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.

Simultaneous hydrolysis of various protein-rich industrial wastes by a naturally evolved protease from tannery wastewater microbiota.

Elimination of protein-rich waste materials is one of the vital environmental protection requirements. Using of non-naturally occurring chemicals for their remediation properties can potentially induce new pollutants. Therefore, enzymes encoded in the genomes of microorganisms evolved in the same environment can be considered suitable alternatives to chemicals. Identification of efficient proteases that can hydrolyze recalcitrant, protein-rich wastes produced by various industrial processes has been widely welcomed as an eco-friendly waste management strategy. In this direction, we attempted to screen a thermo-halo-alkali-stable metagenome-derived protease (PersiProtease1) from tannery wastewater. The PersiProtease1 exhibited high pH stability over a wide range and at 1 h in pH 11.0 maintained 87.59% activity. The enzyme possessed high thermal stability while retaining 76.64% activity after 1 h at 90 °C. Moreover, 65.34% of the initial activity of the enzyme remained in the presence of 6 M NaCl, showing tolerance against high salinity. The presence of various metal ions, inhibitors, and organic solvents did not remarkably inhibit the activity of the discovered protease. The PersiProtease1 was extracted from the tannery wastewater microbiota and efficiently applied for biodegradation of real sample tannery wastewater protein, chicken feathers, whey protein, dehairing sheepskins, and waste X-ray films. PersiProtease1 proved its enormous potential in simultaneous biodegradation of solid and liquid protein-rich industrial wastes based on the results.

Enhancing the ethanol production by exploiting a novel metagenomic-derived bifunctional xylanase/ß-glucosidase enzyme with improved ß-glucosidase activity by a nanocellulose carrier.

Some enzymes can catalyze more than one chemical conversion for which they are physiologically specialized. This secondary function, which is called underground, promiscuous, metabolism, or cross activity, is recognized as a valuable feature and has received much attention for developing new catalytic functions in industrial applications. In this study, a novel bifunctional xylanase/ß-glucosidase metagenomic-derived enzyme, PersiBGLXyn1, with underground ß-glucosidase activity was mined by in-silico screening. Then, the corresponding gene was cloned, expressed and purified. The PersiBGLXyn1 improved the degradation efficiency of organic solvent pretreated coffee residue waste (CRW), and subsequently the production of bioethanol during a separate enzymatic hydrolysis and fermentation (SHF) process. After characterization, the enzyme was immobilized on a nanocellulose (NC) carrier generated from sugar beet pulp (SBP), which remarkably improved the underground activity of the enzyme up to four-fold at 80°C and up to two-fold at pH 4.0 compared to the free one. The immobilized PersiBGLXyn1 demonstrated 12 to 13-fold rise in half-life at 70 and 80°C for its underground activity. The amount of reducing sugar produced from enzymatic saccharification of the CRW was also enhanced from 12.97 g/l to 19.69 g/l by immobilization of the enzyme. Bioethanol production was 29.31 g/l for free enzyme after 72 h fermentation, while the immobilized PersiBGLXyn1 showed 51.47 g/l production titre. Overall, this study presented a cost-effective in-silico metagenomic approach to identify novel bifunctional xylanase/ß-glucosidase enzyme with underground ß-glucosidase activity. It also demonstrated the improved efficacy of the underground activities of the bifunctional enzyme as a promising alternative for fermentable sugars production and subsequent value-added products.