Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Enzymatic production of xylooligosaccharides from corn cobs: Assessment of two different pretreatment strategies.

Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo- and exo-acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via "green" and sustainable XOS production.

Alternative Controlling Agent of Theobroma grandiflorum Pests: Nanoscale Surface and Fractal Analysis of Gelatin/PCL Loaded Particles Containing Lippia origanoides Essential Oil.

A new systematic structural study was performed using the Atomic Force Microscopy (AFM) reporting statistical parameters of polymeric particles based on gelatin and poly-ε-caprolactone (PCL) containing essential oil from Lippia origanoides. The developed biocides are efficient alternative controlling agents of Conotrachelus humeropictus and Moniliophtora perniciosa, the main pests of Theobroma grandiflorum. Our results showed that the particles morphology can be successfully controlled by advanced stereometric parameters, pointing to an appropriate concentration of encapsulated essential oil according to the particle surface characteristics. For this reason, the absolute concentration of 1000 µg·mL-1 (P1000 system) was encapsulated, resulting in the most suitable surface microtexture, allowing a faster and more efficient essential oil release. Loaded particles presented zeta potential around (-54.3 ± 2.3) mV at pH = 8, and particle size distribution ranging from 113 to 442 nm. The hydrodynamic diameter of 90% of the particle population was found to be up to (405 ± 31) nm in the P1000 system. The essential oil release was evaluated up to 80 h, with maximum release concentrations of 63% and 95% for P500 and P1000, respectively. The best fit for the release profiles was obtained using the Korsmeyer-Peppas mathematical model. Loaded particles resulted in 100% mortality of C. humeropictus up to 48 h. The antifungal tests against M. perniciosa resulted in a minimum inhibitory concentration of 250 µg·mL-1, and the P1000 system produced growth inhibition up to 7 days. The developed system has potential as alternative controlling agent, due to its physical stability, particle surface microtexture, as well as pronounced bioactivity of the encapsulated essential oil.

Pulsatile Controlled Release and Stability Evaluation of Polymeric Particles Containing Piper nigrum Essential Oil and Preservatives.

Considerable efforts have been spent on environmentally friendly particles for the encapsulation of essential oils. Polymeric particles were developed to encapsulate the essential oil from Piper nigrum based on gelatin and poly-ε-caprolactone (PCL) carriers. Gas Chromatography ((Flame Ionization Detection (GC/FID) and Mass Spectrometry (GC/MS)), Atomic Force Microscopy (AFM), Nanoparticle Tracking Analysis (NTA), Confocal Laser Scanning Microscopy (CLSM), Attenuated Total Reflectance-Fourier-transform Infrared Spectroscopy (ATR-FTIR), and Ultraviolet-Visible (UV-VIS) spectroscopy were used for the full colloidal system characterization. The essential oil was mainly composed of ß-caryophyllene (~35%). The stability of the encapsulated systems was evaluated by Encapsulation Efficiency (EE%), electrical conductivity, turbidity, pH, and organoleptic properties (color and odor) after adding different preservatives. The mixture of phenoxyethanol/isotialzoni-3-one (PNE system) resulted in enhanced stability of approximately 120 and 210 days under constant handling and shelf-life tests, respectively. The developed polymeric system presented a similar controlled release in acidic, neutral, or basic pH, and the release curves suggested a pulsatile release mechanism due to a complexation of essential oil in the PCL matrix. Our results showed that the developed system has potential as an alternative stable product and as a controlling agent, due to the pronounced bioactivity of the encapsulated essential oil.

Investigation on the in vitro anti-Trichophyton activity of photosensitizers.

Onychomycosis is the most common disease caused by fungal nail infections, and often caused by dermatophytes. This infection is very resistant to antifungal treatments, and promising Photodynamic Therapy (PDT) mediated treatments has been presented as a multitarget tracking. Optimization of PDT guide for uptake time, concentration of photosensitizers (PS) and the light dose to inactivate Trichophyton mentagrophytes. Curcumin derivatives, porphyrin Chlorin e6 (CHL-E6) and Chlorin-P6-6-N-butylamide-7-methyl-ester (CHL-butyl) were evaluated. PS photobleaching was observed on the hyphae photosensitized over the time, correlating the PS concentration and light dose of antifungal PDT. Porphyrin, Curcumin, Chl-e6 and Chl-butyl concentrations of 2.5 µg/mL, 0.025 µg/mL, 10 µg/mL and 5 µg/mL respectively, under illumination of 10.5 J/cm2 were the best antifungal conditions found in the study. Curcumin, in low concentrations, and chlorin were the PSs with higher activity anti-T. mentagrophytes.

Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model.

Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dose-dependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes.

Short-term and long-term effects of osteoporosis on incisor teeth and femoral bones evaluated by Raman spectroscopy and energy dispersive X-ray analysis in ovariectomized rats.

There are few published data on the relationship between loss of bone mass due to osteoporosis and poor tooth quality. This study analyzed the effects of osteoporosis on incisor teeth and femoral bones using optical techniques in rats. Twenty female Wistar rats aged 6 months (n = 20) were randomized into two groups: control group, non-ovariectomized rats (n = 10); ovariectomy group, ovariectomized rats to induce osteoporosis (n = 10). Each group was subdivided randomly into two groups containing five rats each as follows. Control group 1: non-ovariectomized rats euthanized at the age of 9 or 3 months post-ovariectomy (n = 5); Control group 2: non-ovariectomized rats euthanized at the age of 1 year or 6 months post-ovariectomy (n = 5); ovariectomy group 1: ovariectomized rats euthanized at the age of 9 months or 3 months post-ovariectomy (n = 5); ovariectomy group 2: ovariectomized rats euthanized at the age of 1 year or 6 months post-ovariectomy (n = 5). The incisor teeth and femoral bones of Wistar rats were removed to perform Raman spectroscopy using an excitation laser at 785 nm. In addition, an energy-dispersive X-ray spectrometer system was used to evaluate calcium (Ca) and phosphorus (P). The main findings included significant changes (p < 0.05) for phosphate and carbonate band areas for both incisor teeth and femur bones. In addition, there was significant negative correlation between the P concentration and phosphate/carbonate ratio (lower P content-larger ratio, p < 0.05) for incisor teeth and femoral bones. The proline and CH2 wag band areas were significantly reduced only for the incisor teeth (p < 0.05). Therefore, Raman spectroscopy assessed the compositional, physicochemical and structural changes in hard tissue. The current study also pointed out the possible action mechanisms of these changes, bone fracture risk and dental fragility. It is important to emphasize that poor dental quality may also occur due to osteoporosis.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy.

BACKGROUND: Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. RESULTS: Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. CONCLUSIONS: Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.