Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests.

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm).

[Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel].

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined.

[Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family].

A chemoenzymatic syntheses was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are i-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel.

[Properties of post-proline cleaving enzymes from Tenebrio molitor].

Two post-proline cleaving enzymes PRE1 and PRE2 with molecular masses of 101 and 62 kDa, respectively, capable of hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the midgut of the flour beetle Tenebrio molitor and characterized. PRE1 is active only in acidic media, with a maximum at pH 5.6, whereas PRE2, both in acidic and alkaline media with a maximum at pH 7.

Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut.

Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.