Jiro Arima

Production of dipeptidyl peptidase IV inhibitory peptides from defatted rice bran

The insulinotropic hormone glucagon-like peptide-1 is metabolised extremely rapidly by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, human DPP-IV is a key regulator involved in the prevention and treatment of type 2 diabetes. To simplify the method of producing an inhibitory peptide against DPP-IV, we focused on rice bran (RB) as a source and subjected proteins from defatted RB to enzymatic proteolysis using 2 commercial enzymes. The RB peptides produced with Umamizyme G exhibited 10 times the inhibitory activity as those produced with Bioprase SP. The half-maximal inhibitory concentration (IC(50)) value of the RB peptides was 2.3 ± 0.1mg/ml. Leu-Pro and Ile-Pro were identified as the inhibitory peptides among the RB peptides produced with Umamizyme G. Ile-Pro was the strongest DPP-IV inhibitor among the 15 Xaa-Pro dipeptides and Pro-Ile tested. Ile-Pro competitively inhibited DPP-IV (K(i)=0.11 mM). Mass spectrometry indicated that the contents of Leu-Pro and Ile-Pro in the RB peptides were 2.91 ± 0.52 μg/mg.

Study on peptide hydrolysis by aminopeptidases from Streptomyces griseus, Streptomyces septatus and Aeromonas proteolytica.

We developed a spectrophotometric assay for peptide hydrolysis by aminopeptidases (APs). The assay enables the measurement of free amino acids liberated by AP-catalyzed peptide hydrolysis using 4-aminoantipyrine, phenol, peroxidase, and l-amino acid oxidase. We investigated the specificity of bacterial APs [enzymes from Streptomyces griseus (SGAP), Streptomyces septatus (SSAP), and Aeromonas proteolytica (AAP)] toward peptide substrates using this assay method. Although these enzymes most efficiently cleave leucyl derivatives among 20 aminoacyl derivatives, in peptide hydrolysis, the catalytic efficiencies of Phe-Phe hydrolysis by SGAP and SSAP exceed that of Leu-Phe hydrolysis. Furthermore, all enzymes showed the maximum catalytic efficiencies for Phe-Phe-Phe hydrolysis. These results indicate that the hydrolytic activities of bacterial APs are affected by the nature of the penultimate residue or flanking moiety and the length of the peptide substrate.

Structural characterization of l‐glutamate oxidase from Streptomyces sp. X‐119‐6

l‐Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6, which catalyzes the oxidative deamination of l‐glutamate, has attracted increasing attention as a component of amperometric l‐glutamate sensors used in the food industry and clinical biochemistry. The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an α2β2γ2 structure; enzymatic proteolysis of the precursor forms the stable mature enzyme. We solved the crystal structure of mature LGOX using molecular replacement with a structurally homologous model of l‐amino acid oxidase (LAAO) from snake venom: LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of LAAO revealed that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. Furthermore, the arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO. Results of the comparative analyses described herein raise the possibility that such a unique structure of LGOX is associated with its substrate specificity.

pTONA5: A hyperexpression vector in streptomycetes

We constructed the Streptomyces hyperexpression vector pTONA5 based on pIJ702 vector; it includes a metalloendopeptidase (SSMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from Streptomyces aureofaciens TH-3. The vector contains recognition sites for restriction enzymes NdeI and EcoRI/XbaI/HindIII between the promoter and terminator to facilitate heterologous gene cloning. The plasmids were transferred from Escherichia coli to streptomycetes via conjugation from oriT; the transformants were able to be selected using kanamycin and/or thiostrepton. The SSMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. We constructed expression plasmids including three Streptomyces aminopeptidases-leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP)-using the pTONA5 vector and Streptomyces lividans. Although they lack signal peptides for secretion, PAP and APP were secreted at high levels in the culture broth.

Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides

ABSTRACT Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.

Characterization of calcium ion sensitive region for β-Mannanase from Streptomyces thermolilacinus

Despite the widespread industrial applications of β-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on β-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding β-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7-9°C high temperature in the presence of Ca(2+) compared with that in the absence of Ca(2+). These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca(2+). StMan, but not TfMan, required Ca(2+) for the hydrolysis activity. To identify the Ca(2+) sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca(2+) and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca(2+) sensitive site.

Modulation of Streptomyces Leucine Aminopeptidase by Calcium IDENTIFICATION AND FUNCTIONAL ANALYSIS OF KEY RESIDUES IN ACTIVATION AND STABILIZATION BY CALCIUM

Streptomyces griseus leucine aminopeptidase (SGAP), which has two zinc atoms in its active site, is clinically important as a model for understanding the structure and mechanism of action of other metallopeptidases. SGAP is a calcium-activated and calcium-stabilized enzyme, and its activation by calcium correlates with substrate specificity. In our previous study, we found a non-calcium-modulated leucine aminopeptidase secreted by Streptomyces septatus, the primary structure of which showed 71% identity with SGAP. In this study, we constructed chimeras of SGAP and S. septatus aminopeptidase by using an in vivo DNA shuffling system and several mutant enzymes by site-directed mutagenesis to identify the key residues in this modulation by calcium. We identified the key residues Asp-173 and Asp-174 of SGAP associated with both SGAP activation and stabilization by calcium. We also showed that the known calcium-binding site, which is composed of Asp-3, Ile-4, Asp-262, and Asp-266 of SGAP, only contributes to SGAP stabilization by calcium. Furthermore, we identified an important residue, Glu-196, that functions in cooperation with Asp-173, Asp-174, and calcium to increase the catalytic activity of SGAP.

Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

ABSTRACT To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed l-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with l-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.

Glassin, a histidine-rich protein from the siliceous skeletal system of the marine sponge Euplectella, directs silica polycondensation.

Significance Hexactinellid sponges of the genus Euplectella produce highly ordered and mechanically robust skeletal systems of amorphous hydrated silica. The high damage tolerance of their constituent skeletal elements and the environmentally benign conditions under which these sponges form have prompted additional investigations into the characterization of the proteins driving the synthesis of these materials. In the present report, we describe a previously unidentified protein, named “glassin,” extracted from the demineralized skeletal elements of Euplectella. Glassin is a histidine-, aspartic acid-, threonine-, and proline-rich protein and directs silica polycondensation at neutral pH and room temperature. The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure–function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named “glassin,” the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6–8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.

Extracellular production and characterization of two streptomyces L-asparaginases.

Abstractl-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.