Rare Halophilic Nocardiopsis from Algerian Saharan Soils as Tools for Biotechnological Processes in Pharmaceutical Industry.

The Sahara Desert, one of the most extreme ecosystems in the planet, constitutes an unexplored source of microorganisms such as mycelial bacteria. In this study, we investigated the diversity of halophilic actinobacteria in soils collected from five regions of the Algerian Sahara. A total of 23 halophilic actinobacterial strains were isolated by using a humic-vitamin agar medium supplemented with 10% NaCl. The isolated halophilic strains were subjected to taxonomic analysis using a polyphasic approach, which included morphological, chemotaxonomic, physiological (numerical taxonomy), and phylogenetic analyses. The isolates showed abundant growth in CMA (complex medium agar) and TSA (tryptic soy agar) media containing 10% NaCl, and chemotaxonomic characteristics were consistent with their assignment to the genus Nocardiopsis. Analysis of the 16S rRNA sequence of 23 isolates showed five distinct clusters and a similarity level ranging between 98.4% and 99.8% within the Nocardiopsis species. Comparison of their physiological characteristics with the nearest species showed significant differences with the closely related species. Halophilic Nocardiopsis isolated from Algerian Sahara soil represents a distinct phyletic line suggesting a potential new species. Furthermore, the isolated strains of halophilic Nocardiopsis were screened for their antagonistic properties against a broad spectrum of microorganisms by the conventional agar method (agar cylinders method) and found to have the capacity to produce bioactive secondary metabolites. Except one isolate (AH37), all isolated Nocardiopsis showed moderate to high biological activities against Pseudomonas syringae and Salmonella enterica, and some isolates showed activities against Agrobacterium tumefaciens, Serratia marcescens, and Klebsiella pneumoniae. However, no isolates were active against Bacillus subtilis, Aspergillus flavus, or Aspergillus niger. The obtained finding implies that the unexplored extreme environments such as the Sahara contain many new bacterial species as a novel drug source for medical and industrial applications.

Heterologous Expression and Structural Elucidation of a Highly Thermostable Alkaline Serine Protease from Haloalkaliphilic Actinobacterium, Nocardiopsis sp. Mit-7.

A highly thermostable alkaline serine protease gene (SPSPro, MN429015) obtained from haloalkaliphilic actinobacteria, Nocardiopsis sp. Mit-7 (NCIM-5746), was successfully cloned and overexpressed in Escherichia coli BL21 under the control of the T7 promoter in the pET Blue1 vector leading to a 20-kDa gene product. The molecular weight of the recombinant alkaline protease, as determined by SDS-PAGE and the Mass Spectrometer (MALDI-TOF), was 34 kDa. The structural and functional attributes of the recombinant thermostable alkaline serine protease were analyzed by Bioinformatic tools. 3D Monomeric Model and Molecular Docking established the role of the amino acid residues, aspartate, serine, and tryptophan, in the active site of thealkaline protease.The activity of the recombinant alkaline protease was optimal at 65 °C, 5 °C higher than its native protease. The recombinant protease was also active over a wide range of pH 7.0-13.0, with a maximal activity of 6050.47 U/mg at pH 9. Furthermore, the thermodynamic parameters of the immobilized recombinant alkaline protease suggested its reduced vulnerability against adverse conditions under which the enzyme has to undergo varied applications.

Nocardiopsis changdeensis sp. nov., an endophytic actinomycete isolated from the roots of Eucommia ulmoides Oliv.

Strain Mg02T was isolated from roots of Eucommia ulmoides Oliv. collected from Changde City, Hunan Province, China. Strain Mg02T, which exhibited distinct chemotaxonomic characteristics of the genus Nocardiopsis: cell-wall chemotype III/C, i.e., meso-diaminopimelic acid as diagnostic amino acid in whole-cell hydrolysates and menaquinone MK-10 with variable degrees of saturation in the side chain as the predominant isoprenoid quinone, was investigated by a polyphasic approach to determine their taxonomic position. Sequence analysis of the 16S rRNA gene indicated that strain Mg02T is affiliated to the genus Nocardiopsis, having highest sequence similarity to Nocardiopsis flavescens CGMCC 4.5723T (99.1%) and <98.7% to other species of the genus Nocardiopsis with validly published names. Phylogenetic analysis of 16S rRNA gene indicated strain Mg02T formed a separate evolutionary clade, suggesting that it could be a novel Nocardiopsis species. Phylogenomic analysis showed that strain Mg02T was closely related to N. flavescens CGMCC 4.5723T and distinct from the latter according to the clustering patterns. The Average Nucleotide Identity and digital DNA-DNA hybridization values between strain Mg02T and N. flavescens CGMCC 4.5723T were far below the species-level thresholds. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, we think that strain Mg02T should represent a novel Nocardiopsis species, for which the name Nocardiopsis changdeensis sp. nov. is proposed. The type strain is Mg02T (=MCCC 1K06174T = JCM 34709T).

