Aspergillus awamori MK788209 cellulase: production, statistical optimization, pea peels saccharification and textile applications.

BACKGROUND: The demand for low-cost cellulolytic enzyme synthesis is rising in the enzyme market. This work aims to produce cellulase by utilizing various agricultural wastes and investigating the use of enzyme in saccharification and textile industries. RESULTS: Solid state fermentation (SSF) was applied to produce industrial enzymes, particularly cellulase, through utilizing Molokhia (Corchorus olitorius) stems by Aspergillus awamori MK788209 isolate. Two stages of statistical factorial designs Plackett-Burman (PB) and Central Composite Design (CCD) were applied to enhance the A. awamori MK788209 cellulase production from Molokhia stems (MS). The fold increase of enzyme production by PB followed by CCD was 2.51 and 4.86, respectively. Additionally, the A. awamori MK788209 culture filtrate was highly effective in saccharifying various agricultural wastes, particularly pea peels (PP) (yielding 98.33 mg reducing sugar/ml), due to its richness in cellulase, laccase, xylanase, pectinase, and amylase. By optimizing the three main variables; pea peel weight, culture filtrate volume added, and saccharification time by CCD, the sugar recovery from PP was enhanced, leading to a 3.44-fold increase in reducing sugar recovery (338 mg reducing sugar /ml). Furthermore, the A. awamori MK788209 culture filtrate showed high efficacy in textile applications, enhancing the roughness, weight loss, white index, and printing capability of treated cotton fabrics. CONCLUSIONS: A. Awamori MK788209 produced cellulase which was effective in PP saccharification. The enzyme was also capable of enhancing cotton fabric properties.

A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen.

BACKGROUND AND AIM: The purpose of the current study is to isolate a heavily amylase-producing bacteria of the genus Bacillus from soil samples, optimize the production of the enzyme, purify it, and evaluate its activity against biofilm-producing bacteria. A total of 12 soil samples were collected and screened for promising Bacillus species with good amylolytic activity. Isolation was done by serial dilution and plating technique and amylolytic activity was determined by starch agar plate method. Among the 12 Bacillus isolates recovered from soil samples, 7 showed positive α-amylase production. The best isolate that recorded the greatest amylolytic activity was selected for further studies. This isolate was identified by 16S rRNA sequencing as Bacillus cereus and registered under gene bank accession number OP811897. Furthermore, the α-amylase enzyme was produced by a submerged fermentation technique using best production media and partially purified by ammonium sulfate and chilled ethanol and molecular weight had been determined by SDS-PAGE gel electrophoresis. The production of α-amylase was optimized experimentally by one-factor at a time protocol and statistically by Plackett-Burman design as well as RSM CCD design. Data obtained from OFAT and CCD revealed that α-amylase activities were 1.5- and twofold respectively higher as compared to un-optimized conditions. The most significant factors had been identified and optimized by CCD design. RESULTS: Among the eleven independent variables tested by PBD, glucose, peptone, (NH4)2SO4, and Mg SO4 were the most significant parameters for α-amylase production with an actual yield of 250U/ml. The best physical parameters affecting the enzyme production were incubation time at 35 °C, and pH 5.5 for 48 h. The partially purified enzyme with 60% ammonium sulphate saturation with 1.38- fold purification showed good stability characteristics at a storage temperature of 4 °C and pH up to 8.5 for 21 days. Antibiofilm activity of purified α-amylase was determined against Pseudomonas aeruginosa (ATCC 35659) by spectrophotometric analysis and CLSM microscopic analysis. Results demonstrated biofilm inhibition by 84% of the formed Pseudomonas biofilm using a microtiter plate assay and thickness inhibition activity by 83% with live/Dead cells percentage of 17%/83% using CLSM protocol. CONCLUSIONS: A highly stable purified α-amylase from B. cereus showed promising antibiofilm activity against one of the clinically important biofilm-forming MDR organisms that could be used as a cost-effective tool in pharmaceutical industries.

Statistical optimization of enzyme cocktail production using Jew's mallow stalks residues by a new isolate Aspergillus flavus B2 via statistical strategy and enzymes characterization.

This study investigates the production of the enzyme cocktail by the isolated fungi Aspergillus flavus B2 (GenBank accession number OL655454) using agricultural and industrial (AI) residues as the sole substrate. Of all the AI residues tested, Jew's mallow stalk was the best inducer substrate for enzyme cocktail production without adding any nutrients. Statistical optimization using Response Surface Methodology enhanced the production by 5.45, 5.20, and 3.34-fold, respectively for pectinase, xylanase, and CMCase. Optimum temperature, activation energy (Ea), and activation energy for denaturation (Ed) were determined. Michaelis constant (Km) for CMCase, xylanase, and pectinase enzyme was 1.82, 1.23, and 1.05 mg/mL, respectively. Maximum reaction rate (Vmax) was 4.67, 5.29, and 17.13 U/mL, respectively for CMCase, xylanase, and pectinase. Thermal stability revealed that pectinase, CMCase, and xylanase enzymes retained 64.7%, 61.8%, and 53.2% residual activities after incubation for 1 h at 50 °C. Half-life time (t0.5) of pectinase, CMCase, and xylanase at 50 °C were 189.38, 129.8, and 127.89 min, respectively. Thermodynamics of the produced enzymes enthalpy (ΔH*d), free energy (ΔG*d), and entropy (ΔS*d) were determined at 40, 50, and 60 °C. In the presence of EDTA (5 mM), CMCase, xylanase, and pectinase retained 69.5%, 66.2%, and 41.2%, respectively of their activity. This work is significant for the valorization of AI residues and the production of value-added products.

Xylanase covalent binding onto amidated pectin beads: Optimization, thermal, operational and storage stability studies and application.

Amidated pectin-polyethylene imine-glutaraldehyde (AP-PEI-GA) immobilizer was prepared. The ideal protocol that should be adopted during the immobilizer preparation was investigated via Box-Behnken design (BBD), and it comprised processing the AP beads with 3.4 % (w/w) PEI solution of pH 9.65 followed by 5.96 % (v/v) GA solution. The obtained AP-PEI-GA immobilizer was efficient, and it acquired 3.03 U.g-1 of immobilized xylanase (im-xylanase) activity. The computed Km and Vmax values for AP-PEI-GA im-xylanase were 16.67 mg.ml-1 and 20 g.ml-1.min-1, respectively. Through covalent coupling to AP-PEI-GA, Aspergillus niger xylanase thermodynamic properties T1/2 and D-values were increased by 2.05, 3.08, and 1.35 at 40, 50, and 60 °C, respectively. ΔHd and ΔGd for AP-PEI-GA im-xylanase at 40, 50, and 60 °C were higher than those for free form emphasizing more resistance to thermal denaturation. Im-xylanase showed 100 % activity for 20 successive cycles and hydrolyzed different agro-industrial wastes into reducing sugar and xylooligosaccharides (XOS) with more efficiency on pea peel (PP). AP-PEI-GA im-xylanase, PP weight, and hydrolysis time that should be adopted to obtain the highest reducing sugar and XOS yield were optimized through central composite design (CCD). Extracted XOS showed prebiotic and anti-oxidant activities.

Preparation and characterization of sugilite glass from basalt for α-amylase immobilization, statistical optimization of the immobilization process and description of free and immobilized enzyme.

Bacterial α-amylase was immobilized on sugilite from modified basalt rock as a new carrier. A set of glass compositions based on sugilite formula KNa2M2Li3Si12O30 (M = Al or Mn or Fe) were prepared. The glasses were prepared through melting-quenching technique and samples of glass were converted to glass ceramic. Among the tested glasses and glass ceramic only sugilite glass based on M = Fe (BSF) give promising results. The sugilite BSF glass was characterized using DSC analysis, FTIR absorption, and SEM. The sugilite glass revealed high thermal resistant till ∼770 °C. Under optimized conditions of the Central composite design, the immobilization yield improved by 4.7-fold. The affinity to starch increased after enzyme immobilization by 4.3-fold. The lower rate of deactivation constant and the increase of t ½ and D-value confirm the suitability of BSF and immobilization method in enhancing enzyme stability. The improvement in thermostability of immobilized α-amylase was judged by the change in thermodynamic parameters. In conclusion, the prepared sugilite BSF glass can be utilized as a new carrier suitable for stabilization of α-amylase enzyme by immobilization.

