Neodymium adsorption from aqueous solution by ß-cyclodextrin nanosponges and a polymer valorized from potato peels waste: experiments and conventional and statistical physics interpretations.

Using organic waste and residue streams to be turned into valuable and greener materials for various applications has proven an efficient and suitable strategy. In this work, two green materials (nanosponges and a polymer) were synthesized using potato peels and applied for the first time to adsorb and recover Neodymium (Nd3+) from aqueous solutions. The recovery of Nd3+ that belongs to the rare earth elements has attracted important interest due to its/their importance in several industrial and technological applications. The fine potato peel waste (FPPW) polymer presented an irregular shape and porous surface. At the same time, the ß-cyclodextrin (ß-CD) nanosponges had uniform distribution with regular and smooth shapes. ß-CD nanosponges exhibited a much higher total carboxyl content (4.02 mmol g-1) than FPPW (2.50 mmol g-1), which could impact the Nd3+ adsorption performance because carboxyl groups can interact with cations. The adsorption capacity increased with the increase of the pH, reaching its maximum at pHs 6-7 for ß-CD nanosponges and 4-7 for FPPW polymer. The kinetic and equilibrium data were well-fitted by General order and Liu models. ß-CD nanosponges attained adsorption capacity near 100 mg Nd per gram of adsorbent. Thermodynamic and statistical physical results corroborated that the adsorption mechanism was due to electrostatic interaction/complexation and that the carboxyl groups were important in the interactions. ß-CD nanosponges (three cycles of use) were more effective than FPPW (one cycle of use) in the regeneration. Finally, ß-CD nanosponges could be considered an eco-friendly adsorbent to recover Nd3+ from aqueous matrices.

Genome sequence and Carbohydrate Active Enzymes (CAZymes) repertoire of the thermophilic Caldicoprobacter algeriensis TH7C1T.

BACKGROUND: Omics approaches are widely applied in the field of biology for the discovery of potential CAZymes including whole genome sequencing. The aim of this study was to identify protein encoding genes including CAZymes in order to understand glycans-degrading machinery in the thermophilic Caldicoprobacter algeriensis TH7C1T strain. RESULTS: Caldicoprobacter algeriensis TH7C1T is a thermophilic anaerobic bacterium belonging to the Firmicutes phylum, which grows between the temperatures of 55 °C and 75 °C. Next generation sequencing using Illumina technology was performed on the C. algeriensis strain resulting in 45 contigs with an average GC content of 44.9% and a total length of 2,535,023 bp. Genome annotation reveals 2425 protein-coding genes with 97 ORFs coding CAZymes. Many glycoside hydrolases, carbohydrate esterases and glycosyltransferases genes were found linked to genes encoding oligosaccharide transporters and transcriptional regulators; suggesting that CAZyme encoding genes are organized in clusters involved in polysaccharides degradation and transport. In depth analysis of CAZomes content in C. algeriensis genome unveiled 33 CAZyme gene clusters uncovering new enzyme combinations targeting specific substrates. CONCLUSIONS: This study is the first targeting CAZymes repertoire of C. algeriensis, it provides insight to the high potential of identified enzymes for plant biomass degradation and their biotechnological applications.

Highlight on mutations affecting the US132 cyclodextrin glucanotransferase binding specificity, thermal stability, and anti-staling activity.

We have already reported that the triple mutant (K47E-S382P-N655S of Paenibacillus pabuli US132 cyclodextrin glucanotransferase US132 (CGTase)) altered the CGTase specificity. In the current study, the single (K47E, S382P and N655S) and double (K47E+S382P, K47E+N655S, and S382P+N655S) mutants were constructed to elucidate the synergic or antagonist substitutions effect on the enzyme behavior. For the six generated mutants, an improvement of the dextrinization/cyclization ratio from 4.4 to 6-fold was observed when compared to the wild-type enzyme. The mutations effect on enzyme specificity was not attributed to synergy modulation since the single mutant N655S had the highest ratio enhancement. Moreover, the mutant N655S revealed the highest ß-cyclodextrin binding affinity with a high amount of hydrophobic bonds which might be contributed to the apparent decrease in the cyclization activity. On the other hand, mutations N655S, K47E, and (K47E-N655S) showed the same positive effect on thermal activity. The highest stability was attained at 70 °C by N655S to be 3.6-fold higher than the wild-type. The addition of N655S to wheat flour induced a decrease of dough and bread hardness and led to an increase in dough and bread cohesiveness and a rise in bread masticability values compared to the control. This mutant addition also corrected the dough elasticity decrease engendered by the wild-type CGTase indicating that N655S-CGTase could be an alternative anti-staling agent.

