In-Soo Kong

Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermented fish

A Bacillus sp. producing a new fibrinolytic enzyme was screened from a fermented fish known as Jeot-Gal in Korea. The enzyme was purified to electrophoretic homogeneity using consecutive procedures including ammonium sulfate fractionation and column chromatography. The enzyme was highly specific toward fibrin clots and directly degraded them. The molecular weight was 41 kDa and the first 10 amino acids of the N-terminal sequence was Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn. Optimal fibrinolytic activity was observed at pH 7.0 and 40°C, and the specific activity was above 1.4 U/mg when determined with plasmin as a standard. The fibrinolytic activity was stimulated with zinc ions and repressed by various metalloprotease inhibitors, which indicates that the enzyme is a novel metalloprotease.

Purification, characterization and molecular cloning of Vibrio fluvialis hemolysin

Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.

Removal of ammonium-N from a recirculation aquacultural system using an immobilized nitrifier

Abstract Various immobilization methods were evaluated for the removal of ammonium-N from recirculating aquacultural water. Ba-alginate, Ca-alginate, carrageenan, and agar beads were prepared with nitrifer consortium from the activated sludge of a sewage treatment facility in Sooyoung, Pusan, South Korea. Batch bioreactor tests for the determination of the effectiveness of the immobilized nitrifier and the optimum hydraulic retention time (HRT) were carried out. The nitrifiers immobilized in Ba-alginate and Ca-alginate showed the most effective nitrification, while those immobilized in the carrageenan and agar beads showed reduced activities. Ninety five percent of the ammonia (20 mg/l) added to the batch bioreactor was nitrified in 6 h when immobilized Ba-alginate or Ca-alginate nitrifiers were used. In order to apply the immobilized nitrifier to an aquaculture facility, a continuous bioreactor was used with synthetic aquacultural water containing 2 mg/l ammonia. Using immobilized Ba-alginate and Ca-alginate beads, 94 and 87% of loaded ammonia were removed with in 3.4 h of HRT, respectively. The amounts of ammonia removal per day were in the range of 2.8–82 g ammonia/m3 per day depending on HRT. The highest ammonia removal rate of 82 g/m3 per day was observed when HRT was 0.3 h (18 min).

Identification of Vibrio anguillarum outer membrane vesicles related to immunostimulation in the Japanese flounder, Paralichthys olivaceus.

We identified outer membrane vesicle (OMV) production in Vibrio anguillarum O1, a major fish pathogen that causes vibriosis, and characterized the OMVs. They were produced during normal growth, and were appeared as spherical vesicle fractions. The protein profile of the OMVs was similar to that of the outer membrane proteins, and the 38-kDa major protein band of OMV was identified as OmpU. The OMVs had enzyme activity of metalloprotease, hemolysin, and phospholipase, and stimulated the production of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 when injected into the flounder.

A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures

Isochrysis galbana, one of the most widely usedmarine microalgae in the rearing of finfish and shellfish larvae, is masscultured frequently in outdoor tanks. Under prolonged and repeated culture,severe contamination occurs. Axenic isolation of I.galbanafrom such cultures was best achieved by using a ternary procedure involvingpercoll-gradient centrifugation, treatment with antibiotics, and growth on agarmedium. Protozoa and other algae were removed most effectively by isolation ofI. galbana at the 30–40% density layer on apercoll-gradient. Removal of bacteria was accomplished using a mixture of 5antibiotics (250 μg mL−1 ampicillin, 50μg mL−1 gentamycin, 100 μgmL−1 kanamycin, 500 μgmL−1 neomycin, 50 μgmL−1 streptomycin). Axenic colonies were isolated fromasolid medium prepared from 1% purified agar. The ternary procedure isconsideredapplicable to the isolation of other axenic single-celled microalgae fromheavily contaminated cultures.

Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus.

The vmc gene encoding a metalloprotease of Vibrio mimicus (ATCC 33653) was cloned in Escherichia coli and sequenced. The vmc gene contained 1884 nt sequence which codes a polypeptide of 628 amino acids with a predicted molecular mass of 71,275 Da. The deduced amino acid sequence had the similarity of 68.5% with V. parahaemolyticus metalloprotease. The consensus sequence of a zinc binding motif (HEXXH) was identified to be HEYTH. The zymography analysis showed a gelatinolytic protein band around molecular mass of 61 kDa, and this result suggested that the cloned metalloprotease may undergo processing during secretion.

Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio mimicus

The structural gene (vmhA) of hemolysin from Vibrio mimicus (ATCC33653) was cloned and sequenced. The vmhA gene contains an open reading frame consisting of 2232 nucleotides which can code for a protein of 744 amino acids with a predicted molecular mass of 83,059. The similarity of amino acid sequence shows 81.6% identity with Vibrio cholerae El Tor hemolysin.

Characterization of Vibrio mimicus phospholipase A (PhlA) and cytotoxicity on fish cell.

Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lec) of V. cholerae. The recombinant protein (rPhlA) produced from the phlA gene of V. mimicus was expressed in Escherichia coli as His-tag fused protein. The rPhlA was purified by gel filtration and Ni-metal affinity chromatographies. When the action mode was investigated by TLC and GC-MS, the purified rPhlA protein showed a phospholipase A activity, which cleaved the fatty acids at the sn-1 and sn-2 positions of phosphatidylcholine. However, it did not show lysophospholipase, sphingomyelinase, and phospholipase C activities. The rPhlA showed maximum activity at temperature of about 40 degrees C and pH around 8-9. Some divalent cations could affect the activity of PhlA. The addition of Co(2+) increased the activity, whereas Mg(2+) and Zn(2+) did not enhance the enzyme activity. The rPhlA could lyse the erythrocytes obtained from the fish such as rainbow trout and tilapia. A significant cytotoxic activity on a fish cell line, CHSE-214, was observed after 24h exposure to 40 microg rPhlA protein.

Identification of an Iron-Regulated Hemin-Binding Outer Membrane Protein, HupO, in Vibrio fluvialis: Effects on Hemolytic Activity and the Oxidative Stress Response

ABSTRACT In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.

Cloning and identification of a phospholipase gene from Vibrio mimicus

The phospholipase gene phl was identified from Vibrio mimicus (ATCC33653) and sequenced. The entire open reading frame (ORF) was composed of 1410 nucleotides and encoding 470 amino acids. The phl was placed upstream of hemolysin gene (vmhA) with opposite direction of transcription. From the BLAST search program, the deduced amino acids sequence showed 74.4% identity with phospholipase gene (lec) from V. cholerae El Tor. The entire ORF of phospholipase gene was amplified by PCR and inserted into an Escherichia coli expression vector, pET22b(+) and introduced E. coli BL21(DE3). SDS-PAGE demonstrated that a protein corresponding to the phospholipase was overexpressed and migrated at a molecular mass of 53 kDa.