Affinophoresis in two-dimensional agarose gel electrophoresis: specific separation of biomolecules by a moving affinity ligand

Affinophoresis is an electrophoretic separation technique for biomolecules which uses an affinophore. An affinophore is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and consequently the electrophoretic mobility of molecules having affinity for the ligand is specifically changed. This technique has now been incorporated in two-dimensional agarose gel electrophoresis in a procedure which utilizes normal electrophoresis in the first dimension and affinophoresis in the second dimension. Proteins which do not have affinity for the ligand migrate to locations along a diagonal line passing through the origin, whereas proteins which have affinity are carried away from the line by the affinophore. Accordingly, molecules having affinity for the ligand can be readily assigned. Trypsins contained in Pronase and pancreatin were separated by this procedure using an affinophore bearing a competitive inhibitor for trypsin, benzamidine, on a polyanionic molecule (a polyacrylic acid derivative).     

Affinophoresis of trypsins

We devised a new separation technique for the protein, "affinophoresis," which is based on its specific affinity and utilizes electrophoresis. This technique requires a carrier macromolecule, "affinophore," which contains both an affinity ligand for a certain protein and many charges, either positive or negative, in order to migrate rapidly in an electric field. When a mixture of proteins is electrophoresed in the presence of the affinophore, the protein having an affinity with the ligand will form a complex with the affinophore. This results in a change in the apparent electrophoretic mobility. If the protein is sufficiently accelerated, we can separate it from other materials. A cationic affinophore for trypsin was prepared. Soluble dextran MW approximately 10,000) was coupled with a DEAE-group and m-aminobenzamidine, a competitive inhibitor of trypsins. Electrophoresis of trypsins from several origins on agarose gel plates in the presence of the affinophore showed that affinophoresis actually occurred. The electrophoretic mobilities of trypsins increased towards the cathode, the same direction as the affinophore movement. The presence of leupeptin and treatment of the trypsins with TLCK suppressed the effect of the affinophore. Streptomyces griseus trypsin, contained in Pronase, was easily separated and detected. This procedure is distinct from affinity chromatography and so-called affinity electrophoresis in that the support of the affinity ligand moves, and has advantages especially for analytical purposes: for example, the detection of specific molecules regardless of their isoelectric points.     

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6?U?ml^?1 (batch cultivation with flask cultivation) or 14.4?U?ml^?1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.     

Rational Design of a Novel Propeptide for Improving Active Production of Streptomyces griseus Trypsin in Pichia pastoris

Applying in silico simulations and in vitro experiments, the amino acid proline was proved to have a profound influence on Streptomyces griseus trypsinogen, and the hydrogen bond between H⁵⁷ and D¹⁰² was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold.     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene intoβ-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

Engineering protein allostery: 1.05 A resolution structure and enzymatic properties of a Na+-activated trypsin

Some trypsin-like proteases are endowed with Na⁺-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na⁺-binding site were found necessary to generate efficient Na⁺ activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na⁺-activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 Å resolution defines the engineered Na⁺ site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na⁺ activation is interwoven with substrate selectivity in the trypsin scaffold.     

Trypsin inhibitor ofStreptomyces griseus grown in a carbon nutrition medium

Streptomyces griseus grown in beef extractpeptone medium produces trypsin inhibitors which are both peptide and protein in nature. When the strain was grown in carbon nutrition medium, only one trypsin inhibitor was obtained. Similar elution patterns of this inhibitor and the TI-3a inhibitor obtained from the beef extract-peptone medium on Sephadex G-15 and ion-exchange columns and also the R_f values indicate that the inhibitors from both the sources are the same. So, TI-3a is not the breakdown product of the protein inhibitor ofS. griseus.     

Improvement of catalytic efficiency and thermostability of recombinant Streptomyces griseus trypsin by introducing artificial peptide

Streptomyces trypsin is one of the serine proteinases in Streptomyces griseus and acts as a key mediator during cell growth and differentiation. S. griseus trypsin (SGT) could be successfully expressed in Pichia pastoris by engineering the natural propeptide APNP. In this study, the recombinant Exmt with peptide YVEF and the wild-type SGT were comparatively investigated in detail. The recombinant Exmt showed significantly increased thermostability which t _1/2 value was 3.89-fold of that of the SGT at 40 °C. Moreover, the catalytic efficiency (referring to the specificity constant, k _cat/K _m) and pH tolerance of Exmt were also improved. In silico modeling analysis uncovered that introduction of the peptide YVEF resulted in a broadened substrate binding pocket and closer catalytic triad (His⁵⁷, Asp¹⁰² and Ser¹⁹⁵). The intramolecular Hydrogen bonds and the cation π-interactions were also dramatically increased. The results indicated that engineering of the N-terminus with artificial peptides might be an effective approach for optimizing the properties of the target enzymes.     

Properties of lactate dehydrogenase from the isopod, Saduria entomon

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.     