A thermophilic and thermostable xylanase from Caldicoprobacter algeriensis: Recombinant expression, characterization and application in paper biobleaching.

A novel xylanase gene xynBCA, encoding a polypeptide of 439 residues (XynBCA), was cloned from Caldicoprobacter algeriensis genome and recombinantly expressed in Escherichia coli BL21(DE3). The amino acid sequence analysis showed that XynBCA belongs to the glycoside hydrolase family 10. The purified recombinant enzyme has a monomeric structure of 52 kDa. It is active and stable in a wide range of pH from 3 to 10 with a maximum activity at 6.5. Interestingly, XynBCA was highly thermoactive with an optimum temperature of 80 °C, thermostable with a half-life of 20 min at 80 °C. The specific activity was 117 U mg-1, while the Km and Vmax were 1.247 mg ml-1, and 114.7 µmol min-1 mg-1, respectively. The investigation of XynBCA in kraft pulp biobleaching experiments showed effectiveness in releasing reducing sugars and chromophores, with best achievements at 100 U g-1 of pulp and 1 h of incubation. The comparative molecular modeling studies with the less thermostable Xylanase B from Clostridium stercorarium, revealed extra charged residues at the surface of XynBCA potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability.

Immobilization of the glucose isomerase from Caldicoprobacter algeriensis on Sepabeads EC-HA and its efficient application in continuous High Fructose Syrup production using packed bed reactor.

The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation.

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive.

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 NH2-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 sapA 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.

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 (10,500 U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 °C for 20 min) 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.

The optimized production, purification, characterization, and application in the bread making industry of three acid-stable alpha-amylases isoforms from a new isolated Bacillus subtilis strain US586.

A new alpha-amylase-producing strain was assigned as Bacillus subtilis US586. The used statistical methodology indicated that amylase production was enhanced by 5.3 folds. The crude enzyme analysis proved the presence of three amylases isoforms Amy1, Amy2, and Amy3 called Amy586. The purified amylases had molecular masses of 48, 52, and 68 kDa with a total specific activity of 2,133 U/mg. Amy586 generated maltose, maltotriose, and maltopentaose as main final products after starch hydrolysis. It exhibited a large 4-6 optimal pH, a 60°C temperature activity, and a moderate thermostability. Amy586 displayed a high pH stability ranging from 3.5 to 6. The addition of Amy586 to weak wheat flour decreased its P/L ratio from 1.9 to 1.2 and increased its dough baking strength (W) from 138 × 10-4 to 172 × 10-4  J. Amy586 also improved the bread texture parameters by reducing its firmness and boosting the cohesion and elasticity values. PRACTICAL APPLICATIONS: Bacterial alpha-amylases with novel properties have been the major extent of recent research. In this paper, we managed to demonstrate that the addition of a purified amylolytic extract from the new isolated Bacillus subtilis strain US586 to weak local flour improves dough rheological proprieties and bread quality. Therefore, Amy586 can be considered as a bread making improver.

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.

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.

Characterization, high production and antimicrobial activity of exopolysaccharides from Lactococcus lactis F-mou.

A new exopolysaccharide (EPS) was produced by the Lactococcus lactis F-mou strain (LT898177.1) isolated from the Sahrawi camel milk in the Bir-Naam region, Algeria. The most influential production parameters were screened by the Plackett-Burman design for enhancing EPS yield utilizing the Mech-Degla juice as a low-cost raw material. An optimum condition of a 0.49 of inoculum size, a 100 rpm of agitation rate, and a 12 h of incubation period resulted in a 301 g/L. This yield was 47 times higher than the one attained before the application of the Box-Behnken Design. Additionally, the FTIR analysis of the EPS confirmed the presence of hydroxyl, carboxyl, amide and sulphate groups. Furthermore, the SEM image showed a porous structure characterized by a flake-like basic configuration with an extremely dense assembly. The NMR studies indicated that EPS contained a backbone of→4-α-D-galactopyranose-(1→, →4, 6-α-D-glucopyranose-(1→, →6- α -D- galactopyranose -(1→ linkages plus a levan part. The EPS exhibited good water and oil holding capacities, a high antioxidant efficiency, and an excellent anti-clotting activity. EPS also showed a strong inhibitory activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, Bacillus cereus, Proteus mirabilis, Acinetobacter baumannii, Enterobacter cloacae, and Candida albicans. Overall, the mentioned findings indicated that EPS could be utilized as a natural additive in pharmaceutical, food, and cosmetic industries.

Production optimization, characterization, and covalent immobilization of a thermophilic Serratia rubidaea lipase isolated from an Algerian oil waste.

A new thermophilic non-induced lipase producer named Serratia rubidaea strain Nehal-mou was isolated from oil waste in Tissemsilat, Algeria. The most influential lipase production parameters were screened by the Plackett-Burman design for enhancing enzyme yield. An optimum condition of a 1.5% of glucose, a 0.01% of potassium, and a 0.025% of manganese contents resulted in a 41.13 U/mL. This yield was 6.29 times higher than the one achieved before the application of the Box-Behnken Design. Lipase activity showed a high organic solvent tolerance following its exposure to hexane, ethanol, methanol, and acetone. Lipase was also perfectly stable in the presence of 10 mM Fe2+, K+, and Na+ ions with more than 75% of the retaining activity. The enzyme half-life times were 22 h, 90 min, and 25 min at 50, 60, and 70 °C respectively. Polyvinyl alcohol (PVA)/boric acid/Starch/CaCO3 were utilized as a carrier for lipase covalent immobilization in order to be used efficiently. The Scanning Electron Microscopy (SEM) Technique and the Fourier Transform Infrared Spectroscopy (FTIR) Method confirmed the covalent bonding success and the excellent carrier characteristics. Thus, the immobilization yield reached 73.5% and the optimum temperature was shifted from 40 to 65 °C. The immobilized lipase kept 80% of its total activity after 10 cycles and had 3 and 3.2-fold half-lives at 70, and 80 °C respectively compared to the free enzyme.

A novel thermostable and efficient Class II glucose isomerase from the thermophilic Caldicoprobacter algeriensis: Biochemical characterization, molecular investigation, and application in High Fructose Syrup production.

A novel glucose isomerase gene from the thermophilic Caldicoprobacter algeriensis, encoding a polypeptide of 438 residues, was identified, cloned and successfully expressed in E. coli. The purified enzyme (GICA) was a homotetramer of about 200 kDa displaying the highest activity at pH 7.0 and 90 °C and retaining 97% of its maximum activity at pH 6.5. The enzyme showed an excellent thermostability with a half-life of 6 min at 100 °C. Interestingly, GICA had a very high affinity of 40 mM and catalytic efficiency of 194 min-1 mM-1 toward d-glucose at 90 °C. A maximum of 54.7% d-glucose to d-fructose conversion was achieved by GICA at 85 °C making it an attractive candidate for HFCS-55 production. The primary sequence inspection and molecular modeling studies revealed that the thermal stability of GICA could be attributed to the presence of extra charged residues at the surface like E108 and Q408 increasing surface charge interactions. Moreover, a serine at position 56 near to P58 could establish hydrogen bond strengthening the dimer attachment. The high catalytic efficiency and affinity of GICA could be ascribed to the presence of amino acid like E108 and K62 that created more charges around the catalytic site entry.

Gene cloning, expression, molecular modeling and docking study of the protease SAPRH from Bacillus safensis strain RH12.

The sapRH gene, which encodes the serine alkaline protease SAPRH, from Bacillus safensis RH12, was isolated and its DNA sequence was determined. The deduced amino-acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with SAPB from B. pumilus CBS, with only 9 amino-acids of difference. The region, encoding SAPRH was heterologously expressed in E. coli BL21-AI™ cells using GATEWAY™ pDEST™17 expression-vector. The recombinant (His)6-tag enzyme (His6-rSAPRH) was purified in a single affinity chromatography step and its biochemical properties were determined and compared to those of SAPRH and rSAPB. Interestingly, His6-rSAPRH showed improved thermostability compared to SAPRH and rSAPB. The molecular dynamics of SAPRH compared to SAPB revealed a more thermostable structure, thus confirming the in vitro results showing that His6-rSAPRH has a t1/2 of 120 min against 90 and 30 min for SAPRH and rSAPB, respectively, at 70 °C and different kinetic parameters to synthetic peptides. The docking simulations data allow in getting an insight into the involvement of some key amino-acids in substrate binding and account for the selectivity. Overall, this is the first report of a sapRH gene cloned from B. safensis which can be a promising potential candidate for future applications in detergent formulations.

Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties.

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72 h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54 kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 °C against 50 °C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 °C, r-AmyA reached 45 min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4 mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.

