Bacillus licheniformis

Five available proteases with therapeutic importance were used: pure elastase from human leukocytes (Sigma-Aldrich, St. Louis, MO, USA E8140), cathepsin B from bovine spleen (cysteine-peptidase) (Sigma-Aldrich, C6286), α-chymotrypsin (serine peptidase) from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA C3142), collagenase from Clostridium histolyticum (metalloproteinases) (Sigma-Aldrich, St. Louis, MO, USA C2674), and thrombin from bovine plasma (serine protease) (Sigma-Aldrich, St. Louis, MO, USA T7513). Six commercially available proteases were also used: esperase from Bacillus sp. (serine-type protease) (Novozyme, Sigma-Aldrich, St. Louis, MO, USA P5860), proteinase K from Tritirachium album (serine protease) (Sigma-Aldrich, St. Louis, MO, USA P2308), subtilisin from Bacillus licheniformis (Subtilisin A is a member of the Serine S8 endoproteinase family) (Sigma-Aldrich, St. Louis, MO, USA P5380), and Aspergillus oryzae (fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae that contains both endoprotease and exopeptidase activities) (Sigma-Aldrich, St. Louis, MO, USA P6110), Bacillus licheniformis (endoprotease of the serine type) (Sigma-Aldrich, St. Louis, MO, USA P4860), and Bacillus sp. (a serine-type protease) (Sigma-Aldrich, St. Louis, MO, USA P3111).

The BSF was provided by Chapul Farm (McMinnville, OR, USA), and the cricket powder was obtained from Cricktone (Saint Louis, MO, USA). Oyster, mussel, and lugworm were provided from local stores. The enzyme used in this study was commercially available Alcalase^®, an endoprotease enzyme (2.4 AU/g) from Bacillus licheniformis (Sigma–Aldrich Inc., St. Louis, MO, USA). The zebrafish embryonic stem cell line ZEM2S CRL-2147™ was obtained from The American Type Culture Collection (ATCC). The antibiotics which were initially used to cultivate zebrafish embryonic stem cells (ESCs) were obtained from Cytiva (Marlborough, MA, USA).

Commercial α-amylase (Bacillus licheniformis, A3403, Sigma) was added into the 1% (w/v) soluble starch and this mixture was incubated at 90 °C for 30 min. Then the mixture was incubated at 100 °C for 20 min to inactivate the enzyme, and centrifuged at 12,000× g for 15 min. After adding 5 ug recombinant GSJ or commercial α-glucosidase (Bacillus. Stearothermophilus, E-TSAGS, Megazyme, Ireland), the supernatant was incubated at 60°C. Following incubation for 1 h, the mixtures were incubated at 100 °C for 5 min to inactivate the enzyme, and centrifuged at 12,000× g for 15 min. The hydrolysis products were determined by HPAEC.