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Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application. [ABSTRACT FROM PUBLISHER]

Streptomyces sp. strain AH4 exhibited a high ability to produce two extracellular proteases when cultured on a yeast malt-extract (ISP2)-casein-based medium. Pure proteins were obtained after heat treatment (30 min at 70 °C) and ammonium sulphate fractionation (30-60 %), followed by size exclusion HPLC column. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis revealed that the purified enzymes (named SAPS-P1 and SAPS-P2) were monomers with molecular masses of 36,417.13 and 21,099.10 Da, respectively. Their identified N-terminal amino acid displayed high homologies with those of Streptomyces proteases. While SAPS-P1 was optimally active at pH 12.0 and 70 °C, SAPS-P2 showed optimum activity at pH 10.0 and 60 °C. Both enzymes were completely stable within a wide range of temperature (45-75 °C) and pH (8.0-11.5). They were noted to be completely inhibited by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, which confirmed their belonging to the serine proteases family. Compared to SAPS-P2, SAPS-P1 showed high thermostability and excellent stability towards bleaching, denaturing, and oxidizing agents. Both enzymes displayed marked stability and compatibility with a wide range of commercial laundry detergents and significant catalytic efficiencies compared to Subtilisin Carlsberg and Protease SG-XIV. Overall, the results indicated that SAPS-P1 and SAPS-P2 can be considered as potential promising candidates for future application as bioadditives in detergent formulations. [ABSTRACT FROM AUTHOR]

Recombinant Escherichia coli / pAD26 cells were optimally immobilized on cotton to obtain plasmid stability. An experimental design that monitored two major responses, namely stability and reusability, was applied for screening the factors affecting the immobilization of the strain (cylodextrin, sucrose, shape support, mass of cotton matrices, mass of initial biomass, age of biomass, ethylenediamine, glutaraldehyde, and incubation time) and the optimization of the most significant variables. The optimal values recorded for the two criteria were obtained with high glutaraldehyde concentration (0.15 %) and early inoculum age (2 h) in the presence of 0.2 mM cylodextrin. With those optimum conditions, cyclodextrin glucanotransferase production yields were enhanced, reaching up to 39.77 U/mL. The stability and reusability of the immobilized strain were two fold and three fold higher, respectively, than those of the control after three cycles. [ABSTRACT FROM AUTHOR]

Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. 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 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca2+. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry. [ABSTRACT FROM AUTHOR]

Abstract: A new fungal strain that was isolated from old sweet soy sauce was identified, based on subsequent microscopic studies and analyses of rRNA18S gene sequence, intergenic region rRNA 18S-23S, and aflatoxins production tests, as an Aspergillus oryzae strain. The latter was noted to produce two extracellular α-amylases, namely AmyA and AmyB. The monitoring of alpha-amylase production in the presence and absence of various protease inhibitors indicated that AmyB could be formed from the proteolysis of AmyA. The enzymes were purified to homogeneity through fractional acetone precipitation, size exclusion, and anion exchange chromatography. The molecular masses estimated for AmyA and AmyB by SDS-PAGE were 50 and 42kDa, respectively. The NH2-terminal of the purified proteins showed the same amino acid sequences. Further biochemical characterization assays revealed that both enzymes attained maximal activity at pH 5.6 and 50°C. They were activated and stabilized by Ca2+ and were noted to produce maltose and maltotriose as major starch hydrolysis end products. Overall, the findings of the present study indicate that both AmyA and AmyB exhibit a number of promising properties that make them potential strong candidates for application as additives in the bread making industry. [Copyright &y& Elsevier]

The gene encoding for the α-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the α-amylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site-directed mutagenesis and their physicochemical and kinetic properties were compared with those of the wild-type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermosensible as its half-life time at 80C was 13 min, while those of BACAAM and the double mutant were likewise 38 min. The single-mutant amylases exhibited an identically intermediate thermal stability as their half-life times at 80C were roughly 22 min. PRACTICAL APPLICATIONS Of particular interest to the current search is that the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important because they offer opportunities to understand the structure–function relationships of these biocatalysts. [ABSTRACT FROM AUTHOR]

