Production of Recombinant APRP, an Alkaline Protease Derived from Bacillus pumilus TYO-67, by In Vitro Refolding of Pro-enzyme Fixed on a Solid Surface

Bacillus pumilus TYO-67 has been isolated from tofuyo, a traditional fermented food made from soybean milk in Okinawa, Japan. This bacterium secretes a soybean-milk-coagulating enzyme (SMCE), which can be applied for the production of processed foods from soybean milk. Thus, an easy method of producing the recombinant enzyme was developed in this study. SMCE is an alkaline serine protease belonging to the subtilisin family; its candidate gene, aprP, which encodes a prepro-enzyme, was isolated in a previous study. Recombinant APRP was then produced by in vitro refolding of pro-APRP-His, i.e., N-terminally His-tagged pro-APRP. A large amount of pro-APRP-His was produced in Esherichia coli BL21(DE3) (ca. 8 mg from a 20-ml culture), collected as insoluble protein, dissolved in 6 M guanidine-HCl (pH 8.0), bound to Ni-NTA, and refolded on the resin at pH 10.0 to become mature APRP by autocleavage. Then, 0.16 mg of purified mature APRP was obtained through single-step chromatography from the refolded sample using 10 mg of pro-APRP-His. The N-terminal sequence and the enzymatic properties of refolded APRP were identical to those of SMCE. In addition, the pro-sequence was found to be essential for the production of mature APRP, suggesting that it could function as an intramolecular chaperone.