Successive remodeling of IgG glycans using a solid-phase enzymatic platform

The success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering strategies to control the glycan structures. With the accumulation of knowledge about the glycan biosynthesis enzymes, enzymatic glycan remodeling provides a potential strategy to construct highly ordered glycans with improved efficiency and biocompatibility. In this study, we quantitatively evaluate more than 30 enzymes for glycoengineering immobilized immunoglobulin G, an impactful glycoprotein class in the pharmaceutical field. We demonstrate successive glycan remodeling in a solid-phase platform, which enabled IgG glycan harmonization into a series of complex-type N-glycoforms with high yield and efficiency while retaining native IgG binding affinity.SignificanceGlycosylation plays critical functional and structural roles in protein biology. However, our understanding of how discrete glycan structures affect protein behaviors remains extremely limited due to the naturally occurring microheterogeneity. Through the use of characterized glycoengineering enzyme combination, we report a solid-phase glycan remodeling (SPGR) platform that enables efficient IgG glycan harmonization into several glycoforms of interest with high biocompatibility to the substrates. It provides an efficient strategy to screen the biological behavior of distinct glycoforms, building a fundamental understanding of glycosylation.

Effects of a xylanase and beta-glucanase enzyme combination on growth performance of broilers fed maize-soybean meal-based diets

The following study evaluated effects of a xylanase and beta-glucanase combination on growth performance of broilers fed energy reduced versus nutritionally adequate maize-soybean meal-based diets. A total of 648, one-day-old male broilers (Ross 308) were assigned to floor-pens (24 birds/pen, nine pens/treatment, three treatments) in a randomised block design. Treatments included: (1) a nutritionally adequate positive control diet (PC); (2) a negative control (NC) diet in which energy, crude protein and digestible amino acids were reduced by 3.4% (-105 kcal apparent metabolisable energy), 2.3% and 1.2 to 3.0% vs PC, respectively; and (3) NC plus a xylanase and beta-glucanase combination that supplied 1,220 U xylanase and 152 U beta-glucanase per kilogram of final feed. All diets contained a background of 500 FTU/kg phytase and were offered to birds ad libitum. Birds fed NC showed reduced average daily gain (ADG) by -6.1% (P<0.05); increased feed conversion ratio (FCR) by 9.2 points (P<0.05), and overall (d 1-35) body weight corrected FCR which was increased by 9.4 points (P<0.05) vs the PC group. Enzyme supplementation increased final BW (+4.2%, P<0.05), ADG (+5.4%, P<0.05) and tended to reduce FCR (+7.5 points, P=0.054) from d 22-35 vs NC, without affecting average daily feed intake. Improvements in performance due to the enzyme combination were equivalent to performance on the PC diet in all cases. The results suggested that significant improvements in growth performance of broilers fed maize-soybean meal-based diets which had been reduced in energy and nutrients can be realised by supplementation with xylanase in combination with beta-glucanase.

An evaluation of synergistic interactions between feruloyl esterases and xylanases during the hydrolysis of various pre-treated agricultural residues

Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future.

Feruloyl esterase (FAE-1) sourced from a termite hindgut and GH10 xylanases synergy improves degradation of arabinoxylan

Cereal feedstocks have high arabinoxylan content as their main hemicellulose, which is linked to lignin by hydroxycinnamic acids such as ferulic acid. The ferulic acid is linked to arabinoxylan by ester bonds, and generally, the high substitution of ferulic acid leads to a loss of activity of xylanases targeting the arabinoxylan. In the current study, a feruloyl esterase (FAE-1) from a termite hindgut bacteria was functionally characterised and used in synergy with xylanases during xylan hydrolysis. The FAE-1 displayed temperature and pH optima of 60 ℃ and 7.0, respectively. FAE-1 did not release reducing sugars from beechwood xylan (BWX), wheat arabinoxylan (WAX) and oat spelt xylan (OX), however, displayed high activity of 164.74 U/mg protein on p-nitrophenyl-acetate (pNPA). In contrast, the GH10 xylanases; Xyn10 and XT6, and a GH11 xylanase, Xyn2A, showed more than two-fold increased activity on xylan substrates with low sidechain substitutions; BWX and OX, compared to the highly branched substrate, WAX. Interestingly, the FAE-1 and GH10 xylanases (Xyn10D and XT6) displayed a degree of synergy (DS) that was higher than 1 in all enzyme loading combinations during WAX hydrolysis. The 75%XT6:25%FAE-1 synergistic enzyme combination increased the release of reducing sugars by 1.34-fold from WAX compared to the control, while 25%Xyn10D:75%FAE-1 synergistic combination released about 2.1-fold of reducing sugars from WAX compared to controls. These findings suggest that FAE-1 can be used in concert with xylanases, particularly those from GH10, to efficiently degrade arabinoxylans contained in cereal feedstocks for various industrial settings such as in animal feeds and baking.