The mineral phosphate solubilizing (MPS) ability of a Serratia marcescens strain, namely CTM 50650, isolated from the phosphate mine of Gafsa, was characterized on a chemically defined medium (NBRIP broth). Various insoluble inorganic phosphates, including rock phosphate (RP), calcium phosphate (CaHPO4), tri-calcium phosphate (Ca3(PO4)2) and hydroxyapatite were tested as sole sources of phosphate for bacterial growth. Solubilization of these phosphates by S. marcescens CTM 50650 was very efficient. Indeed, under optimal conditions, the soluble phosphorus (P) concentration it produced reached 967, 500, 595 and 326 mg/l from CaHPO4, Ca3(PO4)2, hydroxyapatite and RP, respectively. Study of the mechanisms involved in the MPS activity of CTM 50650, showed that phosphate solubilization was concomitant with significant drop in pH. HPLC-analysis of culture supernatants revealed the secretion of gluconic acid (GA) resulting from direct oxidation pathway of glucose when the CTM 50650 cells were grown on NBRIP containing glucose as unique carbon source. This was correlated with the simultaneous detection by PCR for the first time in a S. marcescens strain producing GA, of a gene encoding glucose dehydrogenase responsible for GA production, as well as the genes pqqA, B, C and E involved in biosynthesis of its PQQ cofactor. This study is expected to lead to the development of an environmental-friendly process for fertilizer production considering the capacity of S. marcescens CTM 50650 to achieve yields of P extraction up to 75% from the Gafsa RP. [ABSTRACT FROM AUTHOR]

Extraction of 25 L fermentation broth of the newly isolated Streptomyces sp. strain TN58 and various separation and purification steps led to the isolation of five bioactive metabolites, namely brevianamide F (C1), reported from a streptomycete for the first time, Nβ-acetyltryptamine (C2), thiazolidomycin (C3), and two rhamnopyranosides (C4 and C5). These two rhamnopyranosides were produced directly, without precursor addition. The chemical structure of these five active compounds was established on the basis of 1H, 13C/APT and 2D NMR spectra, ESI and EI-MS data, and by comparison with data from the literature. According to the biological studies, we show in this work that the compounds C1, C2, C4 and C5 possess antimicrobial activities. [ABSTRACT FROM AUTHOR]

Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the α-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2. [ABSTRACT FROM AUTHOR]

Gene araA encoding thel-arabinose isomerase (l-AI) from Lactobacillus plantarum NC8 was cloned and expressed in Escherichia coli. It encodes a polypeptide of 474 residues having 55% identities withl-AIs from Bacillus stearothermophilus US100 and Thermus sp. IM6501. The active form of the purified recombinantl-AI NC8 enzyme is a hexamer composed of six identical 55-kDa subunits. The purified enzyme was optimally active at 60 °C and pH 7.5. It required divalent cations such as Co2+ and Mn2+ for maximal activity and thermostability. Thel-AI NC8 was exceptionally active and stable at acidic pH. Indeed, it exhibited 68% of its maximal activity at pH 5.5 and retained 89% of activity after a 24-h incubation at pH 5. The apparent Km values of the enzyme forl-arabinose andd-galactose were 43.4 and 69.7 mM, respectively, and its catalytic efficiency was c. 10-fold higher for the physiological substratel-arabinose (15.5 mM−1 min−1) thand-galactose (1.6 mM−1 min−1). The bioconversion yield ofd-galactose tod-tagatose by the purifiedl-AI NC8 after 6 h at 60 °C was 30%. [ABSTRACT FROM AUTHOR]

The dyatic symmetric element (DSE) present in the α-amylase gene promoter region of the thermophilic Streptomyces strain sp. TO1, and the whole α-amylase gene (amy TO1) with a 3-bp change in the DSE, were cloned in the high copy number replicative cloning vector pIJ702 giving pLM10 and pLM11 plasmids respectively. In TO1/pLM10 and Streptomyces lividans TK24/pLM11 strains, the expression of α-amylase TO1 gene became insensible to the negative effect of glucose and glycerol. These results strongly suggest that, in a high copy number system, the negative transcriptional regulator was titrated out by the DSE and the repression of the expression of amy TO1 gene is abolished. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

A series of citrus flavonoids were evaluated for α-amylase and α-glucosidase inhibitory activities in vitro. The inhibitory capacities of flavanone glycosides were greater than those of polymethoxy flavones for α-amylase. Naringin exhibited the most potent α-glucosidase inhibitory activity with IC50 0.55 μM and was about 196.83 times more active than acarbose. Poncirin led to a 43-fold improvement in α-amylase inhibition over acarbose. The double-reciprocal (Lineweaver- Burk) plot confirms a competitive inhibition mode towards α-amylase and α-glucosidase activities. The inhibition activity was significantly lowered when citrus flavonoids were pre-incubated with starch. The binding site for naringin, poncirin, hesperidin, tangeretin in α-amylase showed a polar contact numbers, respectively, of 47, 54, 51, and 44 more than that involving acarbose. The hydrogen bonds formed between nobilitin and the key residue of Lys506 of α-glucosidase, namely Asn475:N-A:Lys506:O, might provide its extra affinity when compared to tangeritin. These findings provided a strong and rational reason to establish the selected citrus flavonoids capability as a therapeutic target for postprandial hyperglycaemia modulation. [ABSTRACT FROM AUTHOR]