Two strains of airborne Nocardiopsis alba producing different volatile organic compounds (VOCs) as biofungicide for Ganoderma boninense.

Nocardiopsis are actinobacteria which produce active compounds, such as antifungals and volatile compounds. Ganoderma boninense is a pathogenic and aggressive fungus that decreases palm oil yield during production. In this study, we isolated two strains of Nocardia (GME01 and GME22) from airborne contaminants on the actinobacteria culture collection in the laboratory. The aim of this study is to identify two strains of Nocardiopsis and to obtain the antifungal potency of volatile organic compounds (VOCs) against G. boninese. We characterized the morphology using Scanning Electrone Microscope (SEM), molecular properties and whole-cell protein spectra using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), antifungal assay on G. boninense and VOCs analysis of Nocardia using solid phase micro extraction/gas chromatography (SPME/GC). The two Nocardiopsis strains had the similar characteristic such as white aerial mycelium and spores, aerobic, grow well on ISP-2, TSA and NA medium without diffusible pigment and had the highest similarity with Nocardiopsis alba DSM 43377 (99.63% and 99.55% similarity for GME01 and GME22, respectively), Different morphological feature was found in aerial mycelium and spores. GME22 has a clearly fragmented mycelium whereas GME01 has none. Other features also showed different on the whole-cell protein spectra, antifungal activity and VOCs profiles. Antifungal activity assay on G. boninense showed that N. alba GME22 has higher antifungal activity than GME01 related with the VOCs abundance in two strains. Almost 38.3% (18 VOCs) of N. alba GME22 and 25.5% (12 VOCs) of N. alba GME01 were found specifically in each strain, and 36.2% (the 17 same VOCs) produced by both. The known volatile antifungal compounds S-methyl ethanethioate, 1,2-dimethyldisulfane, acetic acid, 2-methyl propanoic acid, 3-methyl-butanoic acid, nonan-2-one, undecan-2-one and 2-isopropyl-5-methylcyclohexan-1-ol only produced by N. alba GME22 and 1,3-dimethyltrisulfane only produced by N. alba GME01. A total of two known antifungal compounds 1,2-dimethyldisulfane and 6-methylheptan-2-one were produced by both N. alba. The abundance of antifungal VOCs produced by these bacteria is potentially to be used as biocontrol agent for pathogenic fungi in plants.

Biosynthesis of silver nanoparticles by Nocardiopsis sp.-MW279108 and its antimicrobial activity.

The utilization of microorganisms like bacteria in the biological synthesis of silver nanoparticles (AgNPs) has attracted widespread attention due to their ability to synthesize different shape sizes, states, and morphology nanoparticles. In the current study, the green synthesis of AgNPs by Nocardiopsis sp. 16S ribosomal RNA analysis was used to characterize the Nocardiopsis sp. The synthesized AgNPs were characterized through multi-instrument platforms such as transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), UV-Vis spectroscopy, scanning electron microscope (SEM), and X-ray diffraction analysis (XRD). The antimicrobial activity of the synthesized AgNPs was determined by the agar plate diffusion method. The UV-Vis absorbance analysis of the synthesized AgNPs has a significant absorbance at 384 nm, confirming the AgNPs' surface plasmon resonance. The SEM and TEM characterizations indicate that the particle size ranges from 2 to 10 nm and is spherical. Additionally, the FTIR spectra revealed bands from 476 to 3819cm-1 , respectively. The XRD planes study pronounced strong bands ranging are between 111 and 311 corresponding to cubic face-center of the silver. Also, the antimicrobial activity of AgNPs indicated the biogenic AgNPs could control the growth of the clinical isolates. The AgNPs produced by Nocardiopsis sp. supernatant could be used in different nanomedicinal applications.

Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5.

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.