Nutrient Utilization in Yankasa Rams Fed Crop Residue Supplemented With Xylanase and Glucanase Combinations

Background: There is less information in Nigeria with regard the understanding of the use of exogenous enzymes widely used in monogastric diets in ruminant’s rations. The study evaluated the effects of supplementing xylanase and glucanase in combination in rations of Yankasa rams. Sixteen yearling Yankasa rams (average 21 kg) were used. Four treatments were evaluated: control (without enzyme combination), 50:50, 75:25 and 25:75 xylanase-glucanase combinations denoted as T1, T2, T3, and T4 respectively. The basal roughage was cowpea husk and sorghum husk. The feeding trial was conducted using complete randomize design.Results: There were differences (p<0.05) with regard to nutrient intake and total digestibility coefficient except crude protein and nitrogen free extract digestibility (p>0.05). The intake and digestibility increased with supplementation of xylanase and glucanase combination at 25:75 ratio respectively. It increased DM intake by 211.90 g/d, DM digestibility by 17.73%, ADF digestibility by 1.17% and NDF digestibility by 1.02%. The nitrogen balance in the body did not increase (p>0.05) with supplementation of 50:50 xylanase-glucanase combination. The efficiency of nitrogen utilization did not differ between the control and 50:50 xylanase-glucanase combination. Conclusion: The combination of xylanase and glucanase at ratio 25:75 respectively improved nutrient intake and digestibility but did not influence nitrogen utilization.

Efficient Biofilms Eradication by Enzymatic-Cocktail of Pancreatic Protease Type-I and Bacterial α-Amylase

Removal of biofilms is extremely pivotal in environmental and medicinal fields. Therefore, reporting the new-enzymes and their combinations for dispersal of infectious biofilms can be extremely critical. Herein, for the first time, we accessed the enzyme “protease from bovine pancreas type-I (PtI)” for anti-biofilm properties. We further investigated the anti-biofilm potential of PtI in combination with α-amylase from Bacillus sp. (αA). PtI showed a very significant biofilm inhibition effect (86.5%, 88.4%, and 67%) and biofilm prevention effect (66%, 64%, and 70%), against the E. coli, S. aureus, and MRSA, respectively. However, the new enzyme combination (Ec-PtI+αA) exhibited biofilm inhibition effect (78%, 90%, and 93%) and a biofilm prevention effect (44%, 51%, and 77%) against E. coli, S. aureus, and MRSA, respectively. The studied enzymes were found not to be anti-bacterial against the E. coli, S. aureus, and MRSA. In summary, the PtI exhibited significant anti-biofilm effects against S. aureus, MRSA, and E. coli. Ec-PtI+αA exhibited enhancement of the anti-biofilm effects against S. aureus and MRSA biofilms. Therefore, this study revealed that this Ec-PtI+αA enzymatic system can be extremely vital for the treatment of biofilm complications resulting from E. coli, S. aureus, and MRSA.

Enzyme additives influence bacterial communities of Medicago sativa silage as determined by Illumina sequencing

This study aimed to evaluate the effects of enzymes (cellulase combined with galactosidase),, and the combination of these enzymes with Lactobacillus plantarum (LP) on bacterial diversity using high-throughput sequencing. Alfalfa forages were treated without or with cellulase + ɑ-galactosidase (CEGA), cellulase + LP (CELP), ɑ-galactosidase + LP (GALP). After 56 days of ensiling, All the treated silages exhibited improved fermentation quality as reflecting by decreased pH, ammonium-N and increased lactic acid levels compared to the control silage. Enzymatic treatment improved nutrients value by increased the level of crude protein and decreased the neutral detergent fibre (NDF) level. Treatment of the silage significantly changed the bacterial community, as determined by the PCoA test. LAB dominated the bacterial community of the treated silage after ensiling. The dominant bacteria from Garciella, Enterococcus, Lactobacillus and Pediococcus in control silage changed to Lactobacillus and Pediococcus in CEGA silage, and Lactobacillus in CELP and GALP silages. Collectively, enzymes and enzyme in combination with inoculants both greatly increased the abundance of LAB, with Enterococcus, Lactobacillus and Pediococcus in enzymes only silge (CEGA) and Lactobacillus in enzyme combination with inoculants silage (CELP and GALP).

A Wild Fomes fomentarius for Biomediation of One Pot Synthesis of Titanium Oxide and Silver Nanoparticles for Antibacterial and Anticancer Application

The present study offers an alternative method for green synthesis of the formation of two types of nanoparticles (NPs). These NPs, titanium oxide and silver NPs (TiO2 and Ag NPs, respectively), were obtained from the amalgamation of intracellular extract of a wild mushroom, Fomes fomentarius, with aqueous solutions of titanium isopropoxide and silver nitrate, respectively. F. fomentarius was identified phenotypically and by 18S ribosomal RNA gene sequencing (Gene accession no: MK635351). The biosynthesis of TiO2 and Ag NPs was studied and characterized by X-ray diffraction (XRD), diffuse reflectance UV-Visible spectroscopy (DR-UV), fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and transmission electron microscope (TEM). Success was achieved in obtaining NPs of differing sizes and shapes. The antibacterial and anticancer activity of the NPs was significant with morphological damage being caused by both, although Ag NPs (10–20 nm) were found to have profound effects on bacterial and cancer cells in comparison to TiO2 NPs (100–120 nm). These metal NPs, synthesized using wild mushrooms, hold a great potential in biomedicinedue to an effective enzyme combination, which permits them to modify different chemical compounds to less toxic forms, which is required for ecofriendly and safe biomaterials.