• ALGAE
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• v.18 no.1
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• pp.89-94
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• 2003

• ALGAE
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• v.18 no.1
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• pp.95-100
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• 2003

• BMB Reports
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• v.40 no.3
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• pp.333-340
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• 2007

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower $K_m$ for coupling reaction using cellobiose and cyclodextrins as substrates.

• Journal of Life Science
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• v.14 no.2
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• pp.362-367
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• 2004

For better understanding of the host infection mechanism of Vibrio, a Vibrio parahaemolyticus collagenase mutant was generated by insertional inactivation of a vppC gene encoding extracellular collagenase. A recombinant DNA containing vppC::nptII was cloned into a suicide plasmid pDMS197, resulted in pVCM03. The recombinant suicide plasmid pVCM03 contained in E. coli $\chi$7213 was transferred to a wild-type V. parahaemolyticus 04 through conjugation. The recombinant vppC::nptII DNA in pVCM03 was exchanged with wild-type allele by homologous recombination resulting vppC mutant, V. parahaemolyticus CM. The mutant was selected and screened on TCBS media containing 10% sucrose and kanamycin. The mutation by allele exchange was confirmed with the comparison of the size of DNAs amplified by PCR. V. parahaemolyticus CM showed at least 4-fold less collagen-degrading activity than those of wild-type, and the mutant exhibited less cytotoxicity than that of wild-type in MTT assay.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.789-793
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• 1999

The effect of acetic acid formation deficiency on recombinant E. coli fermentation was investigated using a mutant strain deficient in acetic acid formation. A mutant strain which does not grow under anaerobic conditions was isolated. The acetic acid production in this strain was negligible in aerobic batch fermentation. The cloned-gene expression in the mutant strain was higher than the wild-type strain. Fed-batch fermentations with controlled specific growth rates were carried out in order to compare the cloned-gene expression between the wild-type and the mutant strains. The expression decreased along with the specific growth rate in both strains. The cloned-gene expression in the mutant strain was 60% higher than in the wild-type strain at the same specific growth rate.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1066-1070
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• 2001

The PhyA gene, encoding myo-inositol hexakisphosphate phosphohydrolase in Aspergillus niger var. awamori (wild-type), was cloned and sequenced. The cDNA was overexpressed by a multicopy gene expression system in Pichia pastoris KM71. Recombinant, wild-type and commercial phytase from Aspergilus ficuum NRRL 3135 (Natuphos) were purified. The PhyA gene of Aspergillus niger var awamori showed perfect homology to the phytase of Aspergillus ficcum and $97\%$ homology to A. niger var awamori (L02421). Wild-type phytase was highly glycosylated and more thermostable than the other two, while deglycosylated farms of three phytases showed identical molecular weight, 507 kDa. After heating at $80^{\circ}C$, wild-type, commercial, and recombinant phytases retained $57\%, 32%,\;and\;8\%$ of their original activities, respectively. In conclusion, glycosylation plays a key role in the thermostability of phytase and its enzymatic characterization.

• Journal of Microbiology
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• v.38 no.1
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• pp.48-51
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• 2000

In liquid cultures using sucrose media, the coculture of Monascus with recombinant Saccharomyces cerevisiae expressing the glucoamylase gene from Aspergillus niger enhanced red pigment production by approx. 19％, compared with the coculture of wild type S. cerevisiae. Coculture with recombinant S. cerevisiae was more effective than with wild type S. cerevisiae for Monascus red pigment production. Cocultures of Monascus with commercial amylases of Aspergillus also induced high production of pigment and morphological changes in a solid culture using sucrose media.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• BMB Reports
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• v.31 no.5
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• pp.432-443
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• 1998

The poliovirus Sabin 1 strain has features that make it a particularly attractive live recombinant mucosal vaccine vehicle. Sabin 1 cDNA was manipulated to have multiple cloning sites and a viral specific 3C-protease cutting site at the N-terminal end of the polyprotein. The gene for the N-terminal 169 amino acids of the HIV-1 p24 was cloned into the multiple cloning site of the manipulated Sabin cDNA. A recombinant progeny virus was produced from HeLa cells when it was transfected with the RNA synthesized from the p24-Sabin chimeric cDNA. The recombinant progeny virus expresses substantial amounts of the HIV-1 p24 protein, which was clearly detected in the infected cell lysates and culture supernatants in Western blot experiments with rabbit anti-p24 serum and AIDS patients' sera. Differing from the Mahoney strain, the recombinant Sabin 1 poliovirus maintained the foreign gene stably during the subsequent passages. Replication capacity was about 1 to 1.5 log lower than that of the wild-type Sabin 1. Other physicochemical stability characteristics of the recombinant virus were similar to that of the wild-type Sabin 1. These results suggest that the manipulated Sabin 1 poliovirus can be used as a live viral vaccine vector for the development of mucosal vaccines.

• The Journal of Korean Society of Virology
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• v.29 no.3
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• pp.145-153
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• 1999

We have now constructed a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) CryIAc crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Surprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus particles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantria cunea, was strikingly improved in comparison with the wild-type AcNPV.