Characteristics and kinetics of thermophilic actinomycetes' amylase production on agro-wastes and its application for ethanol fermentation.

Thermophilic actinomycetes are commonly found in extreme environments and can thrive and adapt to extreme conditions. These organisms exhibit substantial variation and garnered significant interest due to their remarkable enzymatic activities. This study evaluated the potential of Streptomyces griseorubens NBR14 and Nocardiopsis synnemataformans NBRM9 strains to produce thermo-stable amylase via submerged fermentation using wheat and bean straw. The Box-Behnken design was utilized to determine the optimum parameters for amylase biosynthesis. Subsequently, amylase underwent partial purification and characterization. Furthermore, the obtained hydrolysate was applied for ethanol fermentation using Saccharomyces cerevisiae. The optimal parameters for obtaining the highest amylase activity by NBR14 (7.72 U/mL) and NBRM9 (26.54 U/mL) strains were found to be 40 and 30 °C, pH values of 7, incubation time of 7 days, and substrate concentration (3 and 2 g/100 mL), respectively. The NBR14 and NBRM9 amylase were partially purified, resulting in specific activities of 251.15 and 144.84 U/mg, as well as purification factors of 3.91 and 2.69-fold, respectively. After partial purification, the amylase extracted from NBR14 and NBRM9 showed the highest activity level at pH values of 9 and 7 and temperatures of 50 and 60 °C, respectively. The findings also indicated that the maximum velocity (Vmax) for NBR14 and NBRM9 amylase were 57.80 and 59.88 U/mL, respectively, with Km constants of 1.39 and 1.479 mM. After 48 h, bioethanol was produced at concentrations of 5.95 mg/mL and 9.29 mg/mL from hydrolyzed wheat and bean straw, respectively, through fermentation with S. cerevisiae. Thermophilic actinomycetes and their α-amylase yield demonstrated promising potential for sustainable bio-ethanol production from agro-byproducts.

Nocardiopsis synnemataformans NBRM9, an extremophilic actinomycete producing extremozyme cellulase, using lignocellulosic agro-wastes and its biotechnological applications.

Actinomycetes are an attractive source of lignocellulose-degrading enzymes. The search for actinomycetes producing extremozyme cellulase using cheap lignocellulosic waste remains a priority goal of enzyme research. In this context, the extremophilic actinomycete NBRM9 showed promising cellulolytic activity in solid and liquid assays. This actinomycete was identified as Nocardiopsis synnemataformans based on its phenotypic characteristics alongside phylogenetic analyses of 16S rRNA gene sequencing (OQ380604.1). Using bean straw as the best agro-waste, the production of cellulase from this strain was statistically optimized using a response surface methodology, with the maximum activity (13.20 U/mL) achieved at an incubation temperature of 40 °C, a pH of 9, an incubation time of 7 days, and a 2% substrate concentration. The partially purified cellulase (PPC) showed promising activity and stability over a wide range of temperatures (20-90 °C), pH values (3-11), and NaCl concentrations (1-19%), with optimal activity at 50 °C, pH 9.0, and 10% salinity. Under these conditions, the enzyme retained >95% of its activity, thus indicating its extremozyme nature. The kinetics of cellulase showed that it has a Vmax of 20.19 ± 1.88 U/mL and a Km of 0.25 ± 0.07 mM. The immobilized PPC had a relative activity of 69.58 ± 0.13%. In the in vitro microtiter assay, the PPC was found to have a concentration-dependent anti-biofilm activity (up to 85.15 ± 1.60%). Additionally, the fermentative conversion of the hydrolyzed bean straw by Saccharomyces cerevisiae (KM504287.1) amounted to 65.80 ± 0.52% of the theoretical ethanol yield. Overall, for the first time, the present work reports the production of extremozymatic (thermo, alkali-, and halo-stable) cellulase from N. synnemataformans NBRM9. Therefore, this strain is recommended for use as a biotool in many lignocellulosic-based applications operating under harsh conditions.

Harnessing Mucor spp. for Xylanase Production: Statistical Optimization in Submerged Fermentation Using Agro-Industrial Wastes.

Xylan is the primary hemicellulosic polymer found in lignocellulosic agricultural wastes and can be degraded by xylanase. In the current research, Mucor circinelloides and M. hiemalis were tested for their ability to produce xylanase from tangerine peel by submerged fermentation. Experiments on five variables were designed with Box-Behnken design and response surface methodology. Analysis of variance was exercised, the xylanase output was demonstrated with a mathematical equation as a function of the five factors, and the quixotic states for xylanase biosynthesis was secured. In addition, xylanase was partially purified, characterized, and immobilized on calcium alginate beads. The optimum parameters for xylanase production by M. circinelloides and M. hiemalis were consisted of incubation temperature (30 and 20°C), pH value (9 and 7) incubation period (9 and 9 days), inoculum size (3 and 3 mL), and substrate concentration (3 and 3 g/100 mL), respectively. M. circinelloides and M. hiemalis demonstrated the highest xylanase activities after RSM optimization, with 42.23 and 35.88 U/mL, respectively. The influence of single, interchange, and quadratic factors on xylanase output was investigated using nonlinear regression equations with significant R 2 and p values. The partial purification of M. circinelloides and M. hiemalis xylanase yielded 1.69- and 1.97-fold purification, and 30.74 and 31.34% recovery with 292.08 and 240.15 U/mg specific activity, respectively. Partially purified xylanase from M. circinelloides and M. hiemalis demonstrated the highest activity at neutral pH and 60 and 50°C, respectively. The immobilized M. circinelloides and M. hiemalis xylanase retained 84.02 and 79.43% activity, respectively. The production of xylanase from M. circinelloides and M. hiemalis utilizing RSM is deemed profitable for the decomposition of the agro-industrial wastes.

Antibacterial and cytotoxic potency of thermophilic Streptomyces werraensis MI-S.24-3 isolated from an Egyptian extreme environment.

The need for novel and active antibiotics specially from actinomycetes is essential due to new and drug-resistant pathogens. In this study, 87 actinomycetes were isolated, and 18 strains among them characterized as thermophilic actinomycetes. Further fractionation and preliminary antibacterial activities indicated that one strain, coded as MI-S.24-3, showed good antibacterial activity. Based on the phenotypic, genomic, phylogenetic, and biochemical analyses, MI-S.24-3 was identified as Streptomyces werraensis. Results demonstrated that the ethyl acetate active fraction showed maximum antibacterial activity against Staphylococcus aureus and Escherichia coli with MIC (12.7 ± 0.1 and 18.3 ± 0.2 mg/mL), and MBC (96.5 ± 1.4 and 91.5 ± 0.7 mg/mL), respectively, with determination of time kill kinetics assay. The active fraction showed moderate-to-weak cytotoxic effects against human lung carcinoma (A549 cells), breast cancer cell line (MCF-7), and human cervical carcinoma (HELA cells) with a IC50 of (23.8 ± 1.2, 54 ± 1.8, 96.4 ± 3.2 µg/mL, respectively). Active components were characterised by different chemically volatile, ester, and lactone compounds, determined by GC-MS coupled with daughter ions of (GC-MS/MS). Notably, erucic acid and reynosin identified compounds are rare metabolites produced by Streptomyces werraensis. Our findings demonstrated that the MI-S.24-3 strain could be a potential source for active compounds of biomedical and pharmaceutical interest.