Copper Ferrite Magnetic Nanoparticles for the Immobilization of Enzyme.

In this study, novel, hollow superparamagnetic copper ferrite (CuFe2O4) nanoparticles (NPs) were synthesized by a low-temperature hydrothermal method. The hollow magnetic spheres were characterized by field emission scanning electron microscopy and high resolution transmission electron microscopy to confirm their morphology and size. The hollow NPs were demonstrated as the support for biological materials by the immobilization of Thermomyces lanuginosus lipase on the inner and outer surfaces of the hollow spheres. The immobilization of the enzyme was confirmed by Fourier Transform Infra-red spectroscopy and confocal laser scanning microscopy. The immobilized enzyme was shown to have an immobilization efficiency of 84.5%, with approximately 176 mg g-1 of enzyme loading, for the hollow-NPs support. The immobilized enzyme exhibited high storage and temperature stability. The reusability of the immobilized lipase was more than 80% after 10 cycles of repeated use.

Immobilization of Xylanase Using a Protein-Inorganic Hybrid System.

In this study, the immobilization of xylanase using a protein-inorganic hybrid nanoflower system was assessed to improve the enzyme properties. The synthesis of hybrid xylanase nanoflowers was very effective at 4°C for 72 h, using 0.25 mg/ml protein, and efficient immobilization of xylanase was observed, with a maximum encapsulation yield and relative activity of 78.5% and 148%, respectively. Immobilized xylanase showed high residual activity at broad pH and temperature ranges. Using birchwood xylan as a substrate, the Vmax and Km values of xylanase nanoflowers were 1.60 mg/ml and 455 µmol/min/mg protein, compared with 1.42 mg/ml and 300 µmol/min/mg protein, respectively, for the free enzyme. After 5 and 10 cycles of reuse, the xylanase nanoflowers retained 87.5% and 75.8% residual activity, respectively. These results demonstrate that xylanase immobilization using a protein-inorganic hybrid nanoflower system is an effective approach for its potential biotechnological applications.

Production of Methanol from Methane by Encapsulated Methylosinus sporium.

Massive reserves of methane (CH4) remain unexplored as a feedstock for the production of liquid fuels and chemicals, mainly because of the lack of economically suitable and sustainable strategies for selective oxidation of CH4 to methanol. The present study demonstrates the bioconversion of CH4 to methanol mediated by Type I methanotrophs, such as Methylomicrobium album and Methylomicrobium alcaliphilum. Furthermore, immobilization of a Type II methanotroph, Methylosinus sporium, was carried out using different encapsulation methods, employing sodium-alginate (Na-alginate) and silica gel. The encapsulated cells demonstrated higher stability for methanol production. The optimal pH, temperature, and agitation rate were determined to be pH 7.0, 30°C, and 175 rpm, respectively, using inoculum (1.5 mg of dry cell mass/ml) and 20% of CH4 as a feed. Under these conditions, maximum methanol production (3.43 and 3.73 mM) by the encapsulated cells was recorded. Even after six cycles of reuse, the Na-alginate and silica gel encapsulated cells retained 61.8% and 51.6% of their initial efficiency for methanol production, respectively, in comparison with the efficiency of 11.5% observed in the case of free cells. These results suggest that encapsulation of methanotrophs is a promising approach to improve the stability of methanol production.

Reduction of Acetate and Lactate Contributed to Enhancement of a Recombinant Protein Production in E. coli BL21.

Acetate and lactate in growth media are detrimental to the production of Thermus maltogenic amylase (ThMA), a heterologous protein, as well as to the growth of recombinant Escherichia coli. Only 50 mM of acetate or 10 mM of lactate reduced 90% of specific ThMA activity. In this study, mutant E. coli strains blocked in the ackA-pta or ackA-pta and ldh pathways were created, characterized, and assessed for their culture performace in 300 L-scale fermentation. The ackApta and ldh double-mutant strain formed significantly less lactate and acetate, and produced a concomitant increase in the excretion of pyruvate (17.8 mM) under anaerobic conditions. The ackA-pta mutant strain accumulated significant acetate but had an approximately 2-fold increase in the formation of lactate. The ackA-pta and ldh double-mutant strain had superior overall performance in large-scale culture under suboptimal conditions, giving 67% higher cell density and 66% higher ThMA activity compared with those of the control strain. The doublemutant strain also achieved a 179% improvement in volumetric ThMA production.

