Macro prep high s column

Squid pens, crab shells and shrimp shells were obtained from Shin-Ma Frozen Food Co. (I-Lan, Taiwan) [45 (link)]. Shrimp head powder (SHP) was acquired from Fwu-Sow Industry (Taichun, Taiwan). Demineralized shrimp shell powder (deSSP) and demineralized crab shell powder (deCSP) were prepared according to the previously described methods [45 (link)]. Acarbose, p-nitrophenyl glucopyranoside and enzymes (yeast α-glucosidase, rat α-glucosidase, porcine pancreatic α-amylase, B. subtilis α-amylase, lysozyme, cellulase, bromelain and papain) were obtained from Sigma Chemical Co., St. Louis, MO, USA. The Macro-Prep High S column was obtained from BioRad (Hercules, CA, USA). All other reagents used were of the highest grade available.

The culture medium was centrifuged at 6000 rpm for 30 min to remove the residual solids and bacterial cells. The liquid was then mixed with ammonium sulfate (60% w/v) and kept at 4 °C for 24 h. The precipitate that appeared in the mixture of culture supernatant and ammonium sulfate was conveniently obtained by centrifugation (9000 rpm, 30 min). A small volume of 20 mM potassium phosphate buffer (PPB) at pH 6 was used to dissolve the obtained precipitate. The residual ammonium sulfate was removed by dialyzing the solution against PPB for 24 h using a cellulose membrane (cellusep T2, Interchim, Montluçon, France). For the enzyme purification, the crude enzyme was loaded onto the Macro Prep High S column (Biorad, Hercules, CA, USA) pre-equilibrated with PPB (20 mM, pH 6). The washing step took place until A_{280 nm} reached a stable value. After the washing step, a linear gradient of NaCl (0 M–0.5 M) was conducted to elute the target enzymes. Fractions exhibiting protease activity were then pooled, and concentrated by the freezing-drying method. Finally, the enzyme was purified by size exclusion chromatography (KW-802.5 column, Showa Denko K.K., Tokyo, Japan). The protein content was estimated by the Lowry method [37 (link)].