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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. [ABSTRACT FROM AUTHOR]

Due to its various biological activities, the roots of Brassica rapa (turnip), has been applied as a therapeutic agent in traditional medicine. The current study aimed to purify a plant peroxidase (POD; designated as TRP) from Algerian white turnip (B. rapa, variety rapa) roots, to homogeneity and to characterise it biochemically. The molecular weight of the purified TRP was determined to be ∼58 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The TRP Reinheitzahl values (RZ) and specific activity were 2.9 and 14,500 U/mg, respectively. Its N-terminal sequence exhibited high identity with those of class III-PODs. TRP showed optimal activity at 55 °C and pH 6 (guaiacol), was completely inhibited by sodium azide (NaN3) and potassium cyanide (KCN) and exhibited greater catalytic efficiency than that of the well-known horseradish peroxidase (HRP) from Armoracia rusticana. TRP demonstrated antioxidant activities with 45% and 61.49% of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•) and hydroxyl (•OH) radical scavenging, respectively, as well as ferrous ion chelating (52.96%) and reducing (48.32%) powers. This study provides information regarding the potential role of TRP in enzyme therapy (as applied in the pharmaceutical and medicinal industries), with biological eradication of free radicals. [ABSTRACT FROM AUTHOR]

Bacillus amyloliquefaciens is a food spoilage spore‐forming bacterium. Its spores are useful for multiple biotechnological applications. Nevertheless, few reports are available regarding the achievement of a high cell density and good sporulation effectiveness under fermentation conditions. Therefore, the current study was designed to optimize a low‐cost fermentation medium allowing the highest sporulation yield by B. amyloliquefaciens strain BS13. Our data revealed that tryptone and starch were the best carbon and energy sources. In addition, two nitrogen sources namely, corn steep liquor (CSL) and yeast extract (YE), allowed a significant enhancement of spore production and they were both retained for further optimization. A combination of CaCl2, MgSO4, and MnSO4 showed a positive impact on spores' production. The composition of the optimized medium was (in g/L); tryptone 3, starch 15, CSL 13.5, YE 1.5, CaCl2 0.1, MgSO4·7H2O 0.012, and MnSO4·7H2O 0.0012. Such medium was further validated in a 400‐L fermentor. The spore yield by B. amyloliquefaciens strain BS13 was enhanced from 3.0 × 1010 spores/mL under flask culture conditions to 6.2 × 1010 spores/mL when cultures were performed on large scale. Therefore, strain BS13 spore preparation could be proposed as a promising probiotic and a biocontrol agent useful for plants, animals, and humans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A novel extracellular lignin peroxidase (LiP) has been produced (65 U/mL) in potato dextrose broth (PDB)-optimized media by a white-rot Agaricomycetes fungus strain KB-DZ15 newly isolated from dead deciduous trees at Zimoula Forest (Tizi-Ouzou, Algeria). This strain was identified as Phlebia radiata based on internal transcribed spacer (ITS) rDNA gene sequencing. The peroxidase enzyme (called LiP PR40) was purified and characterised. The matrix-assisted laser desorption-ionization – time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that LiP PR40 enzyme was a monomer with a molecular mass of 40,125.31 Da. The 25 N-terminal residue sequence of LiP PR40 showed high similarity with those of fungi LiPs. Interestingly, LiP PR40 presents premium activity at pH 3 and 80 °C with specific activity and Reinheitzahl (RZ) level of 510 U/mg and 2.81, respectively. Furthermore, it was completely inhibited by sodium azide (NaN3) and potassium cyanide (KCN), suggesting the presence of haem components in its tertiary structure. More interestingly, LiP PR40 illustrated more upper catalytic efficiency and dye-decolorisation aptitude than that of commercial well-known horseradish peroxidase (HRP) from roots of Armoracia rustanica and the purified reported LiP BA45 from Bjerkandera adusta strain CX-9. Consequently, these properties make LiP PR40 a potential candidate for destaining synthetic-dyes and lignin-biodegradation. [ABSTRACT FROM AUTHOR]

