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). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine.

Subtilisin Carlsberg (SC) was shown to catalyze the solid phase segment coupling of peptides in complex with sodium dodecyl sulfate (SDS) in an organic medium on Aminosilochrom and polyvinyl alcohol (PVA) cryogel activated with glutaraldehyde or divinylsulfone. Diamines of different lengths with a general formula NH 2 -(CH 2 ) n -NH 2 ( n = 2, 4, and 6) were used as spacers between the PVA cryogel and the peptide. A model reaction of enzymatic attachment of the Dnp-Ala-Ala-Leu-OMe tripeptide to the PVA cryogel was carried out by treatment with the SDS-SC complex in a mixture of anhydrous ethanol and DMSO (7 : 3, v/v) using a tenfold excess of the carboxyl component. The molar enzyme-substrate ratio was 1 : 88. The effect of the method of matrix activation, length of a spacer, and reaction time on the coupling efficiency was studied. Hexamethylenediamine was found to be the most effective spacer for the enzymatic coupling on the PVA cryogel activated with glutaraldehyde (the reaction proceeded with the highest yield of 60%). The reaction efficiency was considerably lower in the case of ethylenediamine and tetramethylenediamine (10 and 15%, respectively). The best results were obtained on the PVA cryogel activated by divinylsulfone with hexamethylenediamine as a spacer. A two-step condensation of tripeptides was carried out on this support. The second step of condensation was shown to proceed better (in 85% yield) in comparison with the first step (37% yield).

Two post-proline cleaving peptidases PPCP1 and PPCP2 with molecular masses of 101 and 63 kDa, respectively, hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the larval midgut of the yellow mealworm Tenebrio molitor and characterized. PPCP1 was active only in acidic media, with a maximum at pH 5.6, whereas PPCP2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PPCP1 and PPCP2 were shown to belong to serine peptidases. The data obtained indicate that a Cys residue is located close to the PPCP2 substrate binding site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, completely inhibited PPCP2 and partially PPCP1. The substrate specificities of the isolated enzymes were studied. Z-Ala-Ala-Pro-pNA was the best substrate for PPCP1, and Z-Ala-Pro-pNA, for PPCP2. The combination of the properties allows characterization of PPCP2 as a proplyl oligopeptidase.

A chemoenzymatic synthesis 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 o -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. Hydrolysis of substrate ( I ) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate ( II ), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate ( II ), than for substrate ( I ). The lowest detectable papain concentrations were 2.4 × 10 −10 M for ( I ) and 1.2 × 10 −11 M for ( II ). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.

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. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.

Разработан химико-энзиматический синтез новых высокоспецифичных флуорогенных субстратов цистеиновых протеиназ семейства папаина – Abz-Phe-Ala-pNA (I) и Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp и Amc – o-аминобензоил, n-нитроанилид, пироглутамил и 4-амино-7-метилкумарид соответственно). Субстрат (I) был получен в водно-органической среде с использованием нативного химотрипсина. Субстрат (II) – в смеси DMF–MeCN под действием химотрипсина и субтилизина Карлсберг, иммобилизованных на криогеле поливинилового спирта. Гидролиз субстрата (I) папаином, фицином и бромелаином сопровождался повышением интенсивности флуоресценции в 15 раз, субстрата (II) – изменением спектра флуоресценции. Показана однозначность ферментативного гидролиза субстратов после остатка Ala. Определена удельная активность гидролиза субстратов папаином, бромелаином и фицином. Показано, что наибольшую активность по обоим субстратам проявлял папаин. Активность всех исследованных протеиназ была существенно выше по субстрату (II), чем по субстрату (I). Минимально определяемые концентрации папаина составили по субстрату (I) – 2.4 ? 10-10 М, по (II) – 1.2 ? 10-11 М. Установлена высокая селективность цистеиновых протеиназ по отношению к Glp-Phe-Ala-Amc.

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P 1 and P 1 ′ (Glu, Arg, and Asp) containing unprotected side ionogenic groups.

