Rational engineering S1' substrate binding pocket to enhance substrate specificity and catalytic activity of thermal-stable keratinase for efficient keratin degradation.

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.

Novel Antioxidant Peptides Derived from Feather Keratin Alleviate H2O2-Induced Oxidative Damage in HepG2 Cells via Keap1/Nrf2 Pathway.

Reactive oxygen species (ROS) are crucial for signal transduction and the maintenance of cellular homeostasis. However, superfluous ROS may engender chronic pathologies. Feather keratin is a promising new source of antioxidant peptides that can eliminate excess ROS and potentially treat oxidative stress-related diseases, but the underlying mechanisms have remained elusive. This study investigated the antioxidant effects and mechanisms against H2O2-induced oxidative damage in HepG2 cells of the two latest discovered antioxidant peptides, CRPCGPTP (CP-8) and ANSCNEPCVR (AR-10), first decrypted from feather keratin. The results revealed that CP-8 and AR-10 did not exhibit cytotoxicity to HepG2 cells while reducing intracellular ROS accumulation. Simultaneously, they enhanced the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), thus alleviating H2O2-induced cell apoptosis. Molecular docking analysis demonstrated that CP-8, AR-10 interacted well with the key amino acids in the Kelch domain of Keap1, thereby directly disrupting the Keap1-Nrf2 interaction. The peptides' biosafety and antioxidant activity via Keap1/Nrf2 signaling lay the groundwork for further animal studies and applications as functional food additives.

Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities.

Feather keratin is an underappreciated protein resource of high quality, with limited bioavailability, and it urgently requires eco-friendly methods to enhance its value. Here, we report on the preparation, purification, and identification of novel peptides with antioxidant and xanthine oxidase (XOD) inhibitory activities from fermented feather broth, using Bacillus licheniformis 8-4. Two peptides, namely, DLCRPCGPTPLA (DA-12) and ANSCNEPCVR (AR-10), displayed remarkable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities with half-maximal inhibitory concentrations (IC50) values of 0.048, 0.034, and 0.95, 0.84 mg/mL, respectively. These values exceed those of the previously reported feather keratin-derived antioxidant peptides. Another peptide, GNQQVHLQSQDM (GM-12), demonstrated XOD activity inhibition, with an IC50 value of 12.15 mg/mL, and it quenched the fluorescence of XOD. Furthermore, after simulating gastrointestinal digestion, DA-12, AR-10, and GM-12 retained their biological activities. Meanwhile, DA-12 and GM-12 showed an unexpected synergistic inhibition on XOD activity accompanied by fluorescence quenching. This study provides new insights into the potential applications of feather keratin, including functionalized feed with antioxidative and antigout (anti-hyperuricemia) activities.

Utilization of feather keratin waste to antioxidant and migration-enhancer peptides by Bacillus licheniformis 8-4.

AIMS: Feathers are keratin-rich byproducts of poultry processing, but those are often frequently abandoned as garbage and thus polluting the environment. Therefore, the study focused on the efficient biodegradation, bioactivity, and high-value application of feather keratin. METHODS AND RESULTS: Feather-degrading bacteria were identified, and the degradation properties were characterized. DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) and ABTS (2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid))radical scavenging assays, cytotoxicity assays, intracellular reactive oxygen scavenging assays, and cell migration assays were used to examine the biological activities of the feather keratin hydrolysis peptides (FKHPs). The results showed that we screened a feather-degrading strain of Bacillus licheniformis 8-4, which achieved complete degradation of 2% (w/v) feathers within 48 h. Notably, the feather fermentation broth was particularly high in FKHPs, which exhibited good DPPH and ABTS radical scavenging ability. Further studies revealed that FKHPs had both the ability to scavenge H2O2-induced ROS from HaCat cells and the ability to promote HaCat cell migration, while remaining non-toxic. CONCLUSIONS: The effective feather-degrading ability of B. licheniformis 8-4 allowed for the fermentation of feather medium to yield active peptides that were both antioxidants and cell-migration enhancers.

Production and characterization of novel thermo- and organic solvent-stable keratinase and aminopeptidase from Pseudomonas aeruginosa 4-3 for effective poultry feather degradation.

Feather biodegradation is an important premise for efficient resource development and utilization, in which keratinase plays an important role. However, there are few keratinases that combine the high activity, thermal stability, and organic solvent tolerance required for industrialization. This paper reported an efficient feather-degrading Pseudomonas aeruginosa 4-3 isolated from slaughterhouses. After 48 h of fermentation by P. aeruginosa 4-3 in a feather medium at 40 °C, pH 8.0, keratinase was efficiently produced (295.28 ± 5.42 U/mL) with complete feather degradation (95.3 ± 1.5%). Moreover, the keratinase from P. aeruginosa 4-3 showed high optimal temperature (55 °C), good thermal stability, wide pH tolerance, and excellent organic solvent resistance. In addition, P. aeruginosa 4-3-derived aminopeptidases also exhibit excellent thermal stability and organic solvent tolerance. Encouragingly, the reaction of crude keratinase and aminopeptidase with feathers for 8 h resulted in a 78% degradation rate of feathers. These properties make P. aeruginosa 4-3 keratinase and aminopeptidase ideal proteases for potential applications in keratin degradation, as well as provide ideas for the synergistic degradation of keratin by multiple enzymes.