The ability of microorganisms to grow at high temperature, alkaline pH, and high salinity makes them an attractive target for enzyme‐production with several industrial applications. One strain TN‐X30 has been selected as protease producer and identified as Streptomyces mutabilis after a phenotypic and molecular study. Its production of protease was improved using Taguchi L27 design. The strategy was carried out to identify the optimum levels and the interaction of the screened factors. Following this step, maximum protease activity (10,895 U/ml) was achieved after 6‐days of incubation. The TN‐X30 protease activity had an optimum of pH and temperature of 10 and 65°C, respectively. Thermodynamic parameters at 60°C were enthalpy 14.26 kJ/mol, entropy −220 J/mol/K, and Gibbs free energy 90.53 kJ/mol. TN‐X30 protease production displayed a 16‐fold increase reaching 175,000 U/ml in a 100‐L fermentor. Furthermore, the lyophilization in presence of sorbitol enhanced the stability of the TN‐X30 protease which remained active at 75% after 24‐months of storage. The lyophilized TN‐X30 protease exhibited exceptional stability indexes in presence of some known commercialized detergent components as NEODOL® 25‐7, Dehydol® LT 7, Na2 CMC, Galaxy LAS, Galaxy LES 70, Galaxy 110, Galaxy CAPB Plus, and Sulfacid K. The lyophilized enzyme also displayed high stability with respect to both solid and liquid detergents. Finally, TN‐X30 protease exhibited remarkable destaining of blood, egg, and chocolate stained cloth pieces. These findings may promote TN‐X30 protease for use as bioadditive in detergent formulation, thereby reducing environmental chemical threat. [ABSTRACT FROM AUTHOR]

The effect of dietary Kefir supplementation on the biometric, biochemical, and histological parameters of Nile tilapia (Oreochromis niloticus) exposed to aflatoxin B1 (AFB1, 200 µg/kg diet) contamination was studied. The yeasts were dominant in Kefir followed by lactic and acetic acid bacteria. The Kefir showed relatively interesting antioxidant potential in the DPPH• (IC50 = 0.9 ± 0.02 mg/ml) and ABTS•+ (IC50 = 2.2 ± 0.03 mg/ml) scavenging activities, Fe3+‐reducing power (EC0.5 = 1.2 ± 0.01 mg/ml), and β‐carotene bleaching assay (IC50 = 3.3 ± 0.02 mg/ml). Three hundred and sixty Nile tilapia weighing 23 ± 5 g were divided into four groups (30 fish/group with 3 replicates), and fed with diets containing Kefir (D2), AFB1 (D3), and Kefir+AFB1 (D4) for 4 weeks, whereas D1 was kept as control group where fish were fed with basal diet. The Kefir supplementation in D4 group significantly increased (p <.05) the percent weight gain as compared to D3 group. Moreover, Kefir improved the antioxidant enzymes in the liver, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities, that significantly increased (p <.05) by 2‐, 3‐, and 1.5‐folds, respectively, as compared to D3 group. The Kefir treatment significantly decreased (p <.05) the liver malonaldehyde content by ~50% as compared to D3 group. Histopathological analysis revealed the hepatoprotective effects of Kefir by showing normal liver histological architecture in D4 group, as compared to degenerative changes observed in D3 group. These results suggest that Kefir could be considered as a potential probiotic in Nile tilapia feed to mitigate the AFB1 harmful effects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The current paper reports on the production and characterization of extracellular thermostable proteases produced from thermophilic bacteria namely Aeribacillus pallidus VP3, Anoxybacillus kamchatkensis M1V, Virgibacillus natechei FarDT, and Melghiribacillus thermohalophilus Nari2AT strains isolated from different extremophlic biotopes in Tunisia and Algeria. The key challenge of the use of the different proteases in industrial applications is their efficiency under unconventional conditions. An optimization via one-factor-at-a-time (OFAT) methodology has been used to obtain 3,000 U.mL-1; 4,600 U.mL-1; 15,800 U.mL-1; and 16,000 U.mL-1 of proteases activity from VP3, M1V, FarDT, and Nari2AT strains, respectively. Particularly, VP3 and FarDT proteases showed a high tolerance to several organic solvents. In addition, the protease produced from FarDT strain might be useful as potential candidate for application in detergency. The deproteinization of shrimp wastes by M1V and Nari2AT proteases was successful with a high rate of deproteinization of about 48% and 82%. The obtained proteolytic hydrolysate obtained by M1V strain showed high biological activities with half maximal inhibitory concentration (IC50) of 71.52 µg.mL-1, 107.67 µg.mL-1, and 133.24 µg.mL-1, respectively for the angiotensin, tyrosinase, and amylase inhibitory activities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

