Invitro bioprocessing of corn as poultry feed additive by the influence of carbohydrate hydrolyzing metagenome derived enzyme cocktail

The carbohydrate-hydrolyzing enzymes play a crucial role in increasing the phenolic content and nutritional properties of polysaccharides substrate, essential for cost-effective industrial applications. Also, improving the feed efficiency of poultry is essential to achieve significant economic benefits. The current study introduced a novel thermostable metagenome-derived xylanase named PersiXyn8 and investigated its synergistic effect with previously reported α-amylase (PersiAmy3) to enhance poultry feed utilization. The potential of the enzyme cocktail in the degradation of poultry feed was analyzed and showed 346.73 mg/g poultry feed reducing sugar after 72 h of hydrolysis. Next, the impact of solid-state fermentation on corn quality was investigated in the presence and absence of enzymes. The phenolic content increased from 36.60 mg/g GAE in control sample to 68.23 mg/g in the presence of enzymes. In addition, the enzyme-treated sample showed the highest reducing power OD 700 of 0.217 and the most potent radical scavenging activity against ABTS (40.36%) and DPPH (45.21%) radicals. Moreover, the protein and ash contents of the fermented corn increased by 4.88% and 6.46%, respectively. These results confirmed the potential of the carbohydrate-hydrolyzing enzymes cocktail as a low-cost treatment for improving the phenolic content, antioxidant activity, and nutritional values of corn for supplementation of corn-based poultry feed.

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL−1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL−1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL−1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

Cloning and Functional Identification of SlPG49 in Solanum lycopersicum

The modification and degradation of pectin in cell walls are necessary for the fruit softening process, which involves a series of pectin-modifying enzymes. Polygalacturonases (PGs) are a major group of pectin-hydrolyzing enzymes, which participate in fruit maturation, organ shedding, pollen development, and other processes by catalyzing the degradation of polygalacturonic acid. However, their function in plants has not yet been fully elucidated. In this paper, a full-length cDNA encoding SlPG49 was cloned from a tomato. Sequence alignment and phylogenetic analysis demonstrated that SlPG49 contains four typical conserved domains and belongs to clade E in PG classification. Quantitative real-time PCR analysis showed that SlPG49 was highly expressed in fruits during the softening stage, indicating that SlPG49 may be involved in fruit softening. Subcellular localization results revealed that SlPG49 was located in the cell membrane and the cell wall. In addition, an in vitro enzymatic activity assay confirmed that SlPG49 does have the ability to catalyze the hydrolysis of polygalacturonic acid. These results indicate that SlPG49 is a newly discovered PG gene involved in tomato fruit softening, and provide an experimental basis for elucidating the biological functions of plant PGs during fruit softening.

Antioxidant and Starch-Hydrolyzing Enzymes Inhibitory Properties of Striga-Resistant Yellow-Orange Maize Hybrids

Most of the health benefits derived from cereals are attributed to their bioactive compounds. This study evaluated the levels of the bioactive compounds, and the antioxidant and starch-hydrolyzing enzymes inhibitory properties of six pipeline Striga-resistant yellow-orange maize hybrids (coded AS1828-1, 4, 6, 8, 9, 11) in vitro. The maize hybrids were grown at the International Institute of Tropical Agriculture (IITA), Nigeria. The bioactive compounds (total phenolics, tannins, flavonoids, and phytate) levels, antioxidant (DPPH• and ABTS•+ scavenging capacity and reducing power) and starch-hydrolyzing enzymes (α-amylase and α-glucosidase) inhibitory activities of the maize hybrids were determined by spectrophotometry. At the same time, carotenoids were quantified using a reverse-phase HPLC system. The ranges of the bioactive compounds were: 11.25–14.14 mg GAE/g (total phenolics), 3.62–4.67 mg QE/g (total flavonoids), 3.63–6.29 mg/g (tannins), 3.66–4.31% (phytate), 8.92–12.11 µg/g (total xanthophylls), 2.42–2.89 µg/g (total β-carotene), and 3.17–3.77 µg/g (total provitamin A carotenoids). Extracts of the maize hybrids scavenged DPPH• (SC50: 9.07–26.35 mg/mL) and ABTS•+ (2.65–7.68 TEAC mmol/g), reduced Fe3+ to Fe2+ (0.25 ± 0.64–0.43 ± 0.01 mg GAE/g), and inhibited α-amylase and α-glucosidase, with IC50 ranges of 26.28–52.55 mg/mL and 47.72–63.98 mg/mL, respectively. Among the six clones of the maize hybrids, AS1828-9 had the highest (p < 0.05) levels of tannins and phytate and the strongest antioxidant and starch-hydrolyzing enzymes inhibitory activities. Significant correlations were observed between total phenolics and the following: ABTS•+ (p < 0.01, r = 0.757), DPPH• SC50 (p < 0.01, r = −0.867), reducing power (p < 0.05, r = 0.633), α-amylase IC50 (p < 0.01, r = −0.836) and α-glucosidase IC50 (p < 0.05, r = −0.582). Hence, the Striga-resistant yellow-orange maize hybrids (especially AS1828-9) may be beneficial for alleviating oxidative stress and postprandial hyperglycemia.

On the Offensive: the Role of Outer Membrane Vesicles in the Successful Dissemination of New Delhi Metallo-β-lactamase (NDM-1)

Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles.

Salt leverages polyethylene terephthalate hydrolase (PETase) enzymatic activity via the predicted conformational switch

Plastic pollution spawned a global challenge caused by the environmental accumulation of polyethylene terephthalate (PET) plastics. Ongoing remediation efforts using microbial and engineered PET hydrolyzing enzymes (PETases) are hindered by slow depolymerization activities. Here, we report the optimized reaction conditions that leveraged the PETase hydrolase activity 2 to 3.8-fold in the presence of high NaCl concentrations. Molecular dynamics simulations (MDS) were applied to model salt-dependent conformational changes of the PETase enzyme bound to a 3-unit PET polymer. MDS demonstrated that PETase interaction with flanking polymer units exhibited a striking structural disparity at low and high salt concentrations. At low salt concentrations, flanking polymer units displayed significant bending. In contrast, flanking units extended at high salt concentrations, thus residues D206, H237, and S160 of the catalytic triad positioned in close vicinity of the scissile ester bond of the polymer substrate. The resulting high salt-specific enzyme/substrate geometry can potentially facilitate hydrolysis. We theorized that a salt-dependent conformational switch could attenuate the enzyme to a broad range of natural and artificial polymers consumed as carbon sources. Altogether, new knowledge may advance the engineering of PET hydrolase enzymes and benefit bioconversion programs.