Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

In order to evaluate the genetic variability of the causative agent of cold water disease (CWD), plasmid profiling was used to characterize Flavobacterium (F.) psychrophilum isolates (n = 169). Size analysis of plasmids in F. psychrophilum isolates (n = 128) from several fish species demonstrated that six kinds of plasmids were harbored, and ayu isolates had different profiles compared to other isolates. Moreover, multiple isolates (n = 41) from CWD outbreaks in 2002 to 2003 at a single ayu farm were examined to determine differences between isolates from successive outbreaks and showed different profiles by the sources of seedlings.
Animals ; DNA, Bacterial/genetics ; Disease Outbreaks/*veterinary ; Electrophoresis, Agar Gel/veterinary ; Fish Diseases/genetics/*microbiology ; Flavobacteriaceae Infections/microbiology/*veterinary ; Flavobacterium/genetics/*isolation & purification ; Genetic Variation/*genetics ; Japan ; *Osmeriformes ; Plasmids/genetics

A strain of Flavobacterium lindanitolerans isolated from a sick child's ascites was described. The 16S rRNA gene of the strain was 100% identical to that of Flavobacterium lindanitolerans which was first identified in India in 2008. It was first described that the isolate required X factor (Hemin) for growth in the optimal conditions of 37 °C with 5% CO(2). The isolate produced indole and H(2)S. It did not present hemolytic feature on blood agar.
Ascitic Fluid ; microbiology ; Child, Preschool ; Enterovirus A, Human ; isolation & purification ; Enterovirus Infections ; complications ; microbiology ; virology ; Fatal Outcome ; Flavobacteriaceae Infections ; complications ; microbiology ; virology ; Flavobacterium ; classification ; genetics ; isolation & purification ; Humans ; RNA, Ribosomal, 16S ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

No abstract available.
Asian Continental Ancestry Group ; Flavobacteriaceae Infections ; Flavobacterium/*genetics/isolation & purification ; Humans ; Liver Cirrhosis, Alcoholic/blood/*diagnosis/microbiology ; Male ; Middle Aged ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; Republic of Korea ; Sequence Analysis, DNA

No abstract available.
Aged ; Aneurysm/surgery ; Anti-Bacterial Agents/pharmacology ; Brain Diseases/surgery ; Craniotomy/adverse effects ; DNA, Bacterial/chemistry/genetics/metabolism ; Female ; Flavobacteriaceae Infections/etiology/microbiology ; Flavobacterium/classification/drug effects/*isolation & purification ; Humans ; Meningitis/*diagnosis/microbiology ; Microbial Sensitivity Tests ; Phylogeny ; Postoperative Complications ; Sequence Analysis, DNA