Based on the Komagataella kurtzmanii yeast, a strain producing Tribolium castaneum recombinant procathepsin (rpTcCathL) has been obtained; this strain provided biosynthesis and secretion of at least 0.5 g/L of the target protein as a result of cultivation in flasks on a medium of complex composition. It was determined that the pH of the yeast culture medium (pH ≥ 6.5) is the key factor that influences the biosynthesis and accumulation of procathepsin. A new chromatographic purification scheme was developed, which allows one to obtain recombinant procathepsin with a yield of about 30

The isolation, heterologous expression and characterization of a new thermostable β-glucanase from Paenibacillus jamilae is described. The bgl26 gene from the P. jamilae Bg1 VKPM B-13093 strain consisting of 714 nucleotides encodes endo-1,3-1,4-β-glucanase (EC 3.2.1.73) containing 213 amino acids and 24 residues of the putative signal peptide in N-end area. The nucleotide sequence of the bgl26 gene and the amino acid sequence of the mature Bgl26 protein have the greatest homology with the sequence of the Paenibacillus macerans endo-l,3-l,4-β-glucanase (82 and 88%, respectively). A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. Purified recombinant enzyme Bgl26 was active towards barley β-glucan. The optimal pH for the enzyme to work was 7,0, and the optimum temperature range was 40-45 °C. The specific activity of β-glucanase was at the level of 6650 U/mg of protein, Km and Vmax were equal to 6.4 ± 0.3 mg/mL and 9450.1 ± 471.2 umol/(min-mg), respectively. The recombinant protein Bgl26 was characterized by high pH and thermal stability, as well as resistance to digestive enzymes. It is also shown that Co2+ ions have a positive effect on the activity of the enzyme. β-glucanase, β-glucan, Paenibacillus jamilae, Pichia pastoris The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) and was carried out using the Multipurpose Scientific Installation of National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

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).

The gene phyA encoding phytase was isolated from Obesumbacterium proteus genomic library and sequenced. The cleavage site of the PhyA signal peptide was predicted and experimentally proved. The PhyA protein shows maximum identity of 53% and 47% to phosphoanhydride phosphorylase from Yersinia pestis and phytase AppA from Escherichia coli, respectively. Based on protein sequence similarity of PhyA and its homologs, the phytases form a novel subclass of the histidine acid phosphatase family. To characterize properties of the PhyA protein, we expressed the phyA gene in E. coli. The specific activity of the purified recombinant PhyA was 310 U mg−1 of protein. Recombinant PhyA showed activity at pH values from 1.5 through 6.5 with the optimum at 4.9. The temperature optimum was 40–45 °C at pH 4.9. The Km value for sodium phytate was 0.34 mM with a Vmax of 435 U mg−1.

Functional destination of propeptides and precursors in bacillar secretory proteases remains uncertain. Formerly deletion assay demonstrated folding and secretion of subtilisin E, chymotrypsin-like protease SGPB from S. griseus and B. cereus metalloprotease to depend on full-length propeptide in the precursors. Actually an artificial B. amyloliquefaciens metalloprotease gene with deletion of 51 amino acid residues from N-terminus was constructed with regard to carry out functional mapping of secretory metalloprotease propeptides. B. subtilis wprA gene 5'-terminal region spanning promoter and secretory leader was coupled to provide transcription to the truncated gene and secretion to its product. B. subtilis clones bearing a plasmid with the modified gene synthesised an active mature metalloprotease.

A secretory system based on L-form cells of Proteus mirabilis was developed for production of native Bacillus licheniformis glutamylendopeptidase precursor never formerly available. The produced precursor was stable per se under physiological conditions and in presence of trypsin and glutamylendopeptidase from B. intermedius. Complete conversion of the precursor to the mature glutamylendopeptidase was performed by bacillar metalloproteases and subtilisin. The artificially processed glutamylendopeptidase was purified by affinity chromatography on bacitracin-sepharose. Native tertiary structure in the purified glutamylendopeptidase was confirmed by demonstrating its activity towards a specific chromogenous peptide substrate.

The data on the precursors of bacterial proteases were generalized. The structure and special features of processing of the precursors of bacillary subtilisins, the α-lytic protease from Lysobacter enzymogenes and the related chymotrypsin-like proteases from Streptomyces griseus , and the metalloproteases from bacilli and Pseudomonas aeruginosa were discussed. The approaches to producing the precursors and the protease propeptides and to in vitro characterizing them were particularly analyzed. The following physiological functions of the propeptides within the protease precursors were considered probable: (a) inhibition of the proteases to protect the host cells from the proteolytic damage; (b) participation in the folding of the mature enzyme; and (c) providing for the protease interaction with the bacterial cell surveillance mechanisms, including protease translocation through the cell wall.

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100–150 mg/L of mature protease.

A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or δ-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N-terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight-binding competitive type with Ki 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.

The structural gene of the carboxypeptidase T (cpt) was successfully expressed in cell wall-less L-form cells of Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a truncated cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the lac promoter was transformed into L-form cells of P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l−1 and should be improvable.

The structural gene of the carboxypeptidase T (cpt) was successfully expressed in cell wall-less L-form cells of Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a truncated cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the lac promoter was transformed into L-form cells of P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l−1 and should be improvable.