Simultaneous pretreatment and saccharification: green technology for enhanced sugar yields from biomass using a fungal consortium.

Two different biomasses were subjected to simultaneous pretreatment and saccharification (SPS) using a cocktail of hydrolytic and oxidizing enzymes. Application of a novel laccase as a detoxifying agent caused the removal of 49.8% and 32.6% of phenolic contents from the soaked rice straw and willow, respectively. Hydrolysis of soaked substrates using a newly developed fungal consortium resulted in saccharification yield of up to 74.2% and 63.6% for rice straw and willow, respectively. A high saccharification yield was obtained with soaked rice straw and willow without using any hazardous chemicals. The efficiency of each step related to SPS was confirmed by atomic force microscopy. The suitability of the developed SPS process was further confirmed by converting the hydrolysate from the process into bioethanol with 72.4% sugar conversion efficiency. To the best of our knowledge, this is the first report on the development of a less tedious, single-pot, and eco-friendly SPS methodology.

Characterization of a ß-1,4-mannanase from a newly isolated strain of Pholiota adiposa and its application for biomass pretreatment.

A highly efficient ß-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa ß-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa ß-1,4-mannanase showed the highest activity toward locust bean gum (V max = 1,990 U/mg protein, K m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa ß-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway(®) (113 mg/g-substrate), P. adiposa ß-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa ß-1,4-mannanase a good candidate as a new commercial ß-1,4-mannanase to improve biomass pretreatment.

Immobilization of laccase on SiO2 nanocarriers improves its stability and reusability.

Laccases have a broad range of industrial applications. In this study, we immobilized laccase on SiO2 nanoparticles to overcome problems associated with stability and reusability of the free enzyme. Among different reagents used to functionally activate the nanoparticles, glutaraldehyde was found to be the most effective for immobilization. Optimization of the immobilization pH, temperature, enzyme loading, and incubation period led to a maximum immobilization yield of 75.8% and an immobilization efficiency of 92.9%. The optimum pH and temperature for immobilized laccase were 3.5 and 45°C, respectively, which differed from the values of pH 3.0 and 40°C obtained for the free enzyme. Immobilized laccase retained high residual activities over a broad range of pH and temperature. The kinetic parameter Vmax was slightly reduced from 1,890 to 1,630 µmol/min/mg protein, and Km was increased from 29.3 to 45.6. The thermal stability of immobilized laccase was significantly higher than that of the free enzyme, with a half-life 11- and 18-fold higher at temperatures of 50°C and 60°C, respectively. In addition, residual activity was 82.6% after 10 cycles of use. Thus, laccase immobilized on SiO2 nanoparticles functionally activated with glutaraldehyde has broad pH and temperature ranges, thermostability, and high reusability compared with the free enzyme. It constitutes a notably efficient system for biotechnological applications.

Immobilization of L-arabinitol dehydrogenase on aldehyde-functionalized silicon oxide nanoparticles for L-xylulose production.

L-Xylulose is a potential starting material for therapeutics. However, its translation into clinical practice has been hampered by its inherently low bioavailability. In addition, the high cost associated with the production of L-xylulose is a major factor hindering its rapid deployment beyond the laboratory. In the current study, L-arabinitol 4-dehydrogenase from Hypocrea jecorina (HjLAD), which catalyzes the conversion of L-arabinitol into L-xylulose, was immobilized onto various carriers, and the immobilized enzymes were characterized. HjLAD covalently immobilized onto silicon oxide nanoparticles showed the highest immobilization efficiency (94.7 %). This report presents a comparative characterization of free and immobilized HjLAD, including its thermostability and kinetic parameters. The thermostability of HjLAD immobilized on silicon oxide nanoparticles was more than 14.2-fold higher than free HjLAD; the t1/2 of HjLAD at 25 °C was enhanced from 190 min (free) to 45 h (immobilized). In addition, the immobilized HjLAD retained 94 % of its initial activity after 10 cycles. When the immobilized HjLAD was used to catalyze the biotransformation of L-arabinitol to L-xylulose, 66 % conversion and a productivity of 7.9 g · h(-1) · L(-1) were achieved. The enhanced thermostability and reusability of HjLAD suggest that immobilization of HjLAD onto silicon oxide nanoparticles has the potential for use in the industrial production of rare sugars.

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles.

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and ß-glucosidase activities of 1,270, 146, 34, and 15 U mL(-1), respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k (cat)/K (m) = 1,440 mg mL(-1) s(-1)) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides.

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.