Endo-beta-N-acetylglucosaminidases acting on carbohydrate moieties of glycoproteins: purification and properties of the two enzymes with different specificities from Clostridium perfringens.

Two endo-β-N-acetylglucosaminidases (C_I and C_I) acting on carbohydrate moieties of glycoproteins were highly purified from the culture fluid of Clostridium perfringens. C_I had the substrate specificity indistinguishable from that of endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae. C_II showed the specificity similar to that of endo-β-N-acetylglucosaminidase H from Streptomyces griseus but is distinct from the streptomyces enzyme with respect to the relative activity toward ovalbumin glycopeptides and Unit A glycopeptides of thyroglobulin. Both enzymes from C. perfringens were most active at neutral pH and were inhibited by p-chloromercuriphenylsulfonate.     

Production of lytic enzymes and siderophores,and inhibition of germination of basidiospores of Moniliophthora (ex Crinipellis) perniciosa by phylloplane actinomycetes

Streptomyces albovinaceus, Streptomyces caviscabies, Streptomyces griseus, Streptomyces setonii, and Streptomyces virginiae selected as antagonists of Moniliophthora (ex Crinipellis) perniciosa, the causal agent of cacao Witches’ broom, were examined in vitro to detect production of chitinases, β-1,3-glucanases, and cellulases. All the species produced chitinases, but not β-1,3-glucanases or cellulases, when grown on a liquid mineral medium containing glucose, colloidal chitin, or cell walls of M. perniciosa as a carbon source. There were no quantitative differences among species in the production of chitinase, however, the germination inhibition of basidiospores of M. perniciosa was higher when they were cultivated using glucose as a carbon source, followed by colloidal chitin and cell walls. All the species also produced hydroxymate type siderophores in similar quantities, and the quantity of siderophores did not correlate with the inhibition of basidiospore germination. The germination inhibition was more pronounced when S. albovinaceus, S. griseus, and S. virginiae were cultivated on iron-deficient medium, suggesting involvement of siderophores in the antagonism by these species of actinomycetes.     

Urea denaturation of the immobilized proteases of Streptomyces griseus (pronase)

The protease preparation (pronase, EC 3.4. group) from Streptomyces griseus has been covalently immobilized on porous succinamidopropyl glass using a carbodiimide carboxyl activation procedure. The separate activities of the individual proteases in this preparation were assayed using specific synthetic substrates. Stabilities of both soluble and immobilized preparations were determined and compared by assaying for each activity in urea solutions of various concentration. The loss of activity by the immobilized enzymes was shown to be reversible under most conditions. Analysis of the data in terms of a two-state transition showed that the urea concentration resulting in 50% loss of activity was increased for each enzyme as a result of immobilization. Also the m-value in the relation ΔG_D = ΔG^H₂O_D - m[urea] decreased for each enzyme upon immobilization. Thus, all of the enzymes were stabilized by immobilization and the apparent broadening of the transitions, as measured by the decreased m-value, was interpreted as the formation of a population of molecules with different stabilities. The degree of apparent stabilization upon immobilization varied with the magnitude decreasing as: aminopeptidases > carboxypeptidase ? trypsin > proteases A and B. Furthermore, it is suggested that stabilization may result from multipoint attachment since the magnitude was correlated with the number of potential enzyme reaction sites as reflected by their lysine contents.     

Studies on an Enzyme Capable of Splitting β-d-1,6-Glucosidic Linkage

Examination was made on the morphological and cultural characteristics of the lutease-producing Streptomyces strain No. OP-4-5 isolated from a dust. The strain was identified as Streptomyces griseus. In addition, it was proved that 2 strains of Streptomyces griseus produce lutease in a test for lutease production in Streptomyces species. Streptomyces parvus and Streptomyces niveoruber also produce the same enzyme. However, production of the lutease by these 4 strains was less than that of produced by Streptomyces griseus strain No. OP-4-5 which was isolated by the authors.     

Determination of the affinity constants of recombinant human galectin-1 and -3 for simple saccharides by capillary affinophoresis

The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars. The relative magnitude of the affinity constants for each of the sugars in terms of dissociation constants found to be consistent with previously reported data on the concentrations of sugars that caused a 50% inhibition (I50) in the binding assay of the lectin to oligosaccharide-immobilized agarose beads but the absolute values of the dissociation constants were considerably smaller than the I50 values. Capillary affinophoresis indicated microheterogeneity of the lectin preparations and enabled the separate analysis of the affinity of each component simultaneously showing the advantage in using a separation method for analysis of bioaffinity.     

