Enzymatic activity of the blue light-regulated phosphodiesterase BlrP1 from Klebsiella pneumoniae shows a nonlinear dependence on light intensity.

The blue light-regulated phosphodiesterase BlrP1 from Klebsiella pneumoniae hydrolyzes cyclic dimeric guanosine monophosphate (GMP) in a blue light-dependent manner. It contains a photosensing BLUF domain and a functional EAL domain. Previously, it was reported that conformational changes in the dimer upon light illumination occurred only when both protomers of the dimer were excited. Based on this observation, it was proposed that BlrP1 might be a nonlinear light intensity sensor. To test this, here, the correlation between the turnover number of the hydrolysis reaction (kcat ) and the fraction of the excited protein (fred ) was measured by simultaneously monitoring the reaction rate and fred . Our results show that kcat is proportional to fred2 . Thus, BlrP1 works as a nonlinear light intensity sensor to sense a strong light environment.

Effects of High-Intensity Ultrasound Pretreatment on Structure, Properties, and Enzymolysis of Walnut Protein Isolate.

The purpose of this paper was to investigate the effect of high-intensity ultrasonication (HIU) pretreatment before enzymolysis on structural conformations of walnut protein isolate (WPI) and antioxidant activity of its hydrolysates. Aqueous WPI suspensions were subjected to ultrasonic processing at different power levels (600-2000 W) and times (5-30 min), and then changes in the particle size, zeta (ζ) potential, and structure of WPI were investigated, and antioxidant activity of its hydrolysates was determined. The particle size of the particles of aqueous WPI suspensions was decreased after ultrasound, indicating that sonication destroyed protein aggregates. The ζ-potential values of a protein solution significantly changed after sonication, demonstrating that the original dense structure of the protein was destroyed. Fourier transform infrared spectroscopy indicated a change in the secondary structure of WPI after sonication, with a decrease in ß-turn and an increase in α-helix, ß-sheet, and random coil content. Two absorption peaks of WPI were generated, and the fluorescence emission intensity of the proteins decreased after ultrasonic treatment, indicating that the changes in protein tertiary structure occurred. Moreover, the degree of hydrolysis and the antioxidant activity of the WPI hydrolysates increased after sonication. These results suggest that HIU pretreatment is a potential tool for improving the functional properties of walnut proteins.

Photolabile Linkers: Exploiting Labile Bond Chemistry to Control Mode and Rate of Hydrogel Degradation and Protein Release.

Photolabile moieties have been utilized in applications ranging from peptide synthesis and controlled protein activation to tunable and dynamic materials. The photochromic properties of nitrobenzyl (NB) based linkers are readily tuned to respond to cytocompatible light doses and are widely utilized in cell culture and other biological applications. While widely utilized, little is known about how the microenvironment, particularly confined aqueous environments (e.g., hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment. Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different fluorophores) for either combinatorial or sequential release, respectively. Overall, this work provides new facile chemical approaches for tuning the degradability of NB linkers and an innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized to guide the design of not only tunable materials platforms but also controlled synthetic protocols or surface modification strategies.

Enhancement of chitin suspension hydrolysis by a combination of ultrasound and chitinase.

This study evaluated the degradation kinetics and structural characteristics of chitin suspension (CS) with a combination of ultrasound and chitinase. Compared with the enzymolysis, the degradation degree of sonoenzymolysis was enhanced to the maximum by 27.93 % at an intensity of 25 W/mL for 20 min. According to degradation kinetics, ultrasound intensified molecular collision rate between chitinase and substrate, thereby increasing the degradation degree. What's more, combined with chitinase, ultrasound intensified the rate of the breakage of glycosidic bond and viscosity-average molecular weight (Mv) decrease, but no obvious change in acetylation degree (DA). Additionally, the intra- or inter-hydrogen bindings were weakened by ultrasound during sonoenzymolysis, leading to a slight decrease in crystalline index and a more ordered structure, which increased the accessibility of the substrate to enzyme. In conclusion, combination of chitinase and ultrasound could enhance the hydrolysis of CS while without changing its primary structure.

Study on the mechanism of ultrasound-accelerated enzymatic hydrolysis of starch: Analysis of ultrasound effect on different objects.

