Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest

Background Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration. Results A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 C for 20min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme 500 L, respectively. Conclusions This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations

Characterization of endospore-forming bacteria producing extracellular enzymes isolated from the Djurdjura Mountains in Algeria

Biodiversity in mountains in Algeria appears scanty and has not been thoroughly investigated. However, the mountain soil has been shown as an  almost entire reserve of novel enzymes with interesting properties for industrial and environmental applications. In the present study, thirty  bacterial strains were isolated from the Djurdjura Mountains in Kabylia (Algeria) and were studied for their ability to produce enzymes to be possibly  used in biotechnological processes such as amylase, caseinase, and chitinase. The characterization of these isolates was carried out using  morphological, physiological, and biochemical characteristics. All the data obtained with regards to the phenotypical properties of the isolates,  confirmed that the strains belonged to the Bacillus group. In addition, the 16S rRNA gene of the two retained strains KA15 and LK-DZ15 was also  amplified and sequenced. Phylogenetic tree was, afterwards, constructed. The nucleotide sequences and blast analyses confirmed that the KA15  and LK-DZ15 strains were closely related to those of the Bacillus altitudinis (accession n°.: MK874318) and Paenibacillus timonensis (accession n°.:  MK734103) strains. The presence of amylases, proteases, and chitinases in KA15 and LK-DZ15 isolates are an indicator of their pivotal application in  a variety of biotechnological processes.&nbsp

Statistical Experimental Design Optimization of Microbial Proteases Production under Co-Culture Conditions for Chitin Recovery from Speckled Shrimp Metapenaeus monoceros By-Product

This study was designed with the aim to produce microbial proteases in presence of speckled shrimp by-product. For this reason, three strains belonging to Bacillus genus, namely, Aeribacillus pallidus VP3, Lysinibacillus fusiformis C250R, and Anoxybacillus kamchatkensis M1V were studied under co-culture procedure. A Taguchi L27 experimental design was applied to optimize the co-culture parameters. The experimental design was built with 9 factors (by-product powder concentration, the pH of the medium, the temperature, the sucrose concentration, the agitation speed, the inoculum sizes of VP3, M1V, and C250R strains, and the culture volume) at three different levels. The obtained results showed that a total protease activity of 8,182 U/mL could be achieved after 24 h of incubation in presence of 20 g/L shrimp by-product and 10 g/L sucrose, at an initial pH of 7, a 40°C temperature and absorbance, at 600 nm, of inoculum sizes of 0.1, 0.3, and 0.1 for VP3, M1V, and C250R strains, respectively. The agitation was set at 200 rpm, and the final volume was 25 mL. Taguchi’s design allowed the identification of temperature, the inoculum size for strain VP3, the inoculum size for strain M1V, and the final culture volume as the most influencing variables. A Box–Behnken design with 27 experiments was carried out for the optimization of these four selected factors. Following such design, the highest protease production reached was 11,300 U/mL. This yield was obtained in a final culture volume of 15 mL containing 20 g/L shrimp by-product powder and 10 g/L sucrose and inoculated with VP3, C250R, and M1V strains at 0.05, 0.1, and 0.2, respectively. The flasks were incubated at 45°C for 24 h with shaking at 200 rpm. The efficiency of chitin extraction by co-cultivation was investigated under the latter conditions. The chitin yield from shells by-product was 16.7%. Fourier-Transform Infrared (FTIR) analysis of the obtained chitin displayed characteristic profiles similar to that of the commercial α-chitin

Cloning, expression, and structural modeling of two alkaline serine protease genes from extremophilic Bacillaceae -related species: Application in valorization of invasive crustaceans

Two novel protease genes sapA and sapN from the thermophilic  Anoxybacillus kamchatkensis M1V and Melghiribacillus thermohalophilus Nari2AT strains respectively, encoding a polypeptide of 381 and 379 residues, were identified, cloned and successfully heterologously expressed in Escherichia coli BL21(DE3)pLysS. The deduced putative amino-acid residues of SAPA and SAPN enzymes evinced identity with proteases from Bacillus strains. 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. While, the highest sequence identity (97.10%) of SAPN was observed with Bacillus licheniformis MP1 protease, but with 10 difference residues. rSAPA and rSAPN enzymes were purified until homogeneity, characterized, and compared to wild-type proteases.  The purified recombinant enzymes rSAPA and rSAPN were two monomers of about 28 and 30 kDa, correspondingly. rSAPA displayed the highest activity at pH 11 and 70°C. While, rSAPN displayed the highest activity at pH 10 and 75°C. To initiate structure-function relationships, a 3Dmodel of the Pro-SAPA and Pro-SAPN proteins were thereafter built based on the available structures of common proteases. The comparative molecular modeling studies with the less thermostable protease, revealed extra charged residues at the surface of SAPA and SAPN potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability

Purification, Biochemical and Kinetic Characterization of a Novel Alkaline sn-1,3-Regioselective Triacylglycerol Lipase from Penicilliumcrustosum Thom Strain P22 Isolated from Moroccan Olive Mill Wastewater

A novel extracellular lipase from a filamentous fungus Ascomycota strain, P22, was isolated from olive mill wastewater, then purified and characterized. This strain was identified as Penicillium crustosum Thom based on sequencing analyses. Penicilliumcrustosum Thom strain P22 lipase (PCrL) was purified 63-fold to homogeneity using ammonium sulfate precipitation and chromatography on a Q-Sepharose Fast Flow column, with a total yield of 34%. The purified PCrL had a molecular mass of 28 kDa, estimated by SDS-PAGE. The 20 NH2-terminal amino-acid residues showed a high degree of homology with those of other Penicillium lipases. The specific activity of PCrL at pH 9 and 37 °C were found to be 5000 and 10,000 U/mg on olive oil and trioctanoin emulsions, respectively. PCrL exhibited clear regioselectivity toward the sn-1 position of the surface-coated triglycerides which were esterified with α-eleostearic acid at the sn-1/3 position. PCrL was completely inhibited by 53 µM of Orlistat, 5 mM of phenylmethylsulfonylfluoride, and 2 mM of diiodopropyl fluorophosphate, suggesting that it belonged to the serine lipase family. PCrL showed high activity and stability in the presence of water-immiscible organic solvents, surfactant, and oxidizing agents, and showed considerable compatibility with commercial laundry detergents. Washing performance analysis revealed that it could effectively remove oil stains. Hence, PCrL has several attractive properties that make it a promising potential candidate for detergent formulations

Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 μM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents

Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2AT for chitin extraction from crab and shrimp shell by-products

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