Tris base

The water used in this study was deionized (18 MΩ·cm) from Water Purification System. Sephadex G-100, DEAE-52, 2,2-diphenyl-1-picrylhydrazyl, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, 2,4,6-tri(2-pyridyl)-s-triazine, 6-hydroxy-2,5,7,8-tetramethlchroman-2-carboxylic acid, casein, glacial acetic acid, Tris-base, alcalase, trypsin, papain, neutrase, pepsin, and ammonium persulfate were purchased from Yuanye Biotech Co. Shanghai, China. Absolute ethanol (analytical grade), acetic acid, acetone, ferric chloride, ferrous sulphate, glucose, methanol (analytical grade), perchloric acid, phenol (redistilled reagent grade), potassium per-sulphate, potassium sulphate, sodium acetate, and sodium hydroxide were purchased from DaMao Chemical Co. Tianjin, China. Boric acid, copper sulfate, hydrochloric acid, methylene blue indicator, methyl red indicator, petroleum ether, sulfuric acid, and sodium thiosulfate were purchased from Guangzhou Chemical Co. (Guangzhou, China). Bovine serum albumin, Tris-HCl buffer, SDS, tetramethylethylenediamine were obtained from Sigma Chemical Co. (St. Louis, MO, USA) RIPA lysis buffer, acrylamide/bis-acrylamide mix, ladder protein, were purchased from Thermo Fisher Scientific Co. (Shanghai, China). All other chemicals were of analytical grade.

The strains and plasmids used in this study are summarized in Supplementary Table S1, and the primers used are listed in Supplementary Table S2. Escherichia coli BL21(DE3) and B. subtilis 168 were used as gene expression hosts, while E. coli JM109 was used for gene cloning. The methods for the transformation of the gene to B. subtilis is referred to by You et al. [25 (link)]. Escherichiacoli cells were cultured at 37 °C in LB medium (10 g/L NaCl, 10 g/L peptone, and 5 g/L yeast extract), and B. subtilis 168 cells were grown at 37 °C in fermentation medium (47 g/L glycerin, 35 g/L yeast extract, 1.5 g/L NH₄Cl, 5 g/L NaCl, 15 g/L corn steep liquor, 2.04 g/L KH₂PO₄, 2.61 g/L K₂HPO₄, 1.85 g/L MgSO₄·7H₂O, and 1 g/L l-asparagine). Kanamycin (50 μg/mL) was added when necessary. We used pET-28a and pMA5 for gene expression, which were kept in our laboratory. NdeI, XhoI, DpnI, Ex Taq DNA polymerase, and T4 DNA ligase were purchased from Takara (Dalian, China). Gel recovery kits and plasmid extraction kits were purchased from Jierui Bioengineering (Shanghai, China). Kanamycin, Nessler’s reagent, Tris base and isopropyl β-d-thiogalactopyranoside (IPTG) were purchased from Yuanye Bio-Technology (Shanghai, China). Glycerin, imidazole, sodium chloride, and l-asparagine were obtained from commercial suppliers and used as received.