Hydrolyzation of mogrosides: Immobilized ß-glucosidase for mogrosides deglycosylation from Lo Han Kuo.

An immobilized enzyme system for bioconversion of Lo Han Kuo (LHK) mogrosides was established. ß-Glucosidase which was covalently immobilized onto the glass spheres exhibited a significant bioconversion efficiency from pNPG to pnitrophenol over other carriers. Optimum operational pH and temperature were determined to be pH 4 and 30°C. Results of storage stability test demonstrated that the glass sphere enzyme immobilization system was capable of sustaining more than 80% residual activity until 50 days, and operation reusability was confirmed for at least 10 cycles. The Michaelis constant (K m) of the system was determined to be 0.33 mM. The kinetic parameters, rate constant (K) at which Mogrosides conversion was determined, the τ 50 in which 50% of mogroside V deglycosylation/mogroside IIIE production was reached, and the τ complete of complete mogroside V deglycosylation/mogroside IIIE production, were 0.044/0.017 min-1, 15.6/41.1 min, and 60/120 min, respectively. Formation of the intermediates contributed to the kinetic differences between mogroside V deglycosylation and mogroside IIIE formation.

Reduction of Phytic Acid in Soymilk by Immobilized Phytase System.

In this study, three carriers (glass microsphere, cellulose beads and AlgNa/PVA beads) were evaluated as phytase solid carriers for reduction of phytic acid within soymilk. Phytase was covalently immobilized onto or entrapped within carriers for repeated use. Glass microsphere was chosen due to its high catalytic efficiency. Optimal operating condition (pH 6.0, 60 °C) was determined using 4-Nitrophenyl phosphate disodium salt hexahydrate as an indicator. Operational reusability was confirmed for more than seven batch reactions and the storage stability was capable of sustaining 70% of its catalytic activity for 40 days. The kinetic parameters including rate constant (K), time (τ50 ) in which 50% of phytic acid hydrolysis was reached, and time (τcomplete ) required to achieve complete phytic acid hydrolysis, were 0.023 min-1 , 35.7 min, 110 min. The current procedure provides a cheap as well as an easy way to carry out the reduction of phytic acid in soymilk, which has great potential in practical application.

Improvement on Physical Properties of Pullulan Films by Novel Cross-Linking Strategy.

Pullulan based films possess several advantages, including high transparency, low toxicity, good biodegradability, good mechanical properties, and low oxygen permeability, are preferable for food packaging. The application of pullulan films on food packaging, however, has inherent disadvantage of high water solubility. In this study, glutaraldehyde and glycerol were used as the cross-linking reagent and the plasticizer respectively to improve water resistance and physical properties of the pullulan films. Effects of cross-linking degree on physical properties, including water absorptions, swelling behaviors, water vapor permeability and tensile strengths of films were evaluated. FTIR results demonstrated that the pullulan films were successfully cross-linked by glutaraldehyde. The tensile strength of pullulan films could be enhanced significantly (P < 0.05) when glutaraldehyde was between 1% and 5% (w/w); nevertheless, the amount of glutaraldehyde above 20% (w/w) led to films brittleness. With the addition of glycerol as a plasticizer enhanced the extensibility of films as well as the hydrophilicity, resulting in higher water vapor permeability.

Enrichment of two isoflavone aglycones in black soymilk by Rhizopus oligosporus NTU 5 in a plastic composite support bioreactor.

BACKGROUND: A plastic composite support (PCS) bioreactor was implemented to evaluate the effects on isoflavone deglycosylation in black soymilk fermented by Rhizopus oligosporus NTU 5. RESULTS: Evaluation for the optimal PCS for mycelia immobilisation was conducted, which led to the significant results that the most mycelium weight (0.237 g per PCS, P < 0.05) is held by an S-type PCS; therefore, it was selected for black soymilk fermentation. It was found that the PCS fermentation system without pH control exhibits better efficiency of isoflavone bioconversion (daidzin to daidzein, and genistin to genistein) than the one with pH control at pH 6.5. As for the long-run fermentation, those without pH control indeed accelerate the isoflavone bioconversion by continuously releasing ß-glucosidase into soymilk. Deglycosylation can be completed in 8 to 24 h and sustained for at least 34 days as 26 batches. The non-pH-control fermentation system also exhibits the highest total phenolic content (ranged from 0.147 to 0.340 mg GAE mL(-1) sample) when compared to the pH-controlled and suspended ones. Meanwhile, the black soymilk from the 22nd batch with 8 h fermentation demonstrated the highest DPPH radical scavenging effect (54.7%). CONCLUSION: A repeated-batch PCS fermentation system was established to accelerate the deglycosylation rate of isoflavone in black soymilk. © 2015 Society of Chemical Industry.