Physical and enzymatic properties of a new manganese peroxidase from the white-rot fungus Trametes pubescens strain i8 for lignin biodegradation and textile-dyes biodecolorization.

A new manganese peroxidase-producing white-rot basidiomycete fungus was isolated from symptomatic wood of the camphor trees Cinnamomum camphora (L.) at the Hamma Botanical Garden (Algeria) and identified as Trametes pubescens strain i8. The enzyme was purified (MnP TP55) to apparent electrophoretic homogeneity and biochemically characterized. The specific activity and Reinheitzahl value of the purified enzyme were 221 U/mg and 2.25, respectively. MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 55.2 kDa. The NH2-terminal sequence of the first 26 amino acid residues of MnP TP55 showed high similarity with those of white-rot fungal peroxidases. It revealed optimal activity at pH 5 and 40 °C. This peroxidase was completely inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in its tertiary structure. Interestingly, MnP TP55 showed higher catalytic efficiency, organic solvent-tolerance, dye-decolorization ability, and detergent-compatibility than that of horseradish peroxidase (HRP) from roots of Armoracia rustanica, manganese peroxidase from Bjerkandera adusta strain CX-9 (MnP BA30), and manganese peroxidase from Phanerochaete chrysosporium (MnP PC). Overall, the findings provide strong support for the potential candidacy of MnP TP55 for environmental applications, mainly the development of enzyme-based technologies for lignin biodegradation, textile-dyes biodecolorization, and detergent formulations.

Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12.

A novel extracellular protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28 kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50 °C and 75% at pH 11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.

Effect of Agave americana L. on the human, and Aspergillus oryzae S2 α-amylase inhibitions.

Among phenolic compounds, Agave americana L. extract contained puerarin (38.4%) and p-coumaric acid (12.29%) (pCa). From the Lineweaver-Burk plots, pCa and puerarin demonstrated a competitive and a non competitive inhibitions towards human α-amylase activity, respectively. PCa exhibited a higher human inhibitory activity with an IC50 of 98.8 µM which was about 2.3 times than acarbose. Puerarin (IC50 = 3.87 µM) and pCa (IC50 = 10.16 µM) also showed an excellent inhibition for Aspergillus oryzae S2 α-amylase activity. The inhibitions of the described biocatalysts compounds towards both amylases were significantly decreased when they were pre-incubated with starch. The binding modes of these compounds were evaluated in silico. The binding efficiency order of these molecules in terms of polar contact numbers for both enzymes was in agreement with the in vitro studies. These findings provided a rational reason to establish the isolated compounds capability as therapeutic target for hyperglycaemia modulation and antifungal therapy.

Production, purification, and biochemical characterization of serine alkaline protease from Penicillium chrysogenium strain X5 used as excellent bio-additive for textile processing.

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 NH2-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.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization.

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Modifing Aspergillus Oryzae S2 amylase substrate specificity and thermostability through its tetramerisation using biochemical and in silico studies and stabilization.

We previously reported that Aspergillus oryzae S2 had produced an amylase called AmyC formed by a tetramer of AmyB subunits under solid state fermentation. In this work, we demonstrated that the half-life time of AmyC at 75 °C and 80 °C were remarkably enhanced to reach 53 min and 41 min compared to 6 min and 4 min for AmyB. The Km values of AmyC for maltoheptaose, maltopentaose, and maltotetraose were 2-fold lower than AmyB. AmyC showed a 6.5 fold higher exo-type activity and hydrolyzed the short oligosaccharides more efficiently than AmyB. The AmyC-3D model was generated and showed that a region named T1 was involved in the oligomerization process. The subunits and the RING network interactions insight suggested that AmyC sub-units were bounded by 20 hydrogen bonds, 4 electrostatic interactions, 16 nodes and 836 edges leading to a higher thermal stability. The disordered (ß3-ß4) and (ß7-ß8) loops contained in the AmyC active cleft were presumed to be the recognition sites of the non-reducing end substrate. The docking studies strongly suggested that AmyC easily accommodated the short substrates as it was exhibited in vitro and seemed to look like maltogenic amylases. The Box-Behnken Response Surface Methodology was applied for Amy C immobilization for efficient use. An optimum condition of an aluminum oxide content of 0.25 g, a carrageenan content of 0.1 g, and a glutaraldehyde content of 0.5%/g of carrier resulted in 76.2% of covalent immobilization yield. The immobilized AmyC kept its total activity for three cycles, shifted the optimum temperature from 60 °C to 65 °C, and had two-fold half-life at 85 °C compared to the free enzyme.