ABSTRACT
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.
Subject(s)
Bacillus/enzymology , Bacterial Proteins/chemistry , Endopeptidases/chemistry , Amino Acid Sequence , Caseins/chemistry , DNA Primers/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Hydrolysis , In Vitro Techniques , Kinetics , Molecular Sequence Data , Peptides/chemistry , Protein Denaturation , Protein Folding , Protein Renaturation , Recombinant Proteins/chemistry , Serine Endopeptidases/chemistry , Glycine max/enzymology , Subtilisin/chemistryABSTRACT
Previous work has shown that when the bacterium Stenotrophomonas maltophilia is grown on polypropylene glycol, different dye-linked polypropylene glycol dehydrogenase (PPG-DH) activities are induced during growth. Here the purification and characterization of the dehydrogenase activity induced in the stationary phase, and present in the periplasmic space, is described. The homogeneous enzyme preparation obtained consists of a homodimeric protein with a molecular mass of about 123 kDa and an isoelectric point of 5.9. The cofactor of the enzyme appeared to be pyrroloquinoline quinone (PQQ), no heme c was present, and holo-enzyme contained two PQQ molecules per enzyme molecule. In these respects, PPG-DH described here is similar to already known quinoprotein alcohol dehydrogenases, but in other respects, it is different. Therefore, it is suggested that PPG-DH could be a new type of quinoprotein alcohol dehydrogenase. Based on its strong preference for polyols, PPG-DH seems well fitted to carry out the first step in the degradation of PPGs, synthetic polymers containing a variety of hydroxyl groups.
