Pyrococcus from an Algerian hydrothermal hot spring.

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80-85 degrees C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.