[Display omitted] • UFPPW-β-CD adsorbent was produced by grafting β-CD on ultrafine potato peels. • UFPPW-β-CD was an efficient and reusable adsorbent for perfluorosulfonic acids. • Adsorption was governed by hydrogen bonds and hydrophobic interactions. • The system acetone/ultrasound provided a 96 % desorption percentage in 25 min. • The adsorbent could be used 7 times with the same adsorption capacity. Per- and polyfluoroalkyl substances are widely used in various consumer products. However, these compounds can cause various harm to the environment and health. Considering the high chemical stability, these compounds are not completely removed from the aqueous environment, and consequently, recent studies have detected their presence in water bodies. In this scenario, biomass-based adsorbents are promising. β-cyclodextrin (β-CD) was grafted in an ultrafine potato peel waste (UFPPW) to produce a novel, efficient, and sustainable adsorbent (UFPPW-β-CD) that was used to remove three different perfluorosulfonic acids (PFSAs) from water. The efficient grafting was proved by several characterization techniques, which also demonstrated the main UFPPW-β-CD features. The UFPPW-β-CD was efficient for all PFSAs (perfluorohexanesulfonic acid (PFHxS), perfluoropentanesulfonic acid (PFPeS), and perfluorobutanesulfonic acid (PFBS)), with removal percentage higher than 74 %. The increase in the C F chain of PFSAs favored the adsorption due to the host–guest hydrophobic interactions between the C F chains of the adsorbates and the hydrophobic cavity of the β-CD. Removal percentages and adsorption capacities at pH 3.0 were 74.6 % (49.92 µg g−1) to the PFBS, 82.6 % (65.66 µg g−1) to the PFPeS, and 90 % (100.89 µg g−1) to the PFHxS. The kinetic followed the General order model, while the equilibrium agreed with the Sips isotherm. The adsorption capacity increased with the increase in the C F chain of the adsorbate, but the adsorption rate followed the opposite trend. PFSAs adsorption on the UFPPW-β-CD adsorbent was favorable and exothermic. UFPPW-β-CD could be used seven times, keeping its maximum adsorption capacity constant using ultrasound-assisted desorption. It can be concluded that UFPPW-β-CD is a sustainable adsorbent to uptake PFSAs from water, and this process is dependent on the size of the C F chains. [ABSTRACT FROM AUTHOR]

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 β-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. [Display omitted] • Six mutants were constructed to elucidate the substitutions effect. • The dextrinization/cyclization ratio was improved from 4.4 to 6-fold. • N655S had the highest D/C ratio and thermal stability. • N655S-CGTase could be an alternative anti-staling agent. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Owing to their beneficial functional capabilities, essential oils were largely used. However, their low aqueous solubility, instability, and high volatility urged scientists to their encapsulation with cyclodextrins (CDs) to tackle their shortcomings. In this study, the co-precipitation method was used to prepare β-CD/ Eucalyptus globulus essential oil (EGEO) inclusion complexes (ICs). β-CD/EGEO ICs were prepared at ratios (w:w) 1:2 and 1:4 with an encapsulation efficiency of 93 and 96%, respectively. The ICs characterization using the Fourier transform Infrared spectroscopy, differential scanning calorimetry, X-ray powder diffraction, Dynamic Light Scattering, and Laser Doppler Velocimetry confirmed the formation of β-CD/EGEO ICs. The insecticidal activity of the free EGEO and ICs was explored and displayed that the complex β-CD/EGEO 1:4 had the highest activity with the lowest LC 50 against Ephestia kuehniella larvae (5.03 ± 1.16 mg/g) when compared to the free oil (8.38 ± 1.95 mg/g). Molecular docking simulations stipulated that the compound α-Bisabolene epoxide had the best docking score (ΔG = −7.4 Kcal/mol) against the selected insecticidal target α-amylase. Additionally, toxicity evaluation of the studied essential oil suggested that it could be safely used as a potent bioinsecticide as compared to chemical insecticides. This study reveals that the formation of β-CD/EGEO ICs enhanced the oil activity and stability and could be a promising and safe tool to boost its application in food or pharmaceutical fields. [Display omitted] • Eucalyptus globulus essential oil (EGEO) was effectively encapsulated in the β-CD cavity. • The biological activity of EGEO inclusion complexes is oil dose-dependent. • The encapsulation enhanced the oil insecticidal activity against Ephestia kuehniella larvae. • Toxicity evaluation showed that EGEO is a promised bioinsecticide. [ABSTRACT FROM AUTHOR]

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 K m and V max 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. [ABSTRACT FROM AUTHOR]