Chitosanase

PCL (Schiff or amide link) micelles in 40 mM sodium acetate (pH = 6) were prepared via dialysis method. Chitosanase (1 mg/mL, Sigma-Aldrich, Cat. 220477) was then added into the micelle solution to a final concentration of 2 μM. The mixture was incubated at 37 °C with gentle shaking. The size and count number were examined by DLS at different times following the incubation.

Cellulase, chitinase, chitosanase (25.9 U/mL), drieselase, β-glucosidase, β-glucuronidase (140 U/mL), hyaluronidase, laminarinase, lysozyme, lyticase, macerozyme, pectinase (3,000 U/mL), pectolyase, sulphatase (3.37 mg/mL), and trypsin were purchased from Sigma Aldrich (St. Louis, MO). Zymolyase (10 mg/mL) was purchased from ZymoResearch (Irvine, CA). macerozyme was purchased from RPI corp (Mt Prospect, IL). Stock concentrations of enzymes were 20 mg/mL unless otherwise noted.

Cells were grown on mDixon’s medium and washed with water by centrifugation at 4000 rpm for 5 min. Cells were resuspended in 10 mL of 5% (v/v) 2-mercaptoethanol solution in water and incubated at 30 °C/ 150 rpm for 45 min. The cells were pelleted, washed, and resuspended in 3 mL spheroplasting buffer (40 mM citric acid, 120 mM Na₂HPO₄ and 1.2 M sorbitol) for every 1.5 × 10⁹ cells. Cell clumps were dissociated by mild sonication for 30 s using the medium-intensity setting in a Bioruptor (Diagenode). Lysing enzymes from Trichoderma harzianum (Sigma), chitosanase (Sigma), and zymolyase-20T (MP Biomedicals) were added at 20 mg/mL, 0.2 µg/mL, and 100 µg/mL, respectively. The spheroplasting suspension was incubated at 30°C/65 rpm for 6–8 hr. The suspension was examined under a microscope to estimate the proportion of spheroplasts. Spheroplasts were washed with ice-cold 1xPBS and used as per the experimental design (adapted from Boekhout, 2003 ).

Chitosan of low molecular weight (DD = 75–85%; Mw = 150 × 10⁻³) and Chitosanase (EC 3.2.1.132) from Streptomyces griseus with 50 U/mg activity and lactic acid (85% syrup) were obtained from Sigma-Aldrich, Poznan, Poland. Cellulase (E.C. 3.2.1.4) from Trichoderma reesei with 127.5 U/mg activity was purchased from Dyadic, Jupiter, FL, USA. Cellulase showed side activities typical of beta-glucanase (33 U/mg).

High-molecular-weight chitosan (CAS 9012-76-4; 310 to 375 kDa) was sourced from Hangzhou Simit Chem. & Tech. Co. (Hangzhou, China). Neutrase^® enzyme was provided by Novozymes A/S (Bagsværd, Denmark). Sodium carboxymethylcellulose (CAS 9004-32-4; USP reference standard), sodium alginate (CAS 9005-38-3; pharmaceutical secondary standard), acetic acid (CAS 64-19-7; purum, 80% in H₂O), methacrylic anhydride (CAS 760-93-0; ≥94%), sodium tripolyphosphate (CAS 7758-29-4; ≥98%), carvacrol (CAS 499-75-2, 98%), chitosanase from Streptomyces griseus (Krainsky) Waksman and Henrici (EC 3.2.1.132, CAS 51570-20-8), methanol (UHPLC, suitable for mass spectrometry, CAS 67-56-1), tetrahydrofuran (THF, CAS 109-99-9; ≥99.9%), and Tween^® 20 (CAS 9005-64-5) were procured from Merck (Darmstadt, Germany). Potato dextrose broth (PDB) and potato dextrose agar (PDA) were supplied by Becton, Dickinson, and Company (Franklin Lakes, NJ, USA).
Botrytis cinerea (CECT 20973) and P. expansum (CECT 20906) were obtained from the Spanish Type Culture Collection (Valencia, Spain), while C. coccodes (CRD 246/190) was sourced from the Regional Diagnostic Center of Aldearrubia (Junta de Castilla y León; Castilla y León, Spain).