Silver-Stained Fibrin Zymography: Separation of Proteases and Activity Detection Using a Single Substrate-Containing Gel.

Silver-stained fibrin zymography for separation of protease bands and activity detection using a single substrate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.

A screening method for ß-glucan hydrolase employing Trypan Blue-coupled ß-glucan agar plate and ß-glucan zymography.

A new screening method for ß-(1,3-1,6) glucan hydrolase was developed using a pure ß-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a ß-glucan hydrolase on the Trypan Blue-coupled ß-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-ß-glucan zymography. The ß-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the ß-glucan hydrolase of Paenibacillus sp. was sequenced.

Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel.

A new zymogram method, silver-stained fibrin zymography, for separation of protease bands and activity detection using a single substrate gel, was developed. The method takes advantage of the nanoscale sensitivity of both zymography and silver staining. After SDS-PAGE in a gel containing fibrin, the gel was incubated in enzyme reaction buffer and the zymogram was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined. Furthermore, proteases of high molecular weight were clearly and sharply resolved.

A new fibrinolytic enzyme (55 kDa) from Allium tuberosum: purification, characterization, and comparison.

Chives have been used both as food and as medicine. Previously, two fibrinolytic enzymes, ATFE-I (90 kDa) and ATFE-II (55 kDa), were identified in chives (Allium tuberosum), a perennial herb. In the present work, ATFE-II was purified by ion-exchange chromatography followed by gel filtration. In addition, the enzyme properties of ATFE-I and ATFE-II were compared. The molecular mass and isoelectric point (pI value) of ATFE-II were 55 kDa and pI 4.0, respectively, as revealed using one- or two-dimensional fibrin zymography. ATFE-II was optimally active at pH 7.0 and 45°C. ATFE-II degraded the Aα-chain of human fibrinogen but did not hydrolyze the Bß-chain or the γ-chain, indicating that the enzyme is an α-fibrinogenase. The proteolytic activity of ATFE-II was completely inhibited by 1 mM leupeptin, indicating that the enzyme belongs to the cysteine protease class. ATFE-II was also inhibited by 1 mM Fe²(+). ATFE-II exhibited high specificity for MeO-Suc-Arg-Pro-Tyr-p-nitroaniline (S-2586), a synthetic chromogenic substrate of chymotrypsin. Thus proteolytic enzymes from A. tuberosum may be useful as thrombolytic agents.

Expression and characterization of a novel deoxyribose 5-phosphate aldolase from Paenibacillus sp. EA001.

A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).

Jeongeupia naejangsanensis gen. nov., sp. nov., a cellulose-degrading bacterium isolated from forest soil from Naejang Mountain in Korea.

A Gram-stain-negative, motile, rod-shaped, cellulose-degrading bacterial strain, BIO-TAS4-2(T), which belongs to the Betaproteobacteria , was isolated from forest soil from Naejang Mountain, Korea, and its taxonomic position was investigated by using a polyphasic study. Strain BIO-TAS4-2(T) grew optimally at pH 7.0-8.0, at 30 degrees C and in the presence of 0-1.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain BIO-TAS4-2(T) clustered with members of the genera Andreprevotia, Silvimonas and Deefgea of the family Neisseriaceae, with which it exhibited 16S rRNA gene sequence similarities of 93.5-94.2 %. Strain BIO-TAS4-2(T) contained Q-8 as the predominant ubiquinone and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH) and C(16 : 0) as the major fatty acids. The DNA G+C content was 63.8 mol%. Strain BIO-TAS4-2(T) could be differentiated from members of phylogenetically related genera by differences in fatty acid composition, DNA G+C content and some phenotypic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BIO-TAS4-2(T) is considered to represent a novel species in a new genus, for which the name Jeongeupia naejangsanensis gen. nov., sp. nov. is proposed, with BIO-TAS4-2(T) (=KCTC 22633(T)=CCUG 57610(T)) as the type strain.

Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix.

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.

Production of pure beta-glucan by Aureobasidium pullulans after pullulan synthetase gene disruption.

By disruption of the pullulan synthetase gene (pul) of Aureobasidium pullulans IMS822 KCTC11179BP, we constructed a mutant strain, A. pullulans NP1221, which produced a pure beta-glucan exopolysaccharide. The mutant NP1221 was white, whereas the wild-type strain produced a black dye. When we compared fermentation kinetics between wide-type and mutant strains, the mutant NP1221 did not produce pullulan. Substrate uptake rate and beta-glucan production were similar in both strains. However, the biomass yield of mutant NP1221 was 2.3-fold (9.2 g l(-1)) greater than that of wild-type.

Purification and characterization of a novel glucansucrase from Leuconostoc lactis EG001.

A gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean fermented food. The L. lactis EG001 glucansucrase gene consists of 4503 bp open reading frame (ORF) and encodes an enzyme of 1500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 degrees C and pH 5.0. In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications.