-is 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 coculture parameters. -e 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. -e 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.-e 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. -is yield was obtained in a final culture volume of 15 mL containing 20 g/L shrimp byproduct powder and 10 g/L sucrose and inoculated with VP3, C250R, and M1V strains at 0.05, 0.1, and 0.2, respectively. -e flasks were incubated at 45°C for 24 h with shaking at 200 rpm. -e efficiency of chitin extraction by co-cultivation was investigated under the latter conditions. -e 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. [ABSTRACT FROM AUTHOR]

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists. [ABSTRACT FROM AUTHOR]

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2AT. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH2-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products. Article Highlights: The purification of subtilisin (SAPN) from M. thermohalophilus Nari2A was carried out. The molecular weight and the NH2-terminal sequence of the subtilisin were determined. Optimum pH and temperature values for activity were pH 10 and 75 °C respectively. SAPN was found to be effective in the deproteinization of crab and shrimp by-products. SAPN may be used as candidate for chitin extraction from crustacean by-products. [ABSTRACT FROM AUTHOR]

In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO 4 ) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100 U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680 U/mL). Additionally, a new extracellular 51 kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70 °C and pH 10. Its half-life times at 70 and 80 °C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5 L from Bacillus licheniformis . This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5 L; which offers an interesting potential for its application in the laundry detergent industry. [ABSTRACT FROM AUTHOR]

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22 h of incubation at 45 °C was 3000 U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40–60%)-dialysis followed by heat-treatment (70 °C for 30 min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29 kDa. The sequence of the 25 NH 2 -terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60 °C, respectively. Its half-life times at 70 and 80 °C were 8 and 4 h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5 L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis. [ABSTRACT FROM AUTHOR]

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40–70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH 2 -terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference. [ABSTRACT FROM AUTHOR]

An extracellular acido-thermostable endo -chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60 °C was 3000 U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH 2 -terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85 °C, respectively. The pure enzyme was completely inhibited by p -chloromercuribenzoic acid ( p -CMB) and N -ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo -chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its K m and k cat values were 0.298 mg colloidal chitin/ml and 14400 s −1 , respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo -splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin. [ABSTRACT FROM AUTHOR]

A new serine alkaline protease (designated as SAPGB) from Gracilibacillus boraciitolerans strain LO15, was produced (9000 U/mL), purified, and characterized. SAPGB has a monomer structure with a precise molecular weight of 30,285.03 kDa as learnt from matrix-assisted laser desorption/ionization-time of flight/mass spectroscopy (MALDI-TOF/MS) exploration. The NH 2 -terminal amino-acid succession revealed significant identity with Bacillus proteases. The SAPGB was irreversibly inhibited by diiodopropyl fluorophosphates (DFP) and phenylmethylsulfonyl fluoride (PMSF). The enzyme displayed optimum activity at 65 °C and pH 10. The maximal activity was achieved in the range 0.5–5 M NaCl and about 52% of the activity was preserved across the broad salinity range of 0–30%. SAPGB exhibited a considerable catalytic efficiency (ratio k cat / K m) and degree of hydrolysis (DH). In addition, SAPGB showed a high tolerance to several organic solvents and an excellent detergent compatibility than SAPV, SAPA, Thermolysin type X, and Esperase 8.0 L. These properties make SAPGB a potential candidate for detergent formulations. On the other hand, sapGB gene was cloned and expressed in E. coli BL21(DE3)pLysS and the biochemical properties of the purified extracellular recombinant protease (rSAPGB) were similar to those of SAPGB. Finally, a 3D structural model of SAPGB was constructed by homology modeling. Unlabelled Image • The protease SAPGB from Gracilibacillus boraciitolerans strain LO15 was purified and typified. • The optimum pH and temperature values for activity were pH 10 and 65 °C, respectively. • The sapGB gene encoding SAPGB, was cloned, sequenced, and expressed in E. coli BL21(DE3)pLysS. • Pro-SAPGB 3D-structure model was built using structure of Pro-subtilisin E (PDB ID: 3WHI). • The SAPGB enzyme is a potential candidate for peptide synthesis and detergent formulations. [ABSTRACT FROM AUTHOR]

