Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.
Flavobacterium ; High-Throughput Screening Assays ; Mirabilis ; Saccharomyces cerevisiae/genetics* ; Tyrosine

Various bacteria were isolated from the casing layer soil of the culture bed of P. ostreatus and their role in fruiting body induction of the edible mushroom, P. ostreatus, was investigated. Analysis of the bacterial community isolated from the casing layer soil revealed that the composition of genera and number of cultivable bacteria were different for each sterilizing treatment. Bordetella was predominant in the bulk soil whereas Flavobacterium was predominant after sterilization of the casing layer soil. Fluorescent Pseudomonas was predominant in the non-sterilized casing layer soil. Total number of the bacterial genera in the casing layer soil was higher than that in the bulk soil. In particular, an increase in the fluorescent Pseudomonas population was observed in the non-sterilized casing layer accompanied by induction of fruiting body and enhanced mushroom production yield. The results suggested that specific bacterial populations in the casing layer play an important role in the formation of primodia and the development of basidiome in P. ostreatus.
Agaricales ; Bacteria ; Bordetella ; Flavobacterium ; Fruit ; Pleurotus ; Pseudomonas ; Soil ; Sterilization

The efficacy of using a bacteriophage (phage) to control Flavobacterium psychrophilum (F. psychrophilum) infection of ayu (Plecoglossus altivelis altivelis) was evaluated in this study. Intramuscular challenge failed to induce sufficient infection levels; therefore, a newly designed net-scratch challenge method was also used to induce bacterial infection. Administration of phage PFpW-3 in F. psychrophilum-infected ayu showed notable protective effects, increased survival rates and mean times to death. Additionally, the fate of inoculated bacteria and phage in ayu were investigated. Our results suggest that the phage PFpW-3 could be considered an alternative biocontrol agent against F. psychrophilum infections in ayu culture.
Bacteria ; Bacterial Infections ; Bacteriophages ; Flavobacterium ; Methods ; Osmeriformes ; Survival Rate

Heparinase II (Hep II) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase II (His-Hep II) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 degrees C, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 degrees C for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep II increased to 9 436 U/L.
Fermentation ; Flavobacterium ; metabolism ; Microbiological Techniques ; Polysaccharide-Lyases ; biosynthesis ; Recombinant Proteins ; biosynthesis

PURPOSE: Flavobacterium indologenes is known to cause keratitis very rarely. Authors have experienced 1 case of keratitis from Flavobacterium indologenes with history of diabetes mellitus, thereby reporting it. METHODS: History taking, slit lamp examination, staining and culture, sensitivity test about antibiotics were performed on 1 case of keratitis. RESULTS: Flavobacterium indologenes was detected in staining and culture that was performed on the first visit. Piperacillin was used based on the sensitivity test about antibiotics. Improvement of corneal lesion and symptom was observed with the use of piperacillin. CONCLUSIONS: Flavobacterium indologenes can be considered as a casual pathogen in keratitis with condition susceptible to opportunistic infection such as systemic illness or abnormal ocular immunity.
Anti-Bacterial Agents ; Diabetes Complications ; Diabetes Mellitus ; Flavobacterium* ; Keratitis* ; Opportunistic Infections ; Piperacillin

BACKGROUND: Heparin released from grafted liver immediately after declamping is one of causes of coagulopathy, and its presence has been diagnosed by comparing thromboelastography(TEG) of blood treated with 0.01% of protamine and untreated blood. However, protamine may affect coagulation if the amount of protamine is not optimal to heparin in the blood sample. Heparinase, an enzyme isolated from Flavobacterium Heparinum, neutralizes heparin without adversely affecting coagulation. Therefore we compared the TEGs of blood treated with heparinase and protamine to clarify the sensitivity and reliability of heparinase in reversing the heparin effect. METHODS: Differences in Reaction time(R time), Alpha angle, Maximal Amplitude(MA) between native and heparinase treated TEG on reperfusion in 8 cases of orthotopic liver transplantations were compared with those between native and protamine in 14 cases of OLT. RESULTS: On reperfusion, all of TEGs treated with heparinase showed more improved data rather than native one in R time, Alpha angle and MA. But, in protamine treated blood, R time and Alpha angle in 6 patients and MA in 3 patients were more depressed. The scattergram show that TEGs treated with heparinase on reperfusion have almost positive difference, but TEGs treated with protamine did not have positive results consistently. CONCLUSIONS: Heparinase is a more reliable reagent and activator than protamine on TEG for detecting heparin effects on reperfusion without showing in-vitro anticoagulation. Those results suggest that heparinase on TEGs can make diagnosis of coagulopathy developed immediately after reperfusion efficiently.
Diagnosis ; Flavobacterium ; Heparin Lyase* ; Heparin* ; Humans ; Liver Transplantation* ; Liver* ; Reperfusion* ; Thrombelastography* ; Transplantation ; Transplants

BACKGROUND: Heparin released from grafted liver immediately after declamping is one of causes of coagulopathy, and its presence has been diagnosed by comparing thromboelastography(TEG) of blood treated with 0.01% of protamine and untreated blood. However, protamine may affect coagulation if the amount of protamine is not optimal to heparin in the blood sample. Heparinase, an enzyme isolated from Flavobacterium Heparinum, neutralizes heparin without adversely affecting coagulation. Therefore we compared the TEGs of blood treated with heparinase and protamine to clarify the sensitivity and reliability of heparinase in reversing the heparin effect. METHODS: Differences in Reaction time(R time), Alpha angle, Maximal Amplitude(MA) between native and heparinase treated TEG on reperfusion in 8 cases of orthotopic liver transplantations were compared with those between native and protamine in 14 cases of OLT. RESULTS: On reperfusion, all of TEGs treated with heparinase showed more improved data rather than native one in R time, Alpha angle and MA. But, in protamine treated blood, R time and Alpha angle in 6 patients and MA in 3 patients were more depressed. The scattergram show that TEGs treated with heparinase on reperfusion have almost positive difference, but TEGs treated with protamine did not have positive results consistently. CONCLUSIONS: Heparinase is a more reliable reagent and activator than protamine on TEG for detecting heparin effects on reperfusion without showing in-vitro anticoagulation. Those results suggest that heparinase on TEGs can make diagnosis of coagulopathy developed immediately after reperfusion efficiently.
Diagnosis ; Flavobacterium ; Heparin Lyase* ; Heparin* ; Humans ; Liver Transplantation* ; Liver* ; Reperfusion* ; Thrombelastography* ; Transplantation ; Transplants

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

OBJECTIVE: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. METHODS: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. RESULTS: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. CONCLUSION: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.
Animals ; Antifreeze Proteins* ; Apoptosis ; Deoxyuridine ; Dimethyl Sulfoxide ; DNA Nucleotidylexotransferase ; Eosine Yellowish-(YS) ; Ethylene Glycol ; Female ; Fertility Preservation ; Flavobacterium ; Hematoxylin ; Humans ; Mice* ; Microscopy ; Ovary* ; Vitrification

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; Glycosylation ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism