Yeon-Hee Kim

Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated fromKimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genusLactobacillus brevis. Under controlled conditions in MRS broth (Difco) with 5% monosodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (gad) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of thegad gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD fromL. brevis showed 97.5 and 82.7% identities to theL. brevis OPK-3 GAD andL. plantarum WCFS1 GAD, respectively. Thegad gene was expressed inEscherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies.

Construction of an expression system for the secretory production of recombinant α-agarase in yeast

Abstractα-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than β-agarase. AgaA gene (2.3xa0kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA (ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6xa0unit/ml, the highest being in the SEY2102/pYInu-AgaA transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant production of α-agarase in yeast for industrial use.

Gene expression and characterization of thermostable glutamate decarboxylase from Pyrococcus furiosus

Gene encoding for a putative glutamate decarboxylase (GAD: EC 4.1.1.15) from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (PF1159) from P. furiosus showed some identity with other group II decarboxylases from an archaea and bacteria. The GAD from P. furiosus (PfGAD) was expressed in Escherichia coli, and the recombinant protein has a molecular mass of 41 kDa, determined by SDS-PAGE. The optimum temperature and pH for GAD activity were 75°C and 6.0, respectively. The half-life of heat inactivation was approximately 60 min at 90°C. The GAD activity was found to be dependent on various salts, such as CaCl2, NaCl, KCl, and NaBr, with an optimum concentration of 400 mM, but not (NH4)2SO4. PfGAD demonstrated activity against various substrates, such as l-glutamate, l-aspartate, and l-tyrosine. The results of the kinetics experiment indicated that l-aspartate was a better substrate of PfGAD than l-glutamate and Ltyrosine.

Development of Simultaneous YAC Manipulation-Amplification (SYMA) system by Chromosome Splitting Technique Harboring Copy Number Amplification System

Artificial chromosome manipulation and amplification of single-copy yeast artificial chromosome (YAC) are usually required in order to use YACs for applications such as physical mapping and functional analysis in eukaryotes. We designed and implemented a Simultaneous YAC Manