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.

The catalytic and kinetic characterization of Bacillus subtilis MK775302 milk clotting enzyme: comparison with calf rennet as a coagulant in white soft cheese manufacture.

BACKGROUND: Calf rennet is considered the traditional source of milk clotting enzyme (MCE). However, increasing cheese consumption with decreasing the calf rennet supply had encouraged the quest for new rennet alternatives. The purpose of this study is to acquire more information about the catalytic and kinetic properties of partially purified Bacillus subtilis MK775302 MCE and to assess the role of enzyme in cheese manufacture. RESULTS: B. subtilis MK775302 MCE was partially purified by 50% acetone precipitation with 5.6-fold purification. The optimum temperature and pH of the partially purified MCE were 70 °C and 5.0, respectively. The activation energy was calculated as 47.7 kJ/mol. The calculated Km and Vmax values were 36 mg/ml and 833 U/ml, respectively. The enzyme retained full activity at NaCl concentration of 2%. Compared to the commercial calf rennet, the ultra-filtrated white soft cheese produced from the partially purified B. subtilis MK775302 MCE exhibited higher total acidity, higher volatile fatty acids, and improved sensorial properties. CONCLUSIONS: The partially purified MCE obtained in this study is a promising milk coagulant that can replace calf rennet at a commercial scale to produce better-quality cheese with improved texture and flavor.

Production of a novel laccase from Ceratorhiza hydrophila and assessing its potential in natural dye fixation and cytotoxicity against tumor cells.

BACKGROUND: Flavonoid natural dyes have gained attention because they are nontoxic and eco-friendly. However, they do not work effectively with artificial fibers and require the use of mordants, which are considered as hazardous chemicals. Laccase enzyme catalyzes the oxidation of phenols, forming phenoxyl radicals that undergo a further polymerization process. So, laccase can oxidize flavonoid dyes, and it can be used instead of harmful mordants in flavonoid dye fixation on cotton fabrics. Laccases also are involved in a variety of metabolic processes, and they have anti-proliferative effects toward HepG2 and MCF-7 tumor cells. RESULTS: Among fifteen fungal isolates, the fungus Ceratorhiza hydrophila isolated from the submerged plant Myriophyllum spicatum was selected as the most potent laccase producer. Optimization of the production medium resulted in a 9.9-fold increase in laccase productivity. The partially purified Ceratorhiza hydrophila laccase could successfully improve the affinity of cotton fabrics toward quercetin (flavonoid) dye with excellent color fastness properties. The partially purified laccase also showed anti-proliferative activity against HepG2 and MCF-7 tumor cells. However, high laccase concentration is required to estimate IC50. CONCLUSIONS: Ceratorhiza hydrophila MK387081 is an excellent laccase producer. The partially purified laccase from Ceratorhiza hydrophila can be used in textile dyeing and printing processes as a safer alternative to the conventional hazardous mordants. Also, it can be used in preparation of cancer treatment drugs. However, further studies are needed to investigate IC50 for both cell types at higher laccase concentrations.

Rice straw and orange peel wastes as cheap and eco-friendly substrates: A new approach in ß-galactosidase (lactase) enzyme production by the new isolate L. paracasei MK852178 to produce low-lactose yogurt for lactose-intolerant people.

Converting wastes to valuable products is the main target for many kinds of research nowadays. Wastes represent an environmental problem and getting rid of it is not easy and causes pollution. Accordingly, this study offers production of the valuable enzyme ß-galactosidase using rice straw and orange peel as the main medium constituents. ß-galactosidase converts lactose to glucose and galactose which are simple sugars and can be fermented easily by lactose-intolerant people who represent more than 50% of the world's population. It was produced by Lactobacillus paracasei, a series isolated from fermented milk, identified using 16S ribosomal RNA gene partial sequence and had the accession number MK852178. Plackett-Burman (PB) and Central Composite (CCD) Designs optimized the production scoring 1.683(10)6 U/ml with a difference five times higher than the non-optimized medium. The addition of 0.3 or 0.6% of ß-galactosidase serves as a good fortification for manufacturing nutritional and therapeutic low-lactose yogurt with no significant differences in total protein, total solids, fat, and ash between control and all treatments. The chemical, rheological and sensory properties of the final produced yogurt were evaluated during storage periods up to 9 days at 5 °C. In conclusion, L. paracasei MK852178 ß-galactosidase is a promising additive in manufacturing low lactose yogurt for lactose-intolerant people since it reduces the lactose content and doesn't influence the chemical and sensory properties.

Catalytic, kinetic and thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease.

Bacillus stearothermophilus alkaline protease was conjugated to several oxidized polysaccharides of different chemical structure. The conjugates were evaluated for the kinetic and thermodynamic stability. The conjugated enzyme with oxidized pectin had the highest retained activity (79.5%) and the highest half-life (T1/2) at 50°C and pH 9.0. Compared to the native protease, the conjugated preparation exhibited lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 50-60°C. The thermodynamic parameters for irreversible inactivation of native and conjugated protease indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy (ΔH*) of deactivation. The calculated value of activation energy for thermal denaturation (Ead) for the conjugated enzyme was 20.4KJmole-1 higher over the native one. The results of thermodynamic analysis for substrate hydrolysis indicated that the enthalpy of activation (ΔH*) and free energy of activation (free energy of substrate binding) ΔG*E-S and (ΔG*), (free energy of transition state) ΔG*E-T values were lower for the modified protease. Similarly, there was significant improvement of kcat, kcat/Km values. The enzyme proved to be metalloprotease and significantly stimulated by Ca2+ and Mg2+ whereas Hg2+, Fe3+ Cu2+ and Zn2+ inhibited the enzyme activity. There was no pronounced effect on substrate specificity after conjugation.

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.