Purification, amino acid sequence, and characterization of bacteriocin GA15, a novel class IIa bacteriocin secreted by Lactiplantibacillus plantarum GCNRC_GA15.

The bacteriocins produced by lactic acid bacteria (LAB) are attracting attention due to their promising applications in food and pharmaceuticals fields. Hence, a LAB strain, GCNRC_GA15, was isolated from Egyptian goat cheese, and molecularly identified as Lactiplantibacillus plantarum. This strain showed a wide antimicrobial spectrum, which was found to be of proteineous nature, suggesting that L. plantarum GCNRC_GA15 is a bacteriocin-producer. This bacteriocin (bacteriocin GA15) was partially purified using cation exchange, and hydrophobic interaction chromatography. Tricine SDS-PAGE analysis for the fraction showing bacteriocin activity has estimated the molecular mass to be 4369 Da. Furthermore, amino acid sequencing of this peptide has detected 34 amino acids, and comparing its amino acid sequence with those of some pediocin-like bacteriocins revealed that bacteriocin GA15 has the conserved sequence (YYGNGV/L) in its N-terminal region which identified bacteriocin GA15 as a pediocin-like bacteriocin. Bacteriocin GA15 showed good heat and pH stabilities, and its activity was enhanced after treatment with Tween 80 or Triton X-100. Bacteriocin production medium was statistically optimized using the Plackett-Burman and Central Composite designs. As a result, bacteriocin production increased from 800 to 12,800 AU/ml using the optimized medium in comparison with result recorded for the un-optimized medium.

Characterization of Aspergillus niger MK981235 xylanase with extraction of anti-hepatotoxic, antioxidant, hypocholesterolemic and prebiotic Corchorus olitorius stems xylooligosaccharides.

The object of this study was to utilize agro-industrial waste Corchorus olitorius stems (molokhia stems, MS) as substrate, for Aspergillus niger MK981235 xylanase production and as source of biologically active xylooligosaccharides (XOS). This study succeeded in utilization of Aspergillus niger MK981235 xylanase under different saccharification conditions designed by central composite design (CCD) for extraction of 15 biologically active XOS (anti-hepatotoxic, antioxidant, hypocholesterolemic and prebiotic) with different monosaccharides constituents composition and percent. A. niger MK981235 xylanase showed the highest activity 6.60 U·ml-1 at 50 °C with 1.5% xylan. The kinetics included Km and Vmax were determined to be 6.67 mg·ml-1 and 20 µmol·ml-1·min-1, respectively. Moreover, A. niger MK981235 xylanase thermodynamics Ea (activation energy) and Ed (activation energy of denaturation) were determined to be 21.95 and 39.51 KJ·mol-1, respectively. The highest prebiotic effect (growth promation) was exerted by the central MS XOS on Lactobacillus plantarum and Lactobacillus rhamnosus (125 and 135.3%, respectively). Also, the central MS XOS, exerted the highest cholesterol reduction and antioxidant activities 74.7 and 92%, respectively, showed remarkable in vivo protective role against the hepatic toxicity of lithium carbonate evaluated by changes in body weight, liver function markers (AST, ALT, Alb, total bilirubin) and tissue makers (MDA and GSH).

The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool.

Aspergillus oryzae (A. oryzae) is a filamentous micro-fungus that is used from centuries in fermentation of different foods in many countries all over the world. This valuable fungus is also a rich source of many bioactive secondary metabolites. Moreover, A. oryzae has a prestigious secretory system that allows it to secrete high concentrations of proteins into its culturing medium, which support its use as biotechnological tool in veterinary, food, pharmaceutical, and industrial fields. This review aims to highlight the significance of this valuable fungus in food industry, showing its generosity in production of nutritional and bioactive metabolites that enrich food fermented by it. Also, using A. oryzae as a biotechnological tool in the field of enzymes production was described. Furthermore, domestication, functional genomics, and contributions of A. oryzae in functional production of human pharmaceutical proteins were presented. Finally, future prospects in order to get more benefits from A. oryzae were discussed.

Physiochemical, kinetic and thermodynamic studies on Aspergillus wewitschiae MN056175 inulinase with extraction of prebiotic and antioxidant Cynara scolymus leaves fructo-oligosaccharides.

Utilization of agricultural wastes as cheap natural resources for production of bioactive products is currently attracting global attention. For this purpose, this study focused on isolation of Aspergillus wewitschiae MN056175 as promising producer of inulinase, then investigating physiochemical, kinetics and thermodynamics of the obtained inulinase, and its ability to extract bioactive fructo-oligosaccharides (FOS) from Cynara scolymus leaves (artichoke leaves, AL). A. wewitschiae MN056175 inulinase gave the maximum activity at temperature 60 °C and inulin concentration 1%. The kinetics including Km and Vmax were determined to be 105.26 mg·ml-1 and 83.33 µmol·ml-1·min-1, respectively. The thermodynamics including, Ea (activation energy) and Ed (activation energy for denaturation) were determined to be 21.82 and 73.21 kJ·mol-1, Kd, T1/2, D-value, ΔH°, ΔG° and ΔS° at 40, 50 and 60 °C which indicated the stability of A. wewitschiae MN056175 inulinase. Moreover, this inulinase was capable of hydrolyzing Cynara scolymus leaves into reducing sugar and 15 FOS with different DP, total carbohydrate, and protein content under different conditions designed by central composite design (CCD). The 15 AL FOS showed different high antioxidant and prebiotic activities. Central FOS with probiotic bacteria exhibited significant antimicrobial activity against tested gram positive bacteria in a way higher than those recorded against gram negative bacteria.

Immobilization of xylanase on modified grafted alginate polyethyleneimine bead based on impact of sodium cation effect.

Alginate- polyethyleneimine gel beads modified by using 0.3 M Na+ were used for covalent immobilization of Aspergillus flavus xylanase. SEM images showed distorted structure with addition of Na+ that impaired the egg-box structure formation offered much covalent binding with xylanase. Immobilization onto (Alg+PEI/Na+) showed an enhancement in the operational stability, immobilization efficiency as well as immobilization yield. Covalent immobilization of xylanase onto (Alg+PEI/Na+) enhanced xylanase activity over a wide range of pHs (4-5.5) comparable to its free formula. As well as an increase in reaction temperature up to 60°C. However, immobilized formula of enzyme showed abroad thermal stability that it retained 79.0% of its initial activity at 70°C up to 30 min whereas, free formula completely lost its activity at this temperature. Thermodynamics studies showed an enhancement in thermal stability at high temperature for the immobilized xylanase. i.e. At 70°C the t1/2 and D-value for free formula of enzyme increased from 24 to165 min and from 79.95to 548.23 min, respectively. Moreover, the enzyme stability enhancement for immobilized formula of xylanase was proved with a remarkable increase in enthalpy and free energy. 93% of the immobilized xylanase activity was retained over 6 weeks of storage at -4°C.

Thermodynamics enhancement of Alternaria tenuissima KM651985 laccase by covalent coupling to polysaccharides and its applications.

This study full filed in enhancement of catalytic, thermodynamics and storage stability of Alternaria tenuissima KM651985 laccase by conjugation to sodium periodate oxidized starch. The starch conjugated A. tenuissima KM651985 laccase was active over a wide range of temperatures and pHs with the highest activity at 60 °C and 4, respectively. The thermal stability of conjugated A. tenuissima KM651985 laccase was indicated by, high T1/2 values (half life) 1076.16, 382.42 and 191.23 min at 50, 60 and 70 °C, respectively, low Kd (denaturation rate constant) 6.44 × 10-4, 18.13 × 10-4 and 36.25 × 10-4 min-1 at the same temperatures, high D-values (decimal reduction time) 3575.56, 1270.61 and 635.38 min at the same temperatures. Also, the thermal stability of conjugated A. tenuissima KM651985 laccase was emphasized by high ΔHd (enthalpy), high ΔGd (free energy) and low ΔSd (entropy). The conjugated A. tenuissima KM651985 laccase showed high effectiveness in dyes decolorization of Remazol Brilliant Blue R (RBBR) and Malachite Green (MG). Moreover, the addition of conjugated A. tenuissima KM651985 laccase with hemicellulolytic enzymes cocktail improved the saccharification of corn cobs, rice straw, corn cobs leaves and water hyacinth with the highest reducing sugar production 847.44 ±â€¯19.17 mg from corn cops.

