High-efficiency novel extraction process of target polyphenols using enzymes in hydroalcoholic media.

Agro-industrial by-products are a sustainable source of natural additives that can replace the synthetic ones in the food industry. Grape pomace is an abundant by-product that contains about 70% of the grape's polyphenols. Polyphenols are natural antioxidants with multiple health-promoting properties. They are secondary plant metabolites with a wide range of solubilities. Here, a novel extraction process of these compounds was developed using enzymes that specifically liberates target polyphenols in the appropriate hydroalcoholic mixture. Tannase, cellulase, and pectinase retained 22, 60, and 52% of their activity, respectively, in ethanol 30% v/v. Therefore, extractions were tested in ethanol concentrations between 0 and 30% v/v. Some of these enzymes presented synergistic effects in the extraction of specific polyphenols. Maximum yield of gallic acid was obtained using tannase and pectinase enzymes in ethanol 10% v/v (49.56 ± 0.01 mg L-1 h-1); in the case of p-coumaric acid, by cellulase and pectinase treatment in ethanol 30% v/v (7.72 ± 0.26 mg L-1 h-1), and in the case of trans-resveratrol, by pectinase treatment in ethanol 30% v/v (0.98 ± 0.04 mg L-1 h-1). Also, the effect of enzymes and solvent polarity was analysed for the extraction of malvidin-3-O-glucoside, syringic acid, and quercetin. Previous studies were mainly focused on the maximization of total polyphenols extraction yields, being the polyphenolic profile the consequence but not the driving force of the optimization. In the present study, the basis of a platform for a precise extraction of the desire polyphenols is provided. KEY POINTS: • Enzymes can be used up to ethanol 30% v/v. • The specific enzymes' action determines the polyphenolic profile of the extracts. • The yields obtained of target polyphenols are competitive.

Quantitative Description of a Protein Fitness Landscape Based on Molecular Features.

Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo-ß-lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo-ß-lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo-ß-lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters.

Metallo-ß-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions.

A number of multiresistant bacterial pathogens inactivate antibiotics by producing Zn(II)-dependent ß-lactamases. We show that metal uptake leading to an active dinuclear enzyme in the periplasmic space of Gram-negative bacteria is ensured by a cysteine residue, an unusual metal ligand in oxidizing environments. Kinetic, structural and affinity data show that such Zn(II)-cysteine interaction is an adaptive trait that tunes the metal binding affinity, thus enabling antibiotic resistance at restrictive Zn(II) concentrations.

Exploring soybean cultivar susceptibility to sudden death syndrome: Insights into isoflavone responses and biocontrol potential.

Sudden Death Syndrome (SDS) caused by Fusarium tucumaniae is a significant threat to soybean production in Argentina. This study assessed the susceptibility of SY 3 × 7 and SPS 4 × 4 soybeans cultivars to F. tucumaniae and studied changes in root isoflavone levels after infection. Additionally, the biocontrol potential of plant-growth promoting rhizobacteria (PGPR) against SDS was also examined. Our results demonstrated that the SY 3 × 7 cultivar exhibited higher disease severity and total fresh weight loss than SPS 4 × 4. Both cultivars showed induction of daidzein, glycitein, and genistein in response to infection, with the partially resistant cultivar displaying significantly higher daidzein levels than the susceptible cultivar at 14 days post infection (dpi) (2.74 vs 2.17-fold), declining to a lesser extent at 23 dpi (0.94 vs 0.35-fold, respectively). However, daidzein was not able to inhibit F. tucumaniae growth in in vitro assays probably due to its conversion to an isoflavonoid phytoalexin which would ultimately be an effective fungal inhibitor. Furthermore, the PGPR bacterium Bacillus amyloliquefaciens BNM340 displayed antagonistic activity against F. tucumaniae and reduced SDS symptoms in infected plants. This study sheds light on the varying susceptibility of soybean cultivars to SDS, offers insights into isoflavone responses during infection, and demonstrates the potential of PGPR as a biocontrol strategy for SDS management, providing ways for disease control in soybean production.

Soybean (Glycine max) hull valorization through the extraction of polyphenols by green alternative methods.