The present study investigated the purification, biochemical, and molecular characterization of a novel thermostable α-amylase (TfAmy48) from Tepidimonas fonticaldi strain HB23. MALDI-TOF/MS analysis indicated that the purified enzyme is a monomer with a molecular mass of 48,138.10 Da. The results from amino-acid sequence analysis revealed high homology between the 25 NH 2 -terminal residues of TfAmy48 and those of Gammaproteobacteria α-amylases. The optimum pH and temperature values for α-amylase activity were pH 8 and 80 °C, respectively. Thin-layer chromatography (TLC) analysis showed that the final hydrolyzed products of the enzyme from soluble potato starch were maltopentaose, maltose, and maltotriose, which indicate that TfAmy48 possessed an endo -acting pattern. Compared to Termamyl®300 L, TfAmy48 showed extreme stability and tolerance towards organic solvents and excellent compatibility with some commercial laundry detergents. These proprieties make TfAmy48 enzyme a potential candidate as a cleaning bioadditive in detergent composition. The Tfamy 48 gene encoding TfAmy48 was cloned, sequenced, and heterologously-expressed in the extracellular fraction of Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rTfAmy48) were similar to those of native one. The highest sequence identity value (97%) was obtained with PsAmy1 α-amylase from Pseudomonas sp. strain KFCC10818, with only 16 amino-acid (aa) residues of difference. • A novel α-amylase from T. fonticaldi HB23 was purified (TfAmy48) and characterized. • TfAmy48 was an endo -acting and monomeric amylase with a MW of 48,138.10 Da. • Optimum pH and temperature values for activity were pH 8 and 80 °C, respectively. • The Tfamy 48 gene encoding TfAmy48 was cloned, sequenced, and expressed in E. coli. • TfAmy48 is a potential candidate as a cleaning bioadditive in detergent composition. [ABSTRACT FROM AUTHOR]

Abstract A new ascomycete fungus X5, a hyperproducer (9000 U/mL) of a serine alkaline protease (SAPTEX) was identified as Penicillium chrysogenum. The experimental purification protocol comprises three steps: heat treatment (10 min at 80 °C) followed by an ammonium sulfate precipitation (30–50%)-dialysis, and a UNO Q-12 anion exchange chromatography using the FPLC system. The chemical characterizations performed include physico-chemical determination and spectroscopic analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 43,074.11 Da. The 25 residue NH 2 -terminal sequence of the enzyme showed high homology with Penicillium proteases. The optimum pH and temperature values for protease activity were pH 10 and 80 °C, respectively. Compared to other proteases (SPTC; Flavourzyme® 500 L; Proteinase, type XXIII; Proteinase K; and Alcalase® 2.4 L), SAPTEX has the highest catalytic efficacy, hydrolysis degree, and a powerful stability toward some commercial detergents. According to morphological, physico-chemical [scanning electron microscopy (SEM), energy dispersive X-Ray analysis (EDX), and FTIR-Fourier transform infrared spectroscopy], and mechanical evaluation, SAPTEX has no destructive impact on fibers after the enzyme treatment and a very slight effect on textile support. Obtained results suggested that SAPTEX may be considered as a potential candidate as a protein stain removal product for textile supports. Highlights • A novel protease (called SAPTEX) from Penicillium chrysogenum strain X5 was studied. • SAPTEX with a molecular mass of 43,074.11 Da was purified and characterized. • The optimum pH and temperature values for activity were pH 10 and 80 °C, respectively. • No destructive impact on fibers, using SEM, EDX, FTIR, and mechanical evaluation • It may be considered a candidate as a protein stain removal product from textile supports. [ABSTRACT FROM AUTHOR]