Computer-assisted in vitro reconstitution of purine degradation pathway to lower the purine content in food.

BACKGROUND: With the increasing prevalence of gout and its etiological hyperuricemia, dietary control of gout based on low-purine food according to patients' eating habits is becoming a better choice compared to the existing drug treatment such as allopurinol with notorious side effects. Reconstructing the purine metabolic pathway in vitro to degrade purine substances in food into natural functional allantoin appears to be an innovative method for preparing nutritious and healthy food of low purine content. The present study reports a computer-assisted in vitro reconstruction of four purinolytic enzymes metabolizing adenosine into allantoin to reduce purine content in food for personalized dietary control of hyperuricemia and gout. RESULTS: Under the optimum reaction conditions of 40 °C and pH 7, 0.1 U of enzymes and 0.5 mmol L-1 adenosine determined by an orthogonal test design, 16 different enzyme complexes were experimentally tested. The tested enzyme composition and allantoin production values were used as input and output to build a three-layer back propagation artificial neural network (BP-ANN) model, which was further optimized by a genetic algorithm (GA). The optimum enzyme complex predicted by the GA-BP-ANN model produced 248.08±7.832 µmol L-1 allantoin, which was 19.9% higher than equimolar mixture of enzymes, and also more efficiently lowered purine contents in beer, as well as beef and yeast extracts. CONCLUSION: This is the first in vitro reconstitution of complete purine metabolic pathway by combining ANN and GA technologies, with successful application with respect to lowering the purine content in food, indicating a promising application of computer-assisted in vitro reconstitution of purinolytic pathway in low-purine food to prevent hyperuricemia and gout. © 2022 Society of Chemical Industry.

Characterization of a Novel Shewanella algae Arginine Decarboxylase Expressed in Escherichia coli.

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1-80 mM L-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s-1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s-1) value obtained by assaying at 1-50 mM L-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs.

6-Shogaol from ginger shows anti-tumor effect in cervical carcinoma via PI3K/Akt/mTOR pathway.

PURPOSE: 6-Shogaol, an active phenolic compound from ginger (Zingiber officinale), can inhibit the growth of a variety of human cancer cells. Nevertheless, its underlying molecular mechanisms in cervical cancer remain unclear. In this study, we systematically examine the inhibitory effect of 6-shogaol on cervical cancer in vitro and in vivo. METHODS: Cell proliferation was assessed by CCK8 assay and colony formation assay in HeLa and SiHa cells. We analyzed cell cycle and apoptosis through flow cytometry. GFP-LC3 puncta and transmission electron microscopy were used to observe autophagic bodies. Wound-healing assay and transwell assay were used for evaluating the migration of cells. Western blot was applied to detect protein expression levels. RESULTS: 6-Shogaol could suppress cell proliferation and migration, cause cell cycle arrest in the G2/M phase in HeLa and SiHa cells. Moreover, 6-shogaol triggered the apoptosis process through the mitochondrial pathway by downregulating the expression levels of p-PI3K, p-Akt and p-mTOR. Further research indicated that the induction of apoptosis by 6-shogaol was remarkably decreased after the treatment of ROS scavenger and PI3K agonist. Additionally, 6-shogaol increased the number of LC3-positive puncta and autophagic bodies per cell in both HeLa and SiHa cells. Pretreatment of cells with Bafilomycin A1, an autophagy inhibitor, accelerated 6-shogaol mediated cell apoptosis, suggesting that induction of autophagy by 6-shogaol is suppressive to apoptosis. Furthermore, in vivo data revealed that 6-shogaol significantly inhibited tumor growth and cell proliferation in tumor tissues. CONCLUSION: These findings suggested that 6-shogaol could be developed as a functional food ingredient, which is potentially used as therapeutic agents for patients with cervical cancer.

α-Cyperone inhibits the proliferation of human cervical cancer HeLa cells via ROS-mediated PI3K/Akt/mTOR signaling pathway.

