Tyrosinase ec 1.14.18.1

Chitosan (CH, viscosity: 100-200 mPa·s), sodium alginate (ALG, 180–220 mPa⋅s), GP, KGN, genipin, EGDE and bovine serum albumin (BSA, isoelectric point (pI): ca.4.8) were supplied by Aladdin Inc. (Shanghai, China). CH was treated in a 50 wt% NaOH aqueous solution to increase its deacetylation degree to around 95.3% using the method described in our previous study [51 (link)]. The PDGF-BB (pI: ca.9.8) and the PDGF-BB ELISA kit were bought from R&D Systems (Minneapolis, MN, USA) and Invitrogen (Waltham, MA, USA), respectively. Tyrosinase (EC 1.14.18.1) (≥1000 units/mg, T-3824) was purchased from Sigma-Aldrich (Shanghai, China). Other reagents and chemicals were of analytic grade and obtained from Sinopharm, China (Shanghai, China).
SF was isolated from cocoons using the method described in our previous study [52 (link)]. The obtained SF solution was diluted to 1.0 wt% of final concentration with storage at 4°C for further use. Rhodamine B (RDB) was conjugated onto CH to synthesize some conjugates (RDB-CH) following a reported method [39 (link)]. The selected RDB-CH was used together with CH to image the shell layer of some MPs.

All chemicals were of an analytical grade and were used without further purification. A phosphate buffer solution (0.1 M, pH 6.8), prepared from sodium phosphate dibasic and sodium dihydrogen phosphate, was used as the supporting electrolyte for all measurements. Tyrosinase (EC 1.14.18.1, from mushroom), catechol, L-tyrosine, Bovine Serum Albumin (BSA), Glutaraldehyde (GA), benzoic acid and sodium azide (NaN₃) were purchased from Sigma-Aldrich (Burlington, MA, USA), whereas kojic acid was purchased from Alfa-Aesar (Tewksbury, MA, USA). The carbon black paste electrode was prepared by mixing 50% (w/w) carbon black powder N220 (Ravenna, Italy) with 50% (w/w) mineral oil (from Fluka, Buchs, Switzerland).
All electrochemical measurements were carried out using a PalmSens Potentiostat electrochemical analyzer provided by PalmSens BV (Utrecht, The Netherlands), controlled by PSTrace 4.0 software. A conventional three-electrode system in an electrochemical cell of 5 mL volume, containing the carbon black paste electrode (CBPE) as a working electrode, a platinum electrode as a counter electrode, and an Ag/AgCl electrode as a reference electrode.
An automated microplate reader (Biotek) was used to measure the absorbance of free enzyme at 490 nm, and the data were evaluated with Gen5 software. OriginPro8 was used as data analysis software.

Sixty-five commercial EOs, pure without additive, were purchased from Jingjing Biotechnology Co. (Guangzhou, China). The information of these EOs was listed in Table 1. Tyrosinase (EC 1.14.18.1) and kojic acid were purchased from Sigma-Aldrich (St. Louis, MO). Tyrosinase was dissolved with 0.05 M phosphate buffer solution (PBS, pH 6.81 ± 0.01) and then diluted to 1,500 U/mL. The final concentration of tyrosinase and L-Dopa in PBS was 37.5 U/mL and 1.0 mM, respectively. In addition, kojic acid was used as a standard compound. Other solvents and reagents that had analytical grade were purchased from Tansoole (Shanghai, China).