Development of a novel trypsin affinity material using a recombinant buckwheat trypsin inhibitor mutant with enhanced activity

A buckwheat trypsin inhibitor mutant with enhanced activity (rEBTI) was expressed, purified, and immobilized onto Sepharose CL-4B matrix. The optimum pH for the adsorption by, and desorption of porcine trypsin from the rEBTI-Sepharose CL-4B column were 8 and 3.5, respectively. The rEBTI-Sepharose CL-4B was applied to purify trypsin from grass carp hepatopancreas to verify its practical utility. The enzyme was purified by one-step chromatography and migrated as a single band with molecular mass of 27 kDa from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimal temperature and pH of the purified enzyme were 60 °C and 9.5, respectively, and it was stable within pH 6.0–12.0. The apparent K_m value of the purified enzyme was determined as 3.4 × 10⁻⁵ M using N-benzoyl-DL-arginine-nitroanilide as substrate. The trypsin was activated by Ba²⁺ and Mg²⁺, but was inhibited by Zn²⁺, EDTA and vitamin C. The novel trypsin affinity material rEBTI-Sepharose CL-4B could obtain grass carp trypsin of high purify by one-step purification, which is less tedious and more cost-effective than most current methods. The process can produce the enzyme in high purity to meet high-end biotechnological applications in the biomedical and pharmaceutical industries.