Chitosan

Chitosan (Sangon, Shanghai, China) with the weight of 500 mg was dispersed in 46 ml distilled water and stirred for 5 min, followed by further stirring for 30 min with addition of 348.6 mg HOBT (Sangon, Shanghai, China). NAC with the weight of 842 mg was added and stirred for 5 min after the previous solution become limpidly. EDC-HCl solution (1978.5 mg of EDCI-HCl dissolved in 4 mL of distilled water) was added, and then, the pH was adjusted at about 5.
The reaction product was dialyzed with molecular weight cut off at 7000 in the sequential solution of 5 mM hydrochloric acid and 2 μM EDTA for 3 days, the solution of 5 mM hydrochloric acid, 2 μM EDTA and 0.1% NaCl for 2 days, the solution of 5 mM hydrochloric acid for 1 day, and finally, the distilled water for 1 day. The entire dialysis process was conducted at 4℃ in dark.

P. anomala (strain TL0903) was used for all the experiment, and fresh suspension was prepared according to our previous research method [13 (link)]. Chitosan (90% deacetylation) was bought from Sangon Biotech Co., Ltd. (Shanghai, China).

E. coli JM109 and E. coli BL21 (DE3) were used for cloning of 16 S rDNA fragment and target Chitosanase gene (choe), and expressing choe, respectively. The plasmid pET 28a(+) was used for expression. Takara Bio Inc (China) supplied such as pMD 19-T vector, T4 DNA ligase, BamH I, Hind III, and Extaq. Beyotime Biotechnology (China) offered Bradford protein assayed kit. Chitosan was supplied by Sangon Biotech (China).

Ultrapure water was produced by the TW-D24UV system of Millitrack (Haryana, India). The yeast nitrogen base without amino acids (YNB), yeast extract peptone dextrose (YPD), chitosan, and chloramphenicol were purchased from Sangon Biotech (Shanghai, China). Chinese Baijiu (52%, v/v) was purchased from a supermarket (Dalian, China). EC was procured from Sigma-Aldrich Co. (St. Louis, MO, USA). TES buffer (300 mmol/L NaCl, 50 mmol/L Tris-HCl, 25 mmol/L EDTA, 0.2% (v/v) SDS, 2 mg/mL proteinase K, pH 8.0) was purchased from TransGen Biotech (Beijing, China). Tris-saturated phenol solution (pH 8.0), absolute ethanol, sodium acetate (pH 5.3), CaCl₂, boric acid, and chloroform-isoamyl alcohol were purchased from Sangon Biotech (Shanghai, China). Glucose, glycerol, sucrose, mannose, and sorbose were purchased from Damao Co. (Tianjin, China). 4-methyl-1-pentanol was purchased from Aladdin Biotech (Shanghai, China).

CMC was synthesized according to ref.[27 (link)]. First, 13.5 g NaOH was dissolved in 100 ml of solvent comprising 20 ml water and 80 ml 2-propanol (Sigma-Aldrich Inc., St. Louis, MO, USA), to which 10 g chitosan (85% deacetylated, Sangon Co., Ltd. (Shanghai, China) was gradually added under stirring (1000 rpm). This mixture was allowed to swell and alkalize at 25°C for 2 h. Next, 15 g monochloroacetic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) dissolved in 20 ml 2-propanol was added dropwise to the mixture in 30 min. The activated chitosan was reacted with the acid for 4 h at 50°C, which was stopped by addition of 200 ml of 70% ethanol. After that, the resulting precipitate was desalted by dialysis and then dried under reduced pressure at 25°C to obtain CMC powder.
CMC gel was first prepared by mixing 2.5 g CMC into 40ml water under stirring (1000 rpm) until the CMC powder was completely dissolved. Then, 0.498 g K₂HPO₄ was added into the CMC gel under stirring (500 rpm). Next, 0.555 g CaCl₂ was added into 10 ml deionized water and this solution was added dropwise into the CMC gel under stirring for 5 min to form CMC/ACP gel. The final concentrations of calcium and phosphate ions were 100 mM and 60 mM, respectively. This gel was immediately frozen at -80°C for 2 h and then lyophilized in vacuum freeze dryer (Lgj-B10, Beijing, China) for 6 h to form CMC/ACP scaffolds.