Royal jelly is a nutritious substance secreted from the hypopharyngeal and mandibular glands of worker bees that serves as the only food on which honeybee queen larvae and adults are fed and which causes them to develop into queen bees. Royal jelly is a protein-rich food and one of the most crucial factors for the growth of queen bees. In this study, we characterized the hydrolytic activity of enzymes from the homogenates of honeybee queen larvae on royal jelly proteins. Homogenates of 3-day-old queen bee larvae were capable of hydrolyzing royal jelly proteins under alkaline conditions. Following separation by cation exchange and gel filtration column chromatographies, two proteases of 38 and 28 kDa were found by SDS-PAGE. The protease of 38 kDa had a carboxypeptidase A-like activity and that of 28 kDa a chymotrypsin-like activity. These enzymes may turn out to be useful in the manufacturing of processed royal jelly.

International audiencePreviously, we found three enzyme fractions containing activities for the hydrolysis of royal jelly proteins from honeybee queen larvae. In this study, we identified a honeybee chymotrypsin-like protease (HCLPase) by LC-MS/MS and expressed it as a recombinant protein in Escherichia coli. The protease had an estimated molecular weight of around 26 kDa and showed high specificity for succinyl-Ala-Ala-Pro-Phe p-nitroanilide as a proteolytic substrate. Furthermore, the protease had an optimal pH of 9, and the activity was markedly inhibited by phenylmethylsulfonyl fluoride but not tosyl phenylalanyl chloromethyl ketone, both of which are irreversible inhibitors of chymotrypsin-like serine proteases. These results suggested that this recombinant protease, HCLPase, was a chymotrypsin-like serine protease with different characteristics from mammalian chymotrypsin

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We previously found a novel chymotrypsin-like protease in honeybee, designated as HCLPase. The recombinant enzyme expressed in insect cells was produced and compared to that in Escherichia coli. Both enzymes showed equivalent molecular size and specificity. However, HCLPase produced in insect cells showed higher specific activity. The C-terminal cleavage sites of HCLPase were phenylalanine, leucine, and tyrosine residues.

Inhibitor concentration of figure legends in Fig. 3 and 5 had some errors. Correct legends are shown below: Fig. 3. Effects of protease inhibitors on hydrolytic activities of crude enzyme solution. RJ solution (30-mg/mL proteins), crude enzyme solution (3-mg/mL proteins), and buffer (50-mM phosphate buffer, pH 7.0, or 50-mM Tris-HCl buffer, pH 9.0) were mixed at 1:1:8 (v/v/v). The reactions after adding the following inhibitors were carried out at 37°C for 24 h. The results of adding the following protease inhibitors at pH 7.0 (a) and at pH 9.0 (b) are shown: 1 untreated RJ solution; 2 no inhibitor; 3 3-mM PMSF; 4 9-μg/mL antipain; 5 9-μg/mL pepstatin A; 6 9-μg/mL leupeptin; and 7 3-mM EDTA. The results of adding the following peptidase inhibitors at pH 7.0 (c) and at pH 9.0 (d) are shown: 1 untreated RJ solution; 2 no inhibitor; 3 0.08-mg/mL bestatin; 4 10-μg/mL CPI; 5 3-mM PMSF. Fig. 5. Effects of inhibitors on the hydrolysis of RJ proteins by the fractions obtained from gel filtration chromatography. Inhibitory activities are shown for fraction I (a), fraction II (b), and fraction III (c). RJ solution (30-mg/mL proteins), sample fractions, and buffer (50-mM Tris-HCl buffer, pH 9.0) were mixed at 1:1:8 (v/v/v). The reactions after adding the following inhibitors were carried out at 37°C for 24 h: 1 untreated RJ solution; 2 no inhibitors; 3 5 mM PMSF; 4 5 μg/mL CPI; 5 10 mM EDTA.Everything else in the paper remains correct.The online version of the original article can be found at http://dx