Kinetic and thermodynamic characteristic of Aspergillus awamori EM66 levansucrase.

This study is a new trial aimed to solve levansucrase high cost and levan associated problems during the purification process. Also, kinetic and thermodynamic study was done to compare between the partial pure (PP) and purified forms (PF). Within this context, Aspergillus awamori EM66 levansucrase was produced constitutively (5.44 U.mL-1) using rice straw as the sole medium component. The enzyme was partially purified and was eluted as single protein after two purification steps. Its molecular weight was determined to be 44.5 KDa. The optimum temperature recorded 40 °C for both enzyme forms. While, the purification process lowering the enzyme pH from 5.2 to 4.0. The NaCl concentrations (0.5-3.0 M) pointed to the halophilic nature of the enzyme. The PP form retained about 76% of its original activity after 1 h at 55 °C while the other retained about 57% after 45 min. at the same temperature. The kinetic parameters Km and Vmax concluded that the PF was more efficient than the PP. The thermodynamic parameters such as Ea, Ed, T1/2, D-value, also, ∆G*, ∆H* and ∆ S* for activation recorded that the PP had higher stability than the PF.

Improvement of catalytic, thermodynamics and antifungal activity of constitutive Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to oxidized polysaccharides.

Our study full filled in two main goals preparation of constitutive exochitinase with low cost, utilizing non-chitin containing agricultural wastes, and improving the thermodynamics of purified Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to sodium periodate activated agar. Central composite design (CCD) was used to improve the chemical modification of Trichoderma longibrachiatum KT693225 exochitinase. Optimum temperature for conjugated exochitinase 60 °C was higher than native form 40 °C. Covalent coupling to oxidized agar caused 4.32, 2.75 and 2.44-fold increase in half-life values at 50, 55 and 60 °C, respectively. Also, conjugated exochitinase showed higher D-values (decimal reduction time) 1790.49 compared to 733.08 min for native form at 60 °C. Moreover, conjugated form had lower deactivation constant rate (kd) 0.39 × 10-3 min-1at 60 °C than native form 1.7 × 10-3 min-1. Native exochitinase exhibited higher activation energy (Ea) 3.39 Kcal·mol-1 and lower energy for denaturation (Ed) 6.88 Kcal·mol-1 compared to 3.21 and 13.05 Kcal·mol-1, respectively for conjugated form. The values of thermodynamic parameters for inactivation of native and conjugated exochitinase indicated that conjugation significantly decreased entropy (ΔS°) and increased enthalpy (ΔH°) and free energy (ΔG°) of deactivation. Conjugated exochitinase exhibited higher antifungal effect against Alternaria alternata, Fusarium oxysporium and Aspergillus niger than native form.

Enhancement stability and catalytic activity of immobilized α-amylase using bioactive phospho-silicate glass as a novel inorganic support.

α-Amylase enzyme was immobilized on bioactive phospho-silicate glass (PS-glass) as a novel inorganic support by physical adsorption and covalent binding methods using glutaraldehyde and poly glutaraldehyde as a spacer. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) studies confirmed the glass-enzyme linkage. Dissolution of PS-glass in acidic and neutral pH is higher than that of alkaline pH. Some immobilization variables were optimized using statistical factorial design (Central Composite Design). Optimized immobilization variables enhanced the immobilization yield (IY) from 27.9 to 79.9% (2.9-fold). It was found that the immobilized enzyme had higher optimum temperature, higher half-life time (t1/2), lower activation energy (Ea), lower deactivation constant rate (kd) and higher decimal reduction time (D-values) within the temperature range of 40-60°C. Differential scanning calorimetry analysis (DSC) confirmed the thermalstability of the immobilized enzyme. The immobilized enzyme was stable at a wide pH range (5.0-8.0). Kinetic studies of starch hydrolysis demonstrated that immobilized enzyme had lower Michaelis constant (Km), maximum velocity (Vmax) and catalytic efficiency (Vmax/Km) values. The storage stability and reusability of the immobilized enzyme were found to be about 74.7 and 62.5% of its initial activity after 28days and 11cycles, respectively. Enhanced α-amylase stabilities upon immobilization make it suitable for industrial application.