Enzymatic hydrolysis of starch is an important process in the food industry. In the present work, ultrasound was introduced in glucoamylase pretreatment, starch pretreatment and mixed reaction system treatment to enhance starch hydrolysis efficiency. These different processes were studied to explore the mechanism of ultrasound in promoting enzymatic reactions. The hydrolysis degree of starch was determined via measuring the reducing sugar yield. Ultrasound caused enzyme inactivation under high temperatures, high ultrasonic power and long-time treatment, especially at high temperatures exceeding 65 °C. Ultrasound pretreatment of starch before enzymolysis led to the furtherance of starch hydrolysis degree. Meanwhile, sonicating the mixed enzymatic reaction system below 65 °C promoted starch hydrolysis significantly, inducing more than five- fold growth in the degree of starch hydrolysis as much as the ultrasound pretreatment caused. Molecular weights analysis conducted by the MALLS system reflected the enormous damage of starch molecules caused by ultrasound. The amylose contents and chain length distributions of samples were separately analyzed by iodine binding method and size exclusion chromatography. The results of the two experiments illustrate that ultrasound could promote the enzymatic hydrolysis of amylopectin, which is harder for glucoamylase to hydrolyze compared to amylose.

Effects of gamma ray irradiation-induced protein hydrolysis and oxidation on tenderness change of fresh pork during storage.

This study investigated the effect of irradiation (0, 3, 5 or 7 kGy) on the tenderness changes of pork during storage at 4 °C for two weeks by determining the total carbonyl, sulfhydryl groups, peptidomic profiles, Warner-Bratzler (WB) shear force value and by gel electrophoresis (SDS-PAGE) experiment. The results showed that, irradiation significantly increased total carbonyl content of pork but had no significant effect on sulfhydryl groups. Protein thiol loss induced by irradiation was greatly promoted after storage for 3 days. Increasing irradiation dose level could significantly decrease the WB shear force value of samples (P < .05) by provoking degradation of myofibrillar protein and collagen fragments. During refrigerated conditioning, however, the WB shear force values increased significantly despite irradiation (P < .05). This was attributed to increasing amount of collagen solubilized from muscle, protein aggregation induced by carbonylation of troponin T (at 7 days of storage) and further cross-linking of myosin heavy chain (MHC) (at 14 days of storage).

Elucidation of a non-thermal mechanism for DNA/RNA fragmentation and protein degradation when using Lyse-It.

Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.

Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

Artificial Light at Night Alleviates the Negative Effect of Pb on Freshwater Ecosystems.

Artificial light at night (ALAN) is an increasing phenomenon worldwide that can cause a series of biological and ecological effects, yet little is known about its potential interaction with other stressors in aquatic ecosystems. Here, we tested whether the impact of lead (Pb) on litter decomposition was altered by ALAN exposure using an indoor microcosm experiment. The results showed that ALAN exposure alone significantly increased leaf litter decomposition, decreased the lignin content of leaf litter, and altered fungal community composition and structure. The decomposition rate was 51% higher in Pb with ALAN exposure treatments than in Pb without ALAN treatments, resulting in increased microbial biomass, ß-glucosidase (ß-G) activity, and the enhanced correlation between ß-G and litter decomposition rate. These results indicate that the negative effect of Pb on leaf litter decomposition in aquatic ecosystems may be alleviated by ALAN. In addition, ALAN exposure also alters the correlation among fungi associated with leaf litter decomposition. In summary, this study expands our understanding of Pb toxicity on litter decomposition in freshwater ecosystems and highlights the importance of considering ALAN when assessing environmental metal pollutions.

Photobiological effect of Laser or LED light in a thermophilic microbial consortium.