Enhancement of protein secretion by TatAC overexpression in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

Production of proteins in secretary form is one of the important factors affecting fermentation. The Tat (twin arginine translocation) protein secretion system, which includes the proteins TatA, TatB, and TatC, was identified in the genomic sequence of Streptomyces griseus IFO13350. The tatA and tatC genes were organized into a polycistronic operon, whereas tatB was located separately on the chromosome. Comparison of amino acid sequences suggested that TatC was a membrane-spanning protein, whereas TatA and TatB were found to be cytoplasmic proteins. Analysis of extracellular proteins and N-terminal amino acid sequencing revealed that secretion of SGR5556 was significantly enhanced by overexpression of TatAC in S. griseus HH1. Further, enzymatic study showed that SGR5556 encoded a glycerophosphoryl diester phosphodiesterase. In addition, other hydrolase activities, such as those of amylase, total protease, metalloprotease, trypsin, chymotrypsin, and Leuaminopeptidase, were also enhanced by 3, 3, 2.6, 2.3, 5.4, and 2.5 fold, respectively, in S. griseus upon TatAC overexpression. Overexpression of TatAC induced the production of a greenish-yellow pigment in S. griseus HH1 as well as more abundant sporulation at an earlier stage in Streptomyces coelicolor A3(2). In silico analysis by TatFIND, SignalP, and TMHMM identified 19 binding proteins, 28 enzymatic proteins, and 27 other proteins with unknown functions as putative TatAC-dependent secretary proteins. These results clearly indicate that TatA and TatC constitute a functional Tat system in S. griseus. Additionally, the S. griseus Tat system can be useful for the production of valuable proteins, including many hydrolytic enzymes and candidates of Tat-dependent secretary proteins, under industrial conditions.     

Novel Biotransformations of 7-Ethoxycoumarin by Streptomyces griseus

Biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of two metabolites which were isolated and identified as 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin. A novel series of biotransformation reactions is implicated in the conversion of the ethoxycoumarin substrate to these products, including O-deethylation, 6-hydroxylation to form a 6,7-dihydroxycoumarin catechol, and subsequent O-methylation. Either 7-hydroxycoumarin or 6,7-dihydroxycoumarin was biotransformed to 7-hydroxy-6-methoxycoumarin by S. griseus. Trace amounts of the isomeric 6-hydroxy-7-methoxycoumarin were detected when 6,7-dihydroxycoumarin was used as the substrate. Efforts to obtain a cell-free catechol-O-methyltransferase enzyme system from S. griseus were unsuccessful. However, [methyl-¹⁴C]methionine was used with cultures of S. griseus to form 7-hydroxy-6-[¹⁴C]methoxycoumarin.     

Biosynthesis of Streptomycin

Investigation was carried out on demonstration of two substances constructing a precursor system located at a late stage of streptomycin biosynthesis by Streptomyces griseus. One of them is thought to be a natural precursor of Streptomycin(L) and the other is suggested as an enzymatic substance(H) transforming L to streptomycin. Both substances had no antibiotic potency and H was inactivated at low pH. L was obtained from a cell-free supernatant (active supernatant) prepared from suspension of young mycelium of Streptomyces griseus in glucose solution. H was obtained not only from active supernatant but also from cell-free extract of the organism.

Two ways of isolation were established for L. Active supernatant was adsorbed on a CM-cellulose column equilibrated with 0.05 m Tris-maleate buffer (pH 8.0). Elution of this column with the same buffer as was used for equilibration gave L-containing fraction separated from streptomycin which was eluted with the buffer including 1% of sodium chloride. L was adsorbed also on active carbon in aqueous solution at neutral pH and liberated from it at acidic pH with 95% methyl alcohol. The former method was useful to separate L from streptomycin, and the latter one was so to concentrate L.

H was isolated by using a column chromatography on DEAE-cellulose. After adsorbing active supernatant or cell-free extract of organism on a column equilibrated previously with the same buffer as above, H was eluted with the buffer including 1% of sodium chloride. Cell-free extract of S. griseus was a better source of H supply than the active supernatant.     

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.     

Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

Streptomyces strain K_1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH₂-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Detection of Polygalacturonase in Kiwifruit during Ripening

Oligosaccharides produced during the course of the hydrolysis of 25% N-acetylated chitosan by Streptomyces griseus chitinase were fractionated by CM-Sephadex C-25 and Toyopearl HW-40F column chromatographies. Sugar compositions and sequences of main oligosaccharides were identified by N-acetylation, exo-splitting with β-GlcNAcase and β-GlcNase, and nitrous acid degradation. In addition to N-acetylated saccharides, GlcNAc, (GlcNAc)₂, and (GlcNAc)₃, hetero-chitooligosaccharides such as GlcN · GlcNAc, GlcN · GlcNAc · GlcNAc, GlcN · GlcN · GlcNAc, GlcN · GlcNAc · GlcNAc · GlcNAc, GlcNAc · GlcN · GlcNAc · GlcNAc, GlcN · GlcNAc · GlcN · GlcNAc, and GlcN · GlcN · GlcNAc · GlcNAc were identified. These results indicate that Streptomyces griseus chitinase specifically cleaves the N-acetyl-β-d-glucosaminidic linkages in partially N-acetylated chitosan.