Type 2 diabetes is one the most common metabolic diseases, which is obviously the price of lifestyle changes for many people. While being dangerous in itself, this disease provokes other metabolic disorders, such as obesity and neurodegenerative diseases such as Alzheimer’s disease. Pharmacologists are very active in creating drugs for these illnesses. The design of synthetic highly active and stable analogues of incretins, peptide hormones produced by neuroendocrine cells, is one of the most promising research areas. Glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide are the best known incretin hormones. Analogues of the first peptide have already found application in medical practice. The next step in the creation of drugs for diabetes was the development of polyagonists, which combine the properties of several different peptide hormones responsible for glucose homeostasis. The combination of the properties of incretins gives hope for a synergetic network effect. In the past few years, the creation of such co-agonists has progressed very rapidly. In some cases, the results of clinical trials have already been obtained; however, they often contradict each other. This difficult situation was the main motivation for writing the present review.

The modified asparaginase Was79 was derived from the recombinant wild-type l-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125–8000 IU/kg). At Was79 single doses of 500–8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.

The crystal toxins (Cry) produced by the bacterium Bacillus thuringiensis (Bt) have been successfully used in both spray formulations and transgenic crops to control some of the most problematic insect pests, as has been discussed in previous chapters. The delta-endotoxins of Bt are functionally active in the insect gut and interact with and are processed by proteolytic enzymes. The structure of Cry proteins has specific features that not only permit them to retain their biological activity in the hostile environment of the insect gut, but also to use the process of proteolysis in the solubilization and activation of Cry protoxins. Because the proteolysis of Cry proteins is critical to their biological activity, we review the literature on studies related to insect and mammalian proteases and their effects on toxin structure and toxicity.

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.

130 In the article by V.G. Bogush, K.V. Sidoruk, L.I. Davydova, I.A. Zalunin, D.G. Kozlov, M.M. Moyse novich, I.I. Agapov, Academician M.P. Kirpichnikov, and Corresponding Member of the RAS V.G. Debabov, entitled “Recombinant Analogue of Spidroin 2 for Biomedical Materials,” which was published in Dokl. Biochem. Biophys., 2011, vol. 441, no. 5, pp. 699–702, the number of the third state contract no. 16.552.11.7029 from April 29, 2011, was missed through the fault of the authors. Erratum: “Recombinant Analogue of Spidroin 2 for Biomedical Materials” [Dokl. Biochem. Biophys., 2011, vol. 441, no. 5, pp. 699–702]

Proteins of inclusions in bacteria symbiotic to entomopathogenic nematodes Xenorhabdus spp. and Photorhabdus spp. have a pronounced antibiotic effect suppressing growth of different bacteria on agar media. The toxicity of proteins from Photorhabdus luminescens and entomopathogenic bacteria Bacillus thuringiensis inclusions towards Escherichia coli has been compared. Inclusion proteins from Xenorhabdus bovienii inhibit the growth of two assayed subspecies of B. thuringiensis: kurstaki and israelensis. The vegetative cells of X bovienii, isolated from Steinernema feltiae, proved to be sensitive to the B. thuringiensis ssp. israelensis Cyt 1A crystal protein.

A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene- bis -acrylamide), acid para -nitroanilides (4′-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n -butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7–8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant ( K m ) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0–7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).

Выделена и охарактеризована металлокарбоксипептидаза, продуцируемая штаммом Streptomyces bikiniensis 27 (ВКПМ Ас-1783) (КПSb). Фермент способен c равной эффективностью отщеплять от синтетических пептидов как основные, так и гидрофобные С-концевые остатки, т.е. обладает специфичностью карбоксипептидаз А и В млекопитающих. Фермент также гидролизует пептиды, имеющие на С-конце глутаминовую кислоту. КПSb имеет максимальную активность при рН 7,0-7,6 и 55°. Установлена последовательность участка ДНК, соответствующего зрелому ферменту КПSb в геноме S. bikiniensis 27 (ВКПМ Ас-1783) (Accession No GU362077). Показано, что первичная структура зрелого фермента имеет умеренную степень идентичности с ортологами из Streptomyces griseus (79% идентичности) и Streptomyces avermitilis (85% идентичности).

An expression system for an effective production of recombinant protein Cry9A in bacillary cell has been suggested in the study. The proteins’ immunological properties, ability to proteolysis, and biological activity were identical to natural protein. The ability of recombinant Cry9A to form crystal bodies in sporulating cells of Bacillus thuringiensis has been shown. Thus, the first evidences of the fact that Cry-proteins which in natural strains form the crystal bodies together with other endotoxins are able to independently form the crystals has been received. The introduced system including vector replicative carriers, expression cassettes, and a protocol of obtaining and cultivation of strain-producer allows simple manipulations with the gene of delta-endotoxin of Cry9A in gene-engineering experiments.