Improvement of Aspergillus flavus saponin hydrolase thermal stability and productivity via immobilization on a novel carrier based on sugarcane bagasse.

Soyasapogenol B (SB) is known to have many biological activities such as hepatoprotective, anti-inflammatory, anti-mutagenic, antiviral and anticancer activities. Enzymatic conversion of soyasaponins to SB was carried out using saponin hydrolase (SH) extracted from Aspergillus flavus. The partially purified enzyme was immobilized on different carriers by physical adsorption, covalent binding or entrapment. Among the investigated carriers, Eupergit C and sugarcane bagasse (SCB) activated by DIC and NHS were the most suitable two carriers for immobilization (the immobilized forms recovered 46.5 and 37.1% of the loaded enzyme activity, respectively). Under optimized immobilization conditions, immobilized SH on Eupergit C and on activated SBC recovered 87.7 and 83.3% of its original activity, respectively. Compared to free SH, immobilized SH on Eupergit C and on activated SCB showed higher optimum pH, activation energy, half-lives and lower deactivation constant rate. Also, their SB productivities were improved by 2.3- and 2.2-folds compared to free SH (87.7 and 83.3 vs. 37.5%, respectively). Hence, being SCB more sustainable and an inexpensive material, it can be considered a good alternative to Eupergit C as a support for SH immobilization. SH immobilization on industrially applicable and inexpensive carrier is necessary to improve SB yield and reduce its production cost. The chemical structure of SCB and the resulting cellulose derivatives were studied by ATR-IR spectroscopy. The thermal analysis technique was used to study the chemical treatment of SCB and coupling with the enzyme. This technique confirmed the removal of lignin and hemicellulose by chemical treatment of SCB.

Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988.

In our search for chitinase and chitosanase producer from unconventional sources, the marine-derived fungus Aspergillus griseoaurantiacus KX010988 was obviously the best producer of the highest chitinase and chitosanase activities by solid state fermentation of potato shells. Chitinase was purified in three steps involving ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel chromatography. 12.55 fold increase in purity with a recovery of 17.6 was obtained. The molecular mass of the purified chitinase was found to be 130kDa. It was optimally active at pH 4.5 and 40°C. Km and Vmax values were 0.22mgmL-1 and 19.6µmolemin-1mg-1 respectively. Mn2+ and Zn2+ ions lead to increased chitinase activity. While Fe2+and Cu2+ions strongly inhibited the chitinase activity. The thermodynamics of pure chitinase including activation energy for thermal denaturation (Ea,d), change of free energy (ΔGd), enthalpy(ΔHd), entropy(ΔSd) and half life values (T1/2) at 40, 50 and 60°C were determined. Chitinase showed antifungal activity against pathogenic fungus Fusarium solani. Chitosanase was partially purified by acetone precipitation (50-75%) v/v concentration. The hydrolytic products of moderate molecular weight of chitosan by chitosanase were analyzed by thin layer chromatography (TLC) after 12 and 24h respectively. Chitosan-oligosaccharides showed good antibacterial and antioxidant activities.

Optimization of inulinase production from low cost substrates using Plackett-Burman and Taguchi methods.

Four marine-derived fungal isolates were screened for the production of inulinase enzyme from low cost substrates under solid state fermentation (SSF), one of them identified as Aspergillus terreus showed the highest inulinase activity using artichoke leaves as a solid substrate. Sequential optimization strategy, based on statistical experimental designs was employed to optimize the composition of the medium, including Plackett-Burman and Taguchi's (L9 3(4)) orthogonal array designs. Under the optimized conditions, inulinase activity (21.058 U/gds) reached the predicted maximum activity derived from the taguchi methodology, which increased about 4.79-folds the initial production medium. Fructose was produced, as an end product of inulin hydrolysis proving that the enzyme produced was exoinulinase. The marine-derived A. terreus is suggested as a new potential candidate for industrial enzymatic production of fructose from low cost substrate containing inulin as an economic source.