Soybean is one of the greatest crops in the world, with about 348.7 million tons being produced in 2018. Soybean hull is a by-product produced during the processing of soybean to obtain flour and oil. Though not being actually exploited, it is a source of polyphenols with antioxidant activity. Here, the extraction of polyphenols from soybean hull was performed by means of an alkaline hydrolysis treatment, which was optimized by the response surface methodology. At the optimal region, a total phenolic content of 0.72 g gallic acid equivalents per 100 g of soybean hull was obtained with an antioxidant activity of 2.17 mmoles of Trolox equivalents. Polyphenols responsible for the antioxidant activities were identified by LC-MS, including phenolic acids, anthocyanins, stilbenes, and the two main isoflavones of soybean, daidzein and genistein, in their non-glycosylated form. Other alternative extraction methods based on Aspergillus oryzae fermentation and α-amylase hydrolysis are also proposed.

Valorization of two agroindustrial wastes to produce alpha-amylase enzyme from Aspergillus oryzae by solid-state fermentation.

The global amount of soybean and wheat produced is about 350 and 750 million metric tons every year, respectively. In consequence, huge amounts of waste are produced from them. The aim of this work was to employ two wastes -soybean husk and flour mill waste- to produce high quantities of alpha-amylase enzyme. The substrate composition and the culture conditions were assayed to improve alpha-amylase production by solid-state fermentation employing the fungus Aspergillus oryzae. The maximum productivity of the enzyme was achieved using a culture substrate composed of the two wastes, at 45% soybean husk and 55% flour mill by-product, without pre-treatment, at an incubation temperature of 30 °C. The optimal incubation time (6 days), yielded a very high alpha-amylase activity (47,000 U/g dry substrate) at low-cost. The enzymatic extract obtained was characterized by LC-MS, providing a complete profile of the proteins produced during the solid-state fermentation on these two wastes. Then, the extract was purified in a single-step by size-exclusion chromatography and the recovery and the purification factor of alpha-amylase enzyme were about 83% and 6, respectively. The system was scaled up 50 times and yielded a similar enzymatic activity (45,900 U/g of dry substrate).

Recovery of phenolic antioxidants from Syrah grape pomace through the optimization of an enzymatic extraction process.

Phenolic compounds are highly valuable products that remain trapped in grape pomace, an abundant winery by-product. Therefore, efficient extraction procedures of these compounds represent a route for grape pomace valorisation. Here we performed a screening of the factors affecting the aqueous enzymatic extraction of phenolic compounds from Syrah grape pomace, including the following independent variables: temperature, pH, pectinase, cellulase and tannase; and a subsequent optimization through response surface methodology. At the optimal region, the enzymatic treatment enhanced the extraction yield of phenolics by up to 66% and its antioxidant capacity by up to 80%, reducing the incubation time and enzyme doses in respect to previous studies. We found that tannase raises the antioxidant capacity of the extract by the liberation of gallic acid, while cellulose favours the liberation of p-coumaric acid and malvidin-3-O-glucoside. We also tested the procedure in different grape pomace varieties, verifying its wide applicability.

Overcoming differences: The catalytic mechanism of metallo-ß-lactamases.

Metallo-ß-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide spread of genes coding for these enzymes, together with the lack of a clinically useful inhibitor, have raised a sign of alarm. Inhibitor design has been mostly impeded by the structural diversity of these enzymes. Here we provide a critical review of mechanistic studies of the three known subclasses of metallo-ß-lactamases, analyzed at the light of structural and mutagenesis investigations. We propose that these enzymes present a modular structure in their active sites that can be dissected into two halves: one providing the attacking nucleophile, and the second one stabilizing a negatively charged reaction intermediate. These are common mechanistic elements in all metallo-ß-lactamases. Nucleophile activation does not necessarily requires a Zn(II) ion, but a Zn(II) center is essential for stabilization of the anionic intermediate. Design of a common inhibitor could be therefore approached based in these convergent mechanistic features despite the structural differences.

Evolution of Metallo-ß-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution.

The production of ß-lactamase enzymes is one of the most distributed resistance mechanisms towards ß-lactam antibiotics. Metallo-ß-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-ß-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-ß-lactamases.