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The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC 50 of 5.34 ± 0.65 μM, which is less potent than benzbromarone (2.01 ± 0.36 μM) but more potent than lesinurad (10.36 ± 1.23 μM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC 50 of 1.04 ± 0.23 μM. Its metabolic product, gallic acid, inhibited XOD with an IC 50 of 0.91 ± 0.14 μM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t 1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Some spoilage yeasts are able to develop resistance to commonly used weak-acid preservatives. We studied the trehalose metabolism and its regulation in Saccharomyces cerevisiae in response to propionic acid stress. We show interruption of trehalose synthetic pathway caused the mutant hypersensitive to the acid stress, while its overexpression conferred acid-tolerance to yeast. Interestingly, this acid-tolerance phenotype was largely independent of trehalose but relied on the trehalose synthetic pathway. We demonstrate trehalose metabolism played a vital role in regulation of glycolysis flux and Pi/ATP homeostasis in yeast during acid-adaptation, and the PKA and TOR signaling pathways were involved in regulating trehalose synthesis at transcriptional level. This work confirmed the regulatory function of trehalose metabolism and improved our understanding of molecular mechanism of acid-adaptation of yeast. By exemplifying trehalose metabolism interruption limited the growth of S. cerevisiae exposed to weak acids, and trehalose pathway overexpression conferring acid-resistance to Yarrowia lipolytica enhanced citric acid production, this work provides new insights into the development of efficient preservation strategies and robust organic acid producers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Organophosphorus compounds (OPs) have been widely used in agriculture for decades because of their high insecticidal efficiency, which maintains and increases crop yields worldwide. More importantly, OPs, as typical chemical warfare agents, are a serious concern and significant danger for military and civilian personnel. The widespread use of OPs, superfluous and unreasonable use, has caused great harm to the environment and food chain. Developing efficient and environmentally friendly solutions for the decontamination of OPs is a long-term challenge. Microbial enzymes show potential application as natural and green biocatalysts. Thus, utilizing OP-degrading enzymes for environmental decontamination presents significant advantages, as these enzymes can rapidly hydrolyze OPs; are environmentally friendly, nonflammable, and noncorrosive; and can be discarded safely and easily. Here, the properties, structure and catalytic mechanism of various typical OP-degrading enzymes are reviewed. The methods and effects utilized to improve the expression level, catalytic performance and stability of OP-degrading enzymes were systematically summarized. In addition, the immobilization of OP-degrading enzymes was explicated emphatically, and the latest progress of cascade reactions based on immobilized enzymes was discussed. Finally, the latest applications of OP-degrading enzymes were summarized, including biosensors, nanozyme mimics and medical detoxification. This review provides guidance for the future development of OP-degrading enzymes and promotes their application in the field of environmental bioremediation and medicine., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional of other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Thermomicrobium roseum DSM 5159 lipase (TrLip) is an enzyme with marked thermostability and excellent solvent resistance. However, TrLip reveals relatively high catalytic efficiency on short-chain substrates but poor activity against mid-long or long-chain fatty acids, which would limit its industrial application. In this study, ancestral sequence reconstruction (ASR), a common engineering tool for the evolutionary history of protein families, was employed to identify the natural evolutionary trends within 5 Å around the catalytic center. Two mutation libraries were constructed, one for the catalytic center and the other for the pocket flexibility. A total of 69 mutants were expressed and purified in the Escherichia coli expression system to determine the kinetic parameters, and W219G could significantly enhance the catalytic efficiency against substrates with 12-, 16- and 18-carbon side chains. In addition, the double mutant W219G/F265M could further catalyze the breakdown of the above three substrates up to 6.34-, 4.21- and 4.86-folds compared to the wild-type TrLip, while the initial pH and thermostability were maintained. Through bioinformatics analysis, the significantly enhanced catalytic efficiency against longer-side chain substrates should be associated with the reduction of steric hindrance. With the outstanding stability and the promoted activity, TrLip should be of great potential in chemical and food industry., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Phosphotriesterase (PTE) is considered to be a good biodegradation agent for organophosphorus pesticides. However, the instability of the free PTE limits its application. In this study, the free PTE was hybridized with copper ions (Cu 2+ ) to enhance its catalytic stability and activity. The acquired particles were freeze-dried after precipitation with PO 4 3- at 4 °C for 72 h. Scanning electron microscopy showed that the Cu-PTE complexes formed flower-like nanoparticles after hybridization. The characteristic peaks of both the enzyme and metal material were revealed by Fourier transform-infrared spectroscopy. X-ray diffraction analysis indicated that PTE was encapsulated in the Cu 3 (PO 4 ) 2 ·3H 2 O based hybrid nanoflowers. Compared with free PTE, the catalytic activity of Cu-PTE hybrid nanoflowers was significantly increased about 2.2 fold. The catalytic efficiency (k cat /V max ) of Cu-PTE hybrid nanoflowers was 1.76 fold than that of free PTE. The stability of the immobilized PTE under thermal and pH conditions was improved and the tolerance of it to organic solvents was also enhanced. Moreover, the Cu-PTE hybrid nanoflowers still exhibited 72.3 % relative activity after ten consecutive reactions. In general, this is the first time to use copper based hybrid nanoflowers to immobilize PTE, and the immobilized enzyme shows excellent performance on OPs degradation. The Cu-PTE hybrid nanoflowers may have great potential in the biodegradation of organophosphorus compounds in future., (Copyright © 2021 Elsevier Inc. All rights reserved.)

An affinity ligand was designed from 1-aminocyclohexane based on the crystal structure of Streptomyces albidoflavus phospholipase A 1 (saPLA 1 ) by using Discovery Studio software. The molecular docking results indicated that the designed ligand could interact with the active pocket of saPLA 1 . Epichlorohydrin, cyanuric chloride and 1-aminocyclohexane were used to synthesize the affinity ligand, which was composed to Sepharose beads. The density of the ligand on Sepharose beads was 22.5 ± 1.1 μmol/g wet gel. Adsorption analysis of the sorbent indicated the maximum adsorption (Q max ) of the enzyme was 10.7 ± 0.29 mg/g and the desorption constant (K d ) was 426.6 ± 29.7 μg/mL. The sorbent could bind the enzyme in the supernatant of disrupted recombinant Escherichia coli through one step of affinity adsorption. After the optimization of the purification process, a single band was obtained at approximately 30 kDa, which was confirmed as saPLA 1 by the matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry and activity assay. The purity of the isolated enzyme was about 96.6% with the purify fold at 7.62, and the activity recovery was 52.5%., (Copyright © 2020 Elsevier B.V. All rights reserved.)