A new extracellular thermostable keratinolytic protease, designated KERDZ, was purified and characterized from a thermophilic actinomycetes Actinomadura viridilutea DZ50 isolated from Algerian fishing port. The isolate exhibited high keratinase production when grown in chicken-feather meal media (18,000 U/ml) after 96-h of incubation at 45 °C. The enzyme was purified by ammonium sulfate precipitation (35–55%)-dialysis and heat treatment (30 min at 75 °C) followed by UNO S-1 FPLC cation exchange chromatography and size exclusion HPLC column. The biochemical characterizations carried on include physico-chemical determination and spectroscopic analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 19536.10-Da. The sequence of the 25 N-terminal residues of KERDZ showed high homology with those of actinomycetes keratinases. Optimal activity was achieved at pH 11 and 80 °C. KERDZ was completely inhibited by PMSF and DFP suggested its belonging to the serine keratinase family. KERDZ displayed higher levels of hydrolysis and catalytic efficiency than bacterial keratinases (KERAK-29, Actinase E, and KERAB) and subtilisins (subtilisin Carlsberg and subtilisin Novo). The kerDZ gene encoding KERDZ was isolated and its DNA sequence was determined. These properties make KERDZ a potential, promising and eco-friendly alternative to the conventional chemicals used for industrial applications. [ABSTRACT FROM AUTHOR]

A protease-producing fungus was isolated from an alkaline wastewater of chemical industries and identified as Trametes cingulata strain CTM10101 on the basis of the ITS rDNA gene-sequencing. It was observed that the fungus strongly produce extracellular protease grown at 30 °C in potato-dextrose-broth (PDB) optimized media (13500 U/ml). The pure serine protease isolated by Trametes cingulata (designated SPTC) was purified by ammonium sulfate precipitation-dialysis followed by heat-treatment and UNO S-1 FPLC cation-exchange chromatography. The chemical characterization carried on include phisico-chemical determination and spectroscopie analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 31405.16-Da. The enzyme had an NH 2 -terminal sequence of ALTTQTEAPWALGTVSHKGQAST, thus sharing high homology with those of fungal-proteases. The optimum pH and temperature values of its proteolytic activity were pH 9 and 60 °C, respectively, and its half-life times at 60 and 70 °C were 9 and 5-h, respectively. It was completely inhibited by PMSF and DFP, which strongly suggested its belonging to the serine protease family. Compared to Flavourzyme ® 500 L from Aspergillus oryzae and Thermolysin typeX from Geobacillus stearothermophilus , SPTC displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency as well as elevated organic solvent tolerance and considerable detergent stability. Finally, SPTC could potentially be used in peptide synthesis and detergent formulations. [ABSTRACT FROM AUTHOR]

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2A T . The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH 2 -terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.

The current paper reports the purification and biochemical characterization of two extracellular keratinolytic enzymes, with moderate elastolytic activity, from Bacillus amyloliquefaciens strain S13 newly isolated from the brown alga Zonaria tournefortii. The enzymes were purified to homogeneity by precipitation with (NH 4 ) 2 SO 4 -dialysis, followed by size exclusion HPLC column, and submitted to biochemical characterization assays. The findings revealed that the pure enzymes designated KERZT-A and B were monomers with molecular masses of 28 and 47 kDa, respectively. Their identified NH 2 -terminal amino acid displayed high homologies with those of Bacillus keratinases. While KERZT-A was optimally active at pH 6.5 and 50 °C, KERZT-B showed optimum activity at pH 8 and 60 °C. Both enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests their belonging to the serine keratinases family. Interestingly, KERZT-A displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than KERUS from Brevibacillus brevis strain US575, NUE 12 MG (commercial enzyme), and KERZT-B unhairing keratinases. Above all, the findings indicated that KERZT-A and B enzymes seems to be an effective and an eco-friendly alternative to the conventional chemicals used for the feather keratin-biodegradation and for the unhairing of hides or skins in the leather processing industry., (Copyright © 2018. Published by Elsevier B.V.)