Cellulose has a highly diversified architecture and its enzymatic complexes are studied for achieving an efficient conversion and a high level of efficiency in the deconstruction of cellulolytic biomass into sugars. The aim of this investigation was to evaluate the effect of Laser or LED light in the cellulolytic activity (CMCase) and on the proliferation of the thermophilic microbial consortium used on the degradation process of a lignocellulosic biomass of green coconut shell. The irradiation protocol consisted of six Laser irradiations (λ660 ηm, 40 mW, 270 s, 13 J/cm2) or LED (λ632 ±â€¯2 ηm, 145 mW, 44 s, 13 J/cm2) with 12- h time intervals in nutrient deprivation conditions. After irradiation, the consortium was inoculated into a lignocellulosic biomass (coconut fibers). Non- irradiated consortium was also inoculated and acted as control. Cell proliferation and endoglucanase activity were quantified during the experimental time. Experiments were carried out in triplicate. The results showed an increase of 250 % of thermo-cellulolytic microorganisms for the LED group and 200% for the Laser group when compared to the control. The enzymatic index (red Congo method), showed a statistically significant difference in the process of degradation of the lignocellulosic biomass between the Laser and LED groups compared to the control group [p < 0.0029; p < 0.029, respectively] 48-hs after the inoculation of the microorganisms. At the end of 72-h, this significant difference was maintained for both irradiated groups (p < 0.0212). Based upon the protocol used on the present study, it is possible to concluded that LED light enhanced cell proliferation of the thermophilic microbial consortium while the Laser light increase the enzymatic index of the lignocellulosic biomass of green coconut shell.

Enzyme-mediated photoinactivation of Enterococcus faecalis using Rose Bengal-acetate.

Rose Bengal-acetate (RB-Ac) is a pro-photosensitizer claimed to diffuse into target cells, where the acetate groups are hydrolyzed and the photosensitizing properties of Rose Bengal (RB) are restored. Despite promising results on tumor cells, the interaction of RB-Ac with bacteria has never been investigated. This study aimed to assess the interaction of RB-Ac with Enterococcus faecalis and to evaluate its potential use in antimicrobial photodynamic therapy (aPDT). Spectrofluorometry was used to assess the ability of E. faecalis to hydrolyze the RB-Ac compound. Fluorescence microscopy was employed to observe the distribution and to evaluate the cellular uptake of the RB produced. The antibacterial efficiency of RB-Ac-mediated aPDT was assessed by flow cytometry in combination with the LIVE/DEAD® staining. Results showed that RB-Ac was successfully hydrolyzed in the presence of E. faecalis cells. The RB produced appeared to incorporate the membrane of bacteria. Higher concentrations of RB-Ac resulted in higher incorporation of RB. The blue-light irradiation of RB-Ac-treated samples significantly reduced bacterial viability. Less than 0.01% of E. faecalis survived after incubation with 200 µM RB-Ac during 900 min and blue-light activation. The current report indicates that E. faecalis cells can hydrolyze the RB-Ac compound to produce active RB. The use of RB-Ac did not appear to allow cytoplasmic internalization of the RB produced, which rather incorporated the membrane bilayers of E. faecalis. The use of RB-Ac did not provide additional advantages over RB in terms of PS localization. Nonetheless, sufficient RB was produced and incorporated into the membranes of bacteria to elicit effective aPDT.

Irradiation pretreatment facilitates the achievement of high total sugars concentration from lignocellulose biomass.

This study evaluated the two hydrolysis strategies, involving one thermal and one dilute acid/enzymatic hydrolysis, to produce high xylose and glucose concentrations from lignocellulose assisted with irradiation pretreatment. Prior to hydrolysis, lignocellulose was pretreated by γ-irradiation at 800KGy. The merits of irradiation pretreatment on lignocellulose were contributed to size-reduced particle distributions and low shear rate of material, which allowed high biomass loadings up to 30-40%(w/v, equals to 23-29wt.%) for the consequent hydrolysis process. Results showed that hemicellulose fraction could achieve ∼84g/L of total sugars containing ∼55g/L xylose and ∼21g/L glucose through this two steps hydrolysis. Cellulose fraction would release ∼251g/L of total sugars consisting of ∼235g/L glucose and ∼16g/L xylose in the ultimate enzymatic hydrolysate. To the best of our knowledge, it was the first report of achieving 235g/L glucose in cellulose enzymatic hydrolysate derived from lignocellulose.

Antioxidant activity measurement and potential antioxidant peptides exploration from hydrolysates of novel continuous microwave-assisted enzymolysis of the Scomberomorus niphonius protein.

