A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by
Anoxybacillus kamchatkensis
M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer ...with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH
2
-terminal amino-acid residues showed high homology with those of
Bacillus
proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The s
apA
gene was cloned and expressed in the extracellular fraction of
E. coli
. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from
Bacillus subtilis
WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from
Bacillus pumilus
CBS and subtilisin A from
Bacillus licheniformis
, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.
Graphic Abstract
Highlights
A new extracellular
Anoxybacillus kamchatkensis
protease was purified (SAPA) and characterized.
SAPA was a serine protease and a monomer with a molecular mass of ∼28 kDa.
Optimum pH and temperature values for activity were pH 11 and 70 °C, respectively.
The
sapA
gene encoding SAPA was cloned, sequenced, and expressed in
E. coli
.
rSAPA is a potential candidate for future application as laundry detergent additive.
•The paper reports on two novel extracellular peroxidases from Bjerkandera adusta CX-9.•These enzymes were purified (MnP BA30 and LiP BA45) and biochemically characterized.•The molecular weights and ...the NH2-terminal sequences of the enzymes were determined.•Their catalytic efficiency, solvent-stability, and dye-decolorization were studied.•These enzymes may be considered as potential candidates in distaining synthetic dyes.
Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH2-terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The Km and Vmax for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes.
Caldicoprobacter guelmensis isolated from the hydrothermal hot spring of Guelma (Algeria) produced high amounts of extracellular thermostable serine alkaline protease (called SAPCG) (23,000U/mL). The ...latter was purified by ammonium sulphate precipitation, UNO Q-6 FPLC and Zorbex PSM 300 HPLC, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 55,824.19Da. The 19 N-terminal residue sequence of SAPCG showed high homology with those of microbial proteases. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine protease family. It showed optimum protease activity at pH 10 and 70°C with casein as a substrate. The thermoactivity and thermostability of SAPCG were enhanced in the presence of 2mM Ca2+. Its half-life times at 80 and 90°C were 180 and 60min, respectively. Interestingly, the SAPCG protease exhibited significant compatibility with iSiS and Persil, and wash performance analysis revealed that it could remove blood-stains effectively. Overall, SAPCG displayed a number of attractive properties that make it a promising candidate for future applications as an additive in detergent formulations.
In a previous study, a thermostable α-amylase-producing bacterium (designated HB23) was isolated from an Algerian hydrothermal spring. In the present study, the native strain was subjected to a ...statistical optimization aimed at enhancing the α-amylase production. To achieve this, thirteen factors have been studied, among which are cultural and nutritional parameters. Wheat bran, a by-product of the grain milling industry, was the factor that positively influenced α-amylase production. A modified L27 Taguchi design was used to screen these factors. Furthermore, a Box-Behnken matrix, supplemented by the use of response surface methodology (RSM), allowed for the identification of optimum levels of the following factors: a 1% inoculum size, 15 g/L soluble starch, 5 g/L wheat bran, and 1 g/L tryptone. Optimized conditions resulted in an amylolytic activity of 320 U/mL, which is a tenfold increase when compared with unoptimized production level. Phenotypical and molecular identification of strain HB23 revealed its close relationship to various
Tepidimonas
strains, specifically to
Tepidimonas fonticaldi
. The crude enzyme preparation turned out to be compatible with various laundry detergents and led to a substantial improvement in their washing performance. A comparison of the performance of the crude enzyme preparation with that of the commercial α-amylase (Termamyl
®
300 L) highlights the potential of the HB23 enzyme as a bio-additive in detergent formulations.
A thermophilic anaerobic bacterium (strain TH7C1T) was isolated from the hydrothermal hot spring of Guelma in the northeast of Algeria. Strain TH7C1T stained Gram-positive, was a non-motile rod ...appearing singly, in pairs, or as long chains (0.7-1 × 2-6 μm²). Spores were never observed. It grew at temperatures between 55 and 75°C (optimum 65°C) and at pH between 6.2 and 8.3 (optimum 6.9). It did not require NaCl for growth, but tolerated it up to 5 g l⁻¹. Strain TH7C1T is an obligatory heterotroph fermenting sugars including glucose, galactose, lactose, raffinose, fructose, ribose, xylose, arabinose, maltose, mannitol, cellobiose, mannose, melibiose, saccharose, but also xylan, and pyruvate. Fermentation of sugars only occurred in the presence of yeast extract (0.1%). The end-products from glucose fermentation were acetate, lactate, ethanol, CO₂, and H₂. Nitrate, nitrite, thiosulfate, elemental sulfur, sulfate, and sulfite were not used as electron acceptors. The G+C content of the genomic DNA was 44.7 mol% (HPLC techniques). Phylogenetic analysis of the small-subunit ribosomal RNA (rRNA) gene sequence indicated that strain TH7C1T was affiliated to Firmicutes, order Clostridiales, family Caldicoprobacteraceae, with Caldicoprobacter oshimai (98.5%) being its closest relative. Based on phenotypic, phylogenetic, and genetic characteristics, strain TH7C1T is proposed as a novel species of genus Caldicoprobacter, Caldicoprobacter algeriensis, sp. nov. (strain TH7C1T = DSM 22661T = JCM 16184T).
