Catalase

Lentivirus plasmids, empty backbone of pLenti CMV Blast DEST (plasmid #17451) addgene, pLenti CMV Puro DEST (plasmid #17452) addgene, psPAX2, pMD2.G, 293 T cells, N2A cells, XL 1 E. Coli, DMEM (Gibco), FBS (BioExcel), Blastidine (Invivogen company), puromycin (Invivogen company), prestained marker (Yeaseen), streptomycin/penicillin solution (Gibco), anti-rabbit granzyme B (Abcam, cat. No. ab53097), anti-goat GFAP (Abcam, cat. No. ab53554), anti-hamster Serpina3n (Merck cat. No. MABC1182), anti-chicken MAP-2 (Abcam cat. No. ab5392), Hoechst 33258 (Sigma-Aldrich cat. No. 14530), anti-mouse 6his-tag (Proteintech, cat. No. 66005-1-Ig), anti-rabbit C-Myc (Proteintech, cat. No. 10828-1-AP), glucose oxidase (Sigma, cat. No. G7141), and catalase (Sangon, cat. No. A001847). Cell culture plates (Nest Biotechnology Co., Ltd.). Human granzyme B gene sequences were stored in our laboratory (EC:3.4.21.79).

Cupric chloride (CuCl₂), l-ascorbic acid (AA), and sodium hydroxide (NaOH) were purchased from J&K Scientific (Beijing, China). 3,3′,5,5′-Tetramethylbenzidine (TMB) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was bought from Macklin (Shanghai, China). Acetic acid (HAc), sodium acetate (NaAc), and potassium persulfate (K₂S₂O₈) was obtained from Sigma-Aldrich (Shanghai, China). Thiol-polyethylene glycol-OH (SH-PEG-OH) (Mw: 1000 Da) was purchased from Yare Biotech. Inc. (Shanghai, China). Catalase (purified powder from bovine liver), superoxide dismutase (purified powder from bovine erythrocytes), and peroxidase (purified powder from horseradish roots) were from Sangon Biotech (Shanghai, China). Phosphate-buffered saline (PBS, pH 7.4, Na₂HPO₄–NaH₂PO₄, 10 mM) solution was prepared in the laboratory. All chemicals and reagents were of analytical grade and used as received without further purification. Ultrapure water (18.2 MΩ cm⁻¹ at 25 °C) purified by a Milli-Q system was used throughout the experiment.

Protein samples were prepared as described in protein extraction and denatured by loading buffer. Protein, 25 μg per sample, was electrophoresed on SDS-PAGE gels (10%–12%) and blotted onto PVDF membranes (Amersham Hybond, United States). After blocking with 5% milk, the membranes were incubated at 4°C overnight with the following primary antibodies: β-actin (Cell Signaling, United States), catalase (CAT, Sangon, China), heme oxygenase 1 (HMOX1, Santa Cruz, United States), and stearoyl-CoA desaturase (SCD, Sangon, China). Anti-rabbit secondary antibodies (Cell Signaling, United States) were used. Target protein expressions were detected by chemiluminescence and quantified using ImageJ.

The GST-IDO1 gene was cloned by Shanghai Generay Biotech Co. Ltd (Shanghai, China). The BL21(DE3) cells were obtained from TransGen Biotech Co. Ltd (Shanghai, China). Ampicillin, isopropyl β-D-thiogalactoside (IPTG), 5-Aminolevulinic acid (5-ALA), phenylmethanesulfonyl fluoride (PMSF), DNase I (from bovine pancreas), reduced glutathione, L-tryptophan, ascorbic acid, methylene blue, catalase, and sodium dithionite were obtained from Sangon Biotech (Shanghai, China) Co. Ltd. DMSO was purchased from Sigma-Aldrich. The CCK8 kit was purchased from Beyotime Biotechnology Co. Ltd.(Shanghai, China). Glutathione agarose beads were purchased from Changzhou Smart-Lifesciences Biotechnology Co. Ltd. (Changzhou, China) PD-10 desalting column was purchased from GE Healthcare Biosciences. HRV 3C Protease was purchased from Sino Biological Inc. (Shanghai, China). Cancer cell lines were purchased from the Cell Bank of Chinese Academy of Sciences. The experimental compounds were provided by Professor Zhao (Shanghai Institute of Organic Sciences, Chinese Academy of Sciences). All chemicals of analytical and reagent grade were obtained from commercial sources and used without further purification.

The LAB isolates with higher antimicrobial activity were selected and tested to determine the nature of the produced antimicrobial substances—primarily organic acids, hydrogen peroxides, and bacteriocins. This characterization followed the method described by Reuben et al. [19 (link)] with some modifications. Specifically, the CFS of LAB, as prepared previously, was separated into five portions that underwent different treatments: one remained untreated as a control, the second was adjusted to a pH of 5.0 using NaOH, the third was treated with 1.5 mg/mL catalase (Sangon Biotech Co., Ltd., Shanghai, China) at 37 °C for 2 h, the fourth was treated with 1 mg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 2 h, and the fifth was heat-treated (boiled) for 20 min. Exactly 100 μL of CFS from each treatment was loaded into sterile Oxford cups placed on the agar plates previously inoculated with 200 μL of indicator pathogens (10⁷ CFU/mL). These indicator pathogens included E. coli ATCC 25922 (representative of Gram-negative bacteria [G^-]) and S. aureus ATCC 6538 (representative of Gram-positive bacteria [G⁺]). The plates were then incubated at 37 °C for 18–24 h, and the inhibition zones (including disc size) of the LAB were measured by using a pair of Vernier calipers.