Hydrolysis of isoflavone in black soy milk using cellulose bead as enzyme immobilizer.

The establishment of a catalytic system to enrich isoflavone aglycones in black soybean milk was investigated in this study. Beta-glucosidase, which was covalently immobilized onto cellulose beads, exhibited a significant efficiency for the conversion of 4-nitrophenyl ß-d-glucuronide to p-nitrophenol over the sol-gel method. The Michaelis constant (Km) of the cellulose bead enzymatic system was determined to be 1.50±0.10 mM. Operational reusability of the cellulose bead enzymatic system was justified for more than 10 batch reactions in black soy milk. Moreover, the storage stability verification indicated that the cellulose bead catalytic system was able to sustain its highest catalytic activity for 10 days. High-performance liquid chromatography results demonstrated that this enzymatic system required only 30 minutes to achieve complete isoflavone deglycosylation, and the aglycone content in the total isoflavones in black soy milk was enriched by 67% within 30 minutes by the cellulose bead enzymatic system.

Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

Electrochemically mediated enantioselective reduction of chiral sulfoxides.

The respiratory DMSO reductase from Rhodobacter capsulatus catalyzes the reduction of dimethyl sulfoxide to dimethyl sulfide. Herein, we have utilized this Mo enzyme as an enantioselective catalyst to generate optically pure sulfoxides (methyl p-tolyl sulfoxide, methyl phenyl sulfoxide and phenyl vinyl sulfoxide) from racemic starting materials. A hexaaminecobalt coordination compound in its divalent oxidation state was employed as the mediator of electron transfer between the working electrode and DMSO reductase to continually reactivate the enzyme after turnover. In all cases, chiral HPLC analysis of the reaction mixture revealed that the S-sulfoxide was reduced more rapidly leading to enrichment or isolation of the R isomer.

Enrichment of two isoflavone aglycones in black soymilk by using spent coffee grounds as an immobiliser for ß-glucosidase.

Spent coffee grounds, discarded as environmental pollutants, were adopted as enzyme immobilisation solid carriers instead of commercialised solid supports to establish an economical catalytic system. ß-Glucosidase was covalently immobilised onto spent coffee grounds for the conversion of isoflavone glycosides into their aglycones in black soymilk. Optimum conditions were determined to be 40°C and pH 6 using 4-nitrophenyl ß-D-glucuronide as an indicator. Operational reusability was confirmed for more than 30 batch reactions and the storage stability was capable of sustaining its highest catalytic activity for 20 days. The kinetic parameters including rate constant (K), time (τ(50)) in which 50% of isoflavone deglycosylation was reached, and time (τ(complete)) required to achieve complete isoflavone deglycosylation, were 0.16±0.02 min(-1), 4.54±0.32 min, 60 min for daidzin and 0.16±0.02 min(-1), 2.28±0.11 min, 60 min for genistin, respectively. The total aglycone content in black soymilk was enriched by 67.14±0.60% in the enzymatic treatment of 60 min duration.

Enrichment of two isoflavone aglycones in black soymilk by immobilized ß-glucosidase on solid carriers.

A catalytic system for deglycosylation of isoflavone in black soybean milk was established. ß-Glucosidase which was covalently immobilized onto the glass microspheres exhibited a significant efficiency for the conversion of pNPG to p-nitrophenol over other carriers. The optimum temperature for pNPG hydrolysis was 40 °C, and complete reaction can be reached in 30 min. Operational reusability was confirmed for more than 40 batch reactions. Moreover, the storage stability verification demonstrated that the glass microsphere catalytic system was capable of sustaining its highest catalytic activity for 40 days. The kinetic parameters, including rate constant (K) at which isoflavone glycosides deglycosylation were determined, the time (τ(50)) in which 50% of isoflavone glycosides deglycosylation was reached, and the time (τ(complete)) required to achieve complete isoflavone glycosides deglycosylation, were 0.35 ± 0.04 min(-1), 2.04 ± 0.25 min, and 30 min (for daidzin) and 0.65 ± 0.03 min(-1), 1.19 ± 0.08 min, and 20 min (for genistin), respectively. HPLC results revealed that this enzyme system took only 30 min to reach complete isoflavone deglycosylation and the aglycone content in the total isoflavones in black soymilk was enriched by 51.42 ± 0.17% under a 30 min treatment by the glass microsphere enzymatic system.

