Enzymatically triggered delignification through a novel stable laccase: A mixed in-silico /in-vitro exploration of a complex environmental microbiota.

Laccases have been broadly applied as a multitasking biocatalyst in various industries, but their applications tend to be limited by easy deactivation, lack of adequate stability, and susceptibility under complex conditions. Identifying stable laccase as a green-biocatalyst is crucial for developing cost-effective biorefining processes. In this direction, we attempted in-silico screening a stable metagenome-derived laccase (PersiLac1) from tannery wastewater in a complex environment. The laccase exhibited high thermostability, retaining 53.19% activity after 180 min at 70 °C, and it was stable in a wide range of pH (4.0-9.0). After 33 days of storage at 50°C, pH 6.0, the enzyme retained 71.65% of its activity. Various metal ions, inhibitors, and organic solvents showed that PersiLac1 has a stable structure. The stable PersiLac1 could successfully remove lignin and phenolic from quinoa husk and rice straw. In the separate hydrolysis and fermentation process (SHF) after 72 h, hydrolysis was obtained 100% and 73.4% for quinoa husk and rice straw, and fermentation by the S. cerevisiae was be produced 41.46 g/L and 27.75g/L ethanol, respectively. Results signified that the novel lignin-degrading enzyme was confirmed to have great potential for industrial application as a green-biocatalyst based on enzymatically triggered to delignification and detoxify lignocellulosic biomass.

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.

The novel homologue of the human α-glucosidase inhibited by the non-germinated and germinated quinoa protein hydrolysates after in vitro gastrointestinal digestion.

Quinoa (Chenopodium quinoa Willd) is a potential source of protein with ideal amino acid profiles which its bioactive compounds can be improved during germination and gastrointestinal digestion. The present investigation studies the impact of germination for 24 hr and simulated gastrointestinal digestion on α-glucosidase inhibitory activity of the quinoa protein and bioactive peptides against the novel homologue of human α-glucosidase, PersiAlpha-GL1. The sprouted quinoa after gastroduodenal digestion was the most effective α-glucosidase inhibitor showing 81.10% α-glucosidase inhibition at concentration 4 mg/ml with the half inhibition rate (IC50 ) of 0.07 mg/ml. Based on the kinetic analysis, both the germinated and non-germinated samples before and after digestion were competitive-type inhibitors of α-glucosidase. Results of this study showed the improved α-glucosidase inhibitory activity of the quinoa bioactive peptides after germination and gastrointestinal digestion and highlighted the potential of metagenome-derived PersiAlpha-GL1 as a novel homologue of the human α-glucosidase for developing the future anti-diabetic drugs. PRACTICAL APPLICATIONS: This study aimed to evaluate the effect of germination and gastrointestinal digestion of the quinoa protein and bioactive peptides on α-glucosidase inhibitory activity against the novel PersiAlpha-GL1. Metagenomic data were used to identify the novel α-glucosidase structurally and functionally homologue of human intestinal. The results showed the highest inhibition on PersiAlpha-GL1 by a germinated quinoa after gastroduodenal digestion and confirmed the potential of PersiAlpha-GL1 to enhance the effectiveness of the anti-diabetic drugs for industrial application.

Efficient removal of various textile dyes from wastewater by novel thermo-halotolerant laccase.

A novel thermostable/halotolerant metagenome-derived laccase (PersiLac2) from tannery wastewater was purified to remove textile dyes in this study. The enzyme was highly active over a wide temperature and pH range and maintained 73.35% of its initial activity after 30 days, at 50 °C. The effect of various metal and organic-solvent tolerance on PersiLac2 showed, retaining greater than 53% activity at 800 mM of metal ions, 52.12% activity at 6 M NaCl, and greater than 44.09% activity at 20% organic solvents. PersiLac2 manifested effective removal of eight different textile dyes from azo, anthraquinone, and triphenylmethane families. It decolorized 500 mg/L of Alizarin yellow, Carmine, Congo red and Bromothymol blue with 99.74-55.85% efficiency after 15 min, at 50 °C, without mediator. This enzyme could practically remove dyes from a real textile effluent and it displayed significant detoxification in rice seed germination tests. In conclusion, PersiLac2 could be useful in future for decolorization/detoxification of wastewater.

Improving the quality of gluten-free bread by a novel acidic thermostable α-amylase from metagenomics data.

Development of gluten-free products is important due to their role in gluten related disorders and health improvement. α-Amylase enzymes have shown to have a positive effect on wheat bread quality. This study aimed to screen in-silico a novel acidic-thermostable α-amylase (PersiAmy2) from the sheep rumen metagenome to increase the quality of gluten-free bread. The PersiAmy2 was cloned, expressed, purified and characterized. The enzyme was highly stable at a wide range of pH, temperature and storage conditions. The PersiAmy2 had excellent activity in the presence of ions, inhibitors, and surfactants. Utilization of the acidic thermostable PersiAmy2 in gluten-free bread resulted in a softer crumb, higher specific volume, porosity, moisture content and caused a darker crust color. The rheological measurement showed a solid-elastic behavior in batters. Also the addition of this enzyme reduced the firmness. From the results of this study it can be concluded that the PersiAmy2 can be used to improve the quality of gluten-free bread.

A novel high performance in-silico screened metagenome-derived alkali-thermostable endo-ß-1,4-glucanase for lignocellulosic biomass hydrolysis in the harsh conditions.

BACKGROUND: Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is significant. RESULTS: The primary aim of this study was to discover a novel alkali-thermostable endo-ß-1,4-glucanase from the sheep rumen metagenome. At first, the multi-step in-silico screening approach was utilized to find primary candidate enzymes with superior properties. Among the computationally selected candidates, PersiCel4 was found and subjected to cloning, expression, and purification followed by functional and structural characterization. The enzymes' kinetic parameters, including Vmax, Km, and specific activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH 8.5 and a temperature of 85 °C and was able to retain more than 70% of its activity after 150 h of storage at 85 °C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl and several metal ions contains Mg2+, Mn2+, Cu2+, Fe2+ and Ca2+. Our results showed that treatment with MnCl2 could enhance the enzyme's activity by 78%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72 h in the harsh condition (T = 85 °C, pH = 8.5). CONCLUSION: Considering the urgent demand for stable cellulases that are operational on extreme temperature and pH conditions and due to several proposed distinctive characteristics of PersiCel4, it can be used in the harsh condition for bioconversion of lignocellulosic biomass.