Halophilic archaea are the dominant type of microorganisms in hypersaline environments. The diversity of halophilic archaea in Zehrez-Chergui (Saharian chott) was analyzed and compared by both ...analysis of a library of PCR amplified 16S rRNA genes and by cultivation approach. This work, represents the first of its type in Algeria. A total cell count was estimated at 3.8 × 10
3
CFU/g. The morphological, biochemical, and physiological characterizations of 45 distinct strains, suggests that all of them might be members of the class
Halobacteria
. Among stains, 23 were characterized phylogenetically and are related to 6 genera of halophilic archaea.The dominance of the genus
Halopiger
, has not been reported yet in other hypersaline environments. The 100 clones obtained by the molecular approach, were sequenced, and analyzed. The ribosomal library of 61 OTUs showed that the archaeal diversity included uncultured haloarcheon,
Halomicrobium
,
Natronomonas
,
Halomicroarcula, Halapricum, Haloarcula, Halosimplex, Haloterrigena, Halolamina, Halorubellus, Halorussus
and
Halonotius
. The results of rarefaction analysis indicated that the analysis of an increasing number of clones would have revealed additional diversity. Surprisingly, no halophilic archaea were not shared between the two approaches. Combining both types of methods was considered the best approach to acquire better information on the characteristics of soil halophilic archaea.
The efficiency of the proteolytic strain
Anoxybacillus kamchatkensis
M1V in the fermentation of speckled shrimp by-product was investigated for the recovery of a deproteinized bioactive hydrolysate. ...The biological activities of the resulting hydrolysate were also examined by applying several antioxidant and enzyme inhibitory assays. The strain M1V was found to produce high level of protease activity (2000 U/mL) when grown in media containing only shrimp powder at 25 g/L. The crude protease displayed a significant deproteinization capabiliy, with the best efficiency (48%) being recorded for an enzyme to substrate (E/S) ratio of 30 U/mg. Following the deproteinization, chitin was recovered and the authenticity was confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis. On the other hand, the obtained hydrolysate showed a significant enzymatic inhibitory potential against acetylcholinesterase, tyrosinase, amylase, and angiotensin I convertase, and a strong antioxidant activity.
Graphical Abstract
This paper aims to characterize halophilic bacteria inhabiting Algerian Saline Ecosystems (Sebkha and Chott) located in arid and semi-arid ecoclimate zones (Northeastern Algeria). In addition, ...screening of enzymatic activities, heavy metal tolerance and antagonistic potential against phytopathogenic fungi were tested. A total of 74 bacterial isolates were screened and phylogenetically characterized using 16S rRNA gene sequencing. The results showed a heterogeneous group of microorganisms falling within two major phyla, 52 strains belonging to Firmicutes (70.2%) and 22 strains (30.8%) of
γ
-Proteobacteria. In terms of main genera present, the isolates were belonging to
Bacillus, Halobacillus, Lentibacillus, Oceanobacillus, Paraliobacillus
,
Planomicrobium, Salicola, Terribacillus
,
Thalassobacillus
,
Salibacterium
,
Salinicoccus
,
Virgibacillus
,
Halomonas, Halovibrio,
and
Idiomarina
. Most of the enzymes producers were related to
Bacillus, Halobacillus,
and
Virgibacillus
genera and mainly active at 10% of growing salt concentrations. Furthermore, amylase, esterase, gelatinase, and nuclease activities ranked in the first place within the common hydrolytic enzymes. Overall, the isolates showed high minimal inhibitory concentration values (MIC) for Ni
2+
and Cu
2+
(0.625 to 5 mM) compared to Cd
2+
(0.1 to 2 mM) and Zn
2+
(0.156 to 2 mM). Moreover, ten isolated strains belonging to
Bacillus
,
Virgibacillus
and
Halomonas
genera, displayed high activity against the pathogenic fungi (
Botrytis cinerea
,
Fusarium oxyporum
,
F.
verticillioides
and
Phytophthora capsici
). This study on halophilic bacteria of unexplored saline niches provides potential sources of biocatalysts and novel bioactive metabolites as well as promising candidates of biocontrol agents and eco-friendly tools for heavy metal bioremediation.
The emergence of a large number of antimicrobialresistant organisms is a cause for concern. Nature is historically the source of drugs; indeed a considerable number of drugs have been developed from ...microorganisms, and are now used daily in the treatment of infectious diseases. However, the introduction to the pharmaceutical market of new therapeutic molecules has decreased during the last two decades. In this review, the genus
Micromonospora
is proposed as a biofactory for production of new antibiotics. The genus
Micromonospora
has been investigated extensively and more than 100 antibiotics have been isolated from diverse
Micromonospora
strains. In addition, recent developments in analytical, biological and bioinformatics screening tools used in the discovery of new therapeutic compounds are described. It may be possible to revive formerly used antibiotics produced by
Micromonospora
and study of this genus may facilitate discovery of novel bioactive molecules.
An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2AT gen. nov. sp. nov., was purified and biochemically ...characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI–TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH2-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its Km and kcat values were 0.253 mg colloidal chitin/mL and 47000 s−1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-β-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.
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•Purification of chitinase (ChiA-Mt45) from M. thermohalophilus Nari2AT was carried out.•The molecular weight and the NH2-terminal sequence of the chitinase was determined.•Optimum pH and temperature values for activity were pH 3.5 and 90 °C, respectively.•The kinetic parameters and the TLC of hydrolysis products of the enzyme were studied.•ChiA-Mt45 may be used as potential candidate for the enzymatic degradation of chitin.
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