A novel continuous microwave-assisted enzymatic digestion (cMAED) method is proposed for the digestion of protein from Scomberomorus niphonius to obtain potential antioxidant peptides. In this study, bromelain was found to have a high capacity for the digestion of the Scomberomorus niphonius protein. The following cMAED conditions were investigated: protease species, microwave power, temperature, bromelain content, acidity of the substrate solution, and incubation time. At 400W, 40°C, 1500U·g-1 bromelain, 20% substrate concentration, pH 6.0 and 5min incubation, the degree of hydrolysis and total antioxidant activity of the hydrolysates were 15.86% and 131.49µg·mL-1, respectively. The peptide analyses showed that eight of the potential antioxidant peptide sequences, which ranged from 502.32 to 1080.55Da with 4-10 amino acid residues, had features typical of well-known antioxidant proteins. Thus, the new cMAED method can be useful to obtain potential antioxidant peptides from protein sources, such as Scomberomorus niphonius.

Ozonolysis combined with ultrasound as a pretreatment of sugarcane bagasse: Effect on the enzymatic saccharification and the physical and chemical characteristics of the substrate.

Sugarcane bagasse (SCB) was treated in three stages using ozone oxidation (O), washing in an alkaline medium (B) and ultrasonic irradiation (U). The impact of each pretreatment stage on the physical structure of the SCB was evaluated by its chemical composition, using an infrared technique (FTIR-ATR), and using thermogravimetric analysis (TGA/DTG). The pretreatment sequence O, B, U provided a significant reduction of lignin and hemicellulose, which was confirmed by changes in the absorption bands corresponding to these compounds, when observed using infrared. Thermogravimetric analysis confirmed an increased thermal stability in the treated sample due to the removal of hemicellulose and extractives during the pretreatment. This pretreatment released 391mg glucose/g from treated SCB after the enzymatic hydrolysis, corresponding to a yield of 94% of the cellulose available.

Heavy metals in aromatic spices by inductively coupled plasma-mass spectrometry.

Objective of this study was to determine the content of Cd, Hg, As and Pb in common spices traded in the Italian market, using inductively coupled plasma-mass spectrometry (ICP-MS). The results were compared with the maximum limits established by the national Legislative Decree (LD) no. 107 implementing the Council Directive 88/388/EEC and by international organisations, such as Food and Agriculture Organization (FAO) and World Health Organization (WHO). Food safety for spices was assessed considering the tolerable weekly intake (TWI) and the provisional tolerable weekly intake (PTWI), respectively, for Cd and Hg and the 95% lower confidence limit of the benchmark dose of 1% extra risk (BMDL01) for As and Pb. Investigated elements in all samples were within the maximum limits as set by the national and international normative institutions. Nevertheless, the heavy metal content of some spices exceeded the PTWI, TWI and BMDL01, which needs attention when considering consumer's health.

Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.

The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

Aluminium content of foods originating from aluminium-containing food additives.

Aluminium (Al) levels of 90 food samples were investigated. Nineteen samples contained Al levels exceeding the tolerable weekly intake (TWI) for young children [body weight (bw): 16 kg] when consuming two servings/week. These samples were purchased multiple times at specific intervals and were evaluated for Al levels. Al was detected in 27 of the 90 samples at levels ranging from 0.01 (limit of quantitation) to 1.06 mg/g. Of these, the Al intake levels in two samples (cookie and scone mix, 1.3 and 2 mg/kg bw/week, respectively) exceeded the TWI as established by European Food Safety Authority, although the level in the scone mix was equivalent to the provisional TWI (PTWI) as established by Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives. The Al levels markedly decreased in 14 of the 19 samples with initially high Al levels. These results indicated reductions in the Al levels to below the PTWI limits in all but two previously identified food samples.

Suppressive effect of Sanmiao formula on experimental gouty arthritis by inhibiting cartilage matrix degradation: An in vivo and in vitro study.