Brevundimonas naejangsanensis sp. nov., a proteolytic bacterium isolated from soil, and reclassification of Mycoplana bullata into the genus Brevundimonas as Brevundimonas bullata comb. nov.

A Gram-negative, motile and rod-shaped bacterial strain, BIO-TAS2-2(T), of the class Alphaproteobacteria, was isolated from a soil in Korea and studied using a polyphasic taxonomic approach. Strain BIO-TAS2-2(T) grew optimally at pH 7.5-8.5 and 30 degrees C and in the presence of 0-1.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain BIO-TAS2-2(T) fell within the clade comprising species of the genus Brevundimonas, forming a coherent cluster with Brevundimonas terrae KSL-145(T) and Brevundimonas diminuta LMG 2089(T). It exhibited 16S rRNA gene sequence similarity values of 96.0-98.7 % to members of the genus Brevundimonas and Mycoplana bullata IAM 13153(T). Strain BIO-TAS2-2(T) contained Q-10 as the predominant ubiquinone and cyclo-C(18 : 1)omega7c and C(16 : 0) as the major fatty acids. The DNA G+C content was 67.0 mol%. Strain BIO-TAS2-2(T) exhibited DNA-DNA relatedness levels of 12-19 % with the type strains of phylogenetically related Brevundimonas species and M. bullata. The novel strain could be differentiated from Brevundimonas species and M. bullata by differences in phenotypic characteristics. On the basis of phenotypic, phylogenetic and genetic data, strain BIO-TAS2-2(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas naejangsanensis sp. nov. is proposed. The type strain is BIO-TAS2-2(T) (=KCTC 22631(T)=CCUG 57609(T)). In this study, it is also proposed that Mycoplana bullata be transferred to the genus Brevundimonas as Brevundimonas bullata comb. nov. (type strain TK0051(T)=ATCC 4278(T)=DSM 7126(T)=JCM 20846(T)=LMG 17157(T)).

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015.

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 micromol/min/mg. The Michaelis constant (k(m) value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 degrees C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.

Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel.

A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.

Identification of a serine protease from a Bacillus sp. using multiple loading of O'Farrell-type isoelectric focusing slab two-dimensional gel.

A protease was purified from Bacillus sp. DJ isolated from Doenjang, a traditional Korean fermented food. Its molecular weight (MW) and isoelectric point (pI) were 18-19 kDa and 6.0-6.5 using 1- or 2-D fibrin zymography, respectively. The protease was optimally active at pH 9 and 55 degrees C. Activity was inhibited by 1 mM PMSF, but not by EDTA, EGTA, aprotinin, or leupeptin, indicating that the protease is a serine protease. By using a new electrophoretic technique, multiple loading of O'Farrell-type isoelectric focusing (IEF) slab gel, the first amino acid residues of the N-terminal sequence of the protease were determined as HPLVLVDPIL, which is 80% identical with serine proteases of the subtilase family.

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet.

A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85 degrees C, respectively. AJ kept 85% of the initial activity after heating at 100 degrees C for 20 min on the zymogram gel. The Michaelis constant (K (m)) and K (cat) values of AJ towards alpha-casein were 0.38 mM and 19.73 s(-1), respectively. AJ cleaved the A alpha-chain of fibrinogen but did not affect the B beta- and gamma-chains, indicating that it is an alpha-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100 degrees C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.

Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel.

We have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.

A method for direct application of human plasmin on a dithiothreitol-containing agarose stacking gel system.

A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1 %) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea.

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye.

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4.

Previously, we purified a strong fibrinolytic enzyme (subtilisin DJ-4) from Bacillus sp. DJ-4 and characterized its enzymatic activity. Here, we cloned the gene subtilisin DJ-4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN' from B. amyloliquefacens. Recombinant full-subtilisin DJ-4 (rf-subDJ-4) and mature-subtilisin DJ-4 (rm-subDJ-4) were expressed using a pET29 vector system, and their fibrin (ogen)olytic and plasminogen activator activities were studied. rf-subDJ-4 was found to have a higher stability to heat (60 degrees C) and to acidic conditions (pH 3.0-4.0) than the native subtilisin DJ-4 of Bacillus sp. DJ-4. The plasminogen activator activity of rf-subDJ-4 was 2.75 times greater than that of plasmin on a molar basis. And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively. rf-subDJ-4 rapidly hydrolyzed the Aalpha-, Bbeta-, and gamma-chains of fibrinogen within 5 min. But, unlike subtilisin BPN' at a very low concentration (50 ng), the gamma-chain was not cleaved. On the other hand, rm-subDJ-4 did not show enzyme activity.

Relationship between acrylamide concentration and enzymatic activity in an improved single fibrin zymogram gel system.

Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDSfibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10 kDa to over 100 kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.