Soyfoods and soybean products: from traditional use to modern applications.

Soybean products (soyfoods), reported as potential functional foods, are implicated in several health-enhancing properties, such as easing the symptoms of postmenopausal women, reducing the risk of osteoporosis, preventing cardiovascular disease, and antimutagenic effects. Isoflavone, for example, is one of the most important compounds abundantly found in soybean, mainly accounting for the health-enhancing properties as mentioned earlier. However, most biological activities of isoflavones are mainly attributed to their aglycone forms. It has also been demonstrated that isoflavone aglycones are absorbed faster and in greater amount than their glycosides in human intestines. Fortunately, deglycosylation of isoflavones can be achieved during fermentation process by several strains such as lactic acid bacteria, basidiomycetes, filamentous fungus, and Bacillus subtilis with their ß-glucosidase activity. This article presents an overview of soybean's chemistry, application, state-of-the-art advances in soybean fermentation processing and products as well as their applications in food and pharmaceutical industries. Different compounds, such as isoflavone, dietary fibers, and proteins which exhibit significant bioactivities, are summarized. The roles of different microorganisms in bioconversion and enhancement of bioactivities of fermented soybean are also discussed.

Cobalt hexaamine mediated electrocatalytic voltammetry of dimethyl sulfoxide reductase: driving force effects on catalysis.

The bacterial molybdoenzyme dimethyl sulfoxide (DMSO) reductase from Rhodobacter capsulatus catalyzes the reduction of DMSO to dimethyl sulfide in anaerobic respiration. In its native state, DMSO reductase is reduced to its active state by a pentaheme cytochrome (DorC). Alternatively, we show that DMSO reductase catalysis may be driven electrochemically using a series of homologous coordination compounds as mediating synthetic electron donors. All mediators are macrocyclic hexaaminecobalt(II) complexes in their active form, differing principally in their redox potentials over a range of about 250 mV. Thus, each complex presents a different reductive driving force to DMSO reductase and this leads to pronounced differences in the electrocatalytic behavior as measured by cyclic voltammetry. Digital simulation of the experimental voltammetry enables the critical features of the catalytic cycle to be extracted.

Mediated electrochemistry of dimethyl sulfoxide reductase from Rhodobacter capsulatus.

Electrochemically driven catalysis of the bacterial enzyme dimethyl sulfoxide (DMSO) reductase (Rhodobacter capsulatus) has been studied using the macrocyclic complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) as a mediator. In the presence of both DMSO and DMSO reductase, the normal transient Co(III/II) voltammetric response of the complex is transformed into an amplified and sigmoidal (steady-state) waveform characteristic of a catalytic EC' mechanism. At low concentrations of DMSO (approximately K (M)) or high mediator concentrations (more than the concentration of DMSO reductase), the steady-state character of the voltammetric response disappears and is replaced by more complicated waveforms that are a convolution of transient and steady-state behavior as different steps within the catalytic cycle become rate limiting. Through digital simulation of cyclic voltammetry performed under conditions where the sweep rate, DMSO concentration, DMSO reductase concentration and mediator concentration were varied systematically, we were able to model all voltammograms with a single set of rate and equilibrium constants which provide new insights into the kinetics of the DMSO reductase catalytic mechanism that have hitherto been inaccessible from steady state or stopped flow kinetic studies.

Transition metal complexes as mediator-titrants in protein redox potentiometry.

A selection of nine macrocyclic Fe(III/II) and Co(III/II) transition metal complexes has been chosen to serve as a universal set of mediator-titrants in redox potentiometry of protein samples. The potential range spanned by these mediators is approximately from +300 to -700 mV vs the normal hydrogen electrode, which covers the range of most protein redox potentials accessible in aqueous solution. The complexes employed exhibit stability in both their oxidized and their reduced forms as well as pH-independent redox potentials within the range 6 < pH < 9. The mediators were also chosen on the basis of their very weak visible absorption maxima in both oxidation states, which will enable (for the first time) optical redox potentiometric titrations of proteins with relatively low extinction coefficients. This has previously been impractical with organic mediators, such as indoles, viologens and quinones, whose optical spectra interfere strongly with those of the protein.