Cervical cancer is the fourth leading killer of female cancer patients worldwide. Each year more than half a million women are diagnosed with cervical cancer and the disease results in over 300, 000 deaths. α-Cyperone is known as the principal active ingredient in the Cyperus rotundus (Family: Cyperaceae). However, the effects of α-Cyperone on cancers, especially on cervical cancer, are yet to be explored. In the present study, the underlying mechanism of the anti-tumor activity of α-Cyperone against HeLa cells was investigated. The results showed that α-Cyperone inhibited proliferation and induced apoptosis in HeLa cells. Mechanistically, α-Cyperone promoted HeLa cells apoptosis via a mitochondrial apoptotic pathway, which was proved by increased level of intracellular reactive oxygen species (ROS) and upregulated expression of cytochrome c, cleaved caspase-3, PARP, and Bax. Further RNA-sequencing revealed α-Cyperone inhibited the activation of PI3K/Akt/mTOR signaling pathway in HeLa cells, which confirmed by PI3K inhibitor and agonist. The PI3K inhibitor (LY294002) synergized with α-Cyperone in arresting the growth of HeLa cells, whereas the PI3K agonist (IGF-1) abrogated such an effect. Interestingly, the expression of PD-L1 was attenuated by both α-Cyperone and LY294002, while the supplement of IGF-1 rescued the low expression of PD-L1. In conclusion, our results reveal that the inhibitory effect of α-Cyperone on HeLa cell growth is triggered via the ROS-mediated PI3K/Akt/mTOR signaling pathway and closely related to a decline in the PD-L1 expression.

14,15ß-dihydroxyklaineanone inhibits HepG2 cell proliferation and migration through p38MAPK pathway.

OBJECTIVES: Eurycoma longifolia Jack (Simaroubaceae) is commonly distributed in the Southeast Asia and Indo China, which has been shown to possess antianxiety, antibacterial, anticancer, antifungal, anti-inflammatory, antimalarial and antioxidant biological activities. 14,15ß-dihydroxyklaineanone is a diterpene isolated from E. longifolia Jack, which is cytotoxic against human lung cancer and human breast cancer cell lines. However, the effects and underlying mechanisms of 14,15ß-dihydroxyklaineanone on hepatocellular carcinoma remain unknown. METHODS: Cell viability assay and colony formation assay were used to measure HepG2 cell proliferation. Flow cytometry was used to analyse cell cycle and apoptosis. Wound-healing assay and transwell assay were used to observe cells migration. RNA sequencing and the enrichment of differentially expressed genes (DEGs) in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to find and determine underlying pathways. KEY FINDINGS: We found that 14,15ß-dihydroxyklaineanone inhibited the growth and migration of HepG2 cells but did not induce cell apoptosis. 14,15ß-dihydroxyklaineanone induced S cell cycle arrest by downregulating the expression levels of cyclin A, p-CDK2, cyclin B1, p21, E2F-1 and PCNA. In addition, RNA sequencing showed that 14,15ß-dihydroxyklaineanone regulated MAPK pathway by increasing the expression levels of phosphor-p38. Downregulating of p38 via both p38 inhibitor (SB203580) and p38-siRNA could antagonize the inhibition of cell proliferation and migration and reverse the changes in p-p38, E-cadherin, N-cadherin and PCNA expression induced by 14,15ß-dihydroxyklaineanone treatment. CONCLUSIONS: 14,15ß-dihydroxyklaineanone inhibited cell proliferation and migration through regulating p38 MAPK pathway in HCC cells.

[Study on the risk factors of induced abortion among floating women of childbearing age in 5 cities of China].

OBJECTIVE: To understand the status of abortion and risk factors among floating women of childbearing age and to provide reference for further improvement of induced abortion services in this population. METHODS: Data on demography, working, and living conditions as well as the use of contraceptive among 4687 persons from a reproductive health survey regarding floating population in five cities in 2005, were involved, while multivariate logistic regression model was used to find out the relationship. RESULTS: The risks of abortion among the younger than 30 age group and the 30 - 39 age groups were 2.21 times (95%CI: 1.47 - 3.34) and 2.38 times (95%CI: 1.53 - 3.70) of the 40 - 49 age group, respectively. The higher education degree these women had, the higher the risk of abortion was. The risks of abortion among groups having elementary, junior high, senior high school and above, were 2.15 times (95%CI: 1.15 - 4.03), 2.47 times (95%CI: 1.33 - 4.57) and 2.61 times (95%CI: 1.34 - 5.11) of those illiterate women. Those having working experience of 2 - 4 years, 5 years or above at the places where the survey was completed, the risks were 2.62 times (95%CI: 1.83 - 3.76) and 7.78 times (95%CI: 5.63 - 10.75) of the less than 2-year-experienced group. The abortion risk of floating women at childbearing age who were living together with their spouses was 1.49 (95%CI: 1.05 - 2.11) times of those women who were not. CONCLUSION: The demographic and lifestyle as well as working features of floating women at childbearing age might increase their risk of abortion. Providing health education regarding these risk factors on the floating women at childbearing age could effectively reduce and prevent the risk of abortion risk among them.