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate.

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200mM.

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content >80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N -acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p -nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, Lys, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70–98% in DMF–MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.

Covalent immobilization of subtilisin and thermolysin on cryogel of poly(vinyl alcohol) was carried out. The biocatalysts obtained are characterized by high stability in water and in DMF—MeCN mixtures of various compositions. The synthetic efficiency of immobilized subtilisin in the multiple iterative synthesis of the peptide Z—Ala—Ala—Leu—Phe—pNA was examined in organic mixtures of different solvent compositions. Immobilized subtilisin exhibits high synthetic activity in organic media. A series of N-acylated p-nitroanilides of tetrapeptides of the general formula Z—Ala—Ala—Xaa—Yaa—pNA (Z is benzyloxycarbonyl, Xaa = Leu, Lys, or Glu; Yaa = Phe or Asp; pNA = 4-NO2—C6H4NH-) were synthesized in 70—98% yields using immobilized subtilisin as a biocatalyst without activation and protection of the ionogenic groups of polyfunctional amino acids. Immobilized thermolysin in a DMF—MeCN mixture catalyzed the formation of the peptide Z—Ala—Ala—Leu—pNA, which was obtained in 90% yield (during 1 h). It was demonstrated that the biocatalyst can be used repeatedly and that it retained activity after storage in an aqueous buffer during 6 months.

Исследованы активность и стабильность нативных субтилизинов Карлсберг и 72 и их комплексов с додецилсульфатом натрия (SDS) в средах, содержащих органические растворители. Определены кинетические константы гидролиза специфических хромогенных пептидных субстратов Z-Ala-Ala-Leu-pNA и Glp-Ala-Ala-Leu-pNA нативными субтилизинами Карлсберг и 72. Установлено, что субтилизин Карлсберг в комплексе с SDS в средах безводных органических растворителей является эффективным катализатором синтеза пептидов с полифункциональными аминокислотами в Р1 и Р'1 -положениях (Glu, Arg, Asp), содержащими незащищенные боковые ионогенные группы.

The segment condensation of peptides on a solid phase (Aminosilochrom) in organic medium catalyzed by a subtilisin complex with sodium dodecylsulfate was studied. The dependence of the efficiency of the enzymatic coupling of tripeptides with the basic structure X-Ala-Ala-Y-OMe [where X = Z, Boc, or Dnp and Y = Leu or Glu(OMe)] on the spacer (Phe-Met-Gly-Gly) content on the support and on the structure of the acylating component was investigated. The tripeptide segments were successively coupled to Aminosilochrom containing the Met-Ala-Gly spacer, and the peptidylaminosilochroms Dnp-Ala-Ala-Leu-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly- A and Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly- A ( A is the Aminosilochrom residue) were obtained in satisfactory yields. It was shown by these examples that the second and third segments are attached in yields higher than that for the first segment and the coupling efficiency does not depend on the amino acid composition of the acylating component.

The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction ZAlaAlaLeuOR+HPhepNA→ZAlaAlaLeuPhepNA, nearly quantitative product yields were observed after 2h, regardless of whether an activated (R=CH3, p-C6H5Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P1-position. Tetra-peptides of general formula ZAlaAlaP1P1′pNA, where P1=Glu, Asp, Lys, Arg or His and P1′=Phe, Arg or Glu have been obtained in good yield.

Предложена серия флуорогенных тетра-, пента- и гексапептидных субстратов с внутримолекулярным тушением флуоресценции общей формулы Abz-X-Phe-Phe-Y-Ded или (-pNA), где X = Ala, Ala-Ala, Val-Ala; Υ = -, Ala, Ala-Ala. Определены кинетические параметры гидролиза субстратов пепсином, катепсином D, гастриксином человека, пепсином свиньи, химозином теленка, аспергиллопепсином А. Показано, что синтезированные соединения - эффективные субстраты аспартильных протеиназ различного происхождения.A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Υ = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.