Sanmiao formula (SM) is a compound prescription, which has been used in traditional Chinese medicine since the Ming Dynasty for gouty and rheumatoid arthritis treatments. However, no evidence has been unfolded to show the relationship between SM and gouty arthritis (GA), particularly inhibiting cartilage matrix degradation. In the present study, we undertook a characterization of anti-GA activity of SM using an in vivo rat model induced by potassium oxonate and cold bath together with in vitro studies with chondrocytes for further molecular characterization. Potassium oxonate and cold bath rats were treated with SM at doses of 7.2g/kg per day for 5days. SM treatments significantly suppressed the swelling rate and the severe pathologic changes in the joints of the animals in gout model. Inflammatory factors count by ELISA analysis, SM exhibited inhibition on IL-1ß and TNF-α. Moreover, histological analysis of the joints and SM-serum substantially interfered with the MSU-induced expression of glycosaminoglycans (GAG), up-regulated the content of proteoglycan. Importantly, SM interfered with GA-augmented expression of matrix metalloproteinases (MMPs) -3 and aggrecanases (ADAMTS)-4, which are considered to be key enzymes in cartilage matrix degradation, and simultaneously augmented GA-reduced tissue inhibitors of metalloproteinases (TIMPs) -1 and -3 expression in the joints and chondrocytes. Therefore, SM is looking forward to be a potential novel agent that could prevent cartilage matrix degradation effectively in gouty arthritis, and this provides a new target for development of new medicines.

Lactic acid and thermal treatments trigger the hydrolysis of myo-inositol hexakisphosphate and modify the abundance of lower myo-inositol phosphates in barley (Hordeum vulgare L.).

Barley is an important source of dietary minerals, but it also contains myo-inositol hexakisphosphate (InsP6) that lowers their absorption. This study evaluated the effects of increasing concentrations (0.5, 1, and 5%, vol/vol) of lactic acid (LA), without or with an additional thermal treatment at 55°C (LA-H), on InsP6 hydrolysis, formation of lower phosphorylated myo-inositol phosphates, and changes in chemical composition of barley grain. Increasing LA concentrations and thermal treatment linearly reduced (P<0.001) InsP6-phosphate (InsP6-P) by 0.5 to 1 g compared to the native barley. In particular, treating barley with 5% LA-H was the most efficient treatment to reduce the concentrations of InsP6-P, and stimulate the formation of lower phosphorylated myo-inositol phosphates such as myo-inositol tetraphosphate (InsP4) and myo-inositol pentaphosphates (InsP5). Also, LA and thermal treatment changed the abundance of InsP4 and InsP5 isomers with Ins(1,2,5,6)P4 and Ins(1,2,3,4,5)P5 as the dominating isomers with 5% LA, 1% LA-H and 5% LA-H treatment of barley, resembling to profiles found when microbial 6-phytase is applied. Treating barley with LA at room temperature (22°C) increased the concentration of resistant starch and dietary fiber but lowered those of total starch and crude ash. Interestingly, total phosphorus (P) was only reduced (P<0.05) in barley treated with LA-H but not after processing of barley with LA at room temperature. In conclusion, LA and LA-H treatment may be effective processing techniques to reduce InsP6 in cereals used in animal feeding with the highest degradation of InsP6 at 5% LA-H. Further in vivo studies are warranted to determine the actual intestinal P availability and to assess the impact of changes in nutrient composition of LA treated barley on animal performance.

Radiofrequency treatment enhances the catalytic function of an immobilized nanobiohybrid catalyst.

Biocatalysis, the use of enzymes in chemical transformation, has undergone intensive development for a wide range of applications. As such, maximizing the functionality of enzymes for biocatalysis is a major priority to enable industrial use. To date, many innovative technologies have been developed to address the future demand of enzymes for these purposes, but maximizing the catalytic activity of enzymes remains a challenge. In this study, we demonstrated that the functionality of a nanobiocatalyst could be enhanced by combining immobilization and radiofrequency (RF) treatment. Aminopeptidase PepA-encapsulating 2 nm platinum nanoparticles (PepA-PtNPs) with the catalytic activities of hydrolysis and hydrogenation were employed as multifunctional nanobiocatalysts. Immobilizing the nanobiocatalysts in a hydrogel using metal chelation significantly enhanced their functionalities, including catalytic power, thermal-stability, pH tolerance, organic solvent tolerance, and reusability. Most importantly, RF treatment of the hydrogel-immobilized PepA-PtNPs increased their catalytic power by 2.5 fold greater than the immobilized PepA. Our findings indicate that the catalytic activities and functionalities of PepA-PtNPs are greatly enhanced by the combination of hydrogel-immobilization and RF treatment. Based on our findings, we propose that RF treatment of nanobiohybrid catalysts immobilized on the bulk hydrogel represents a new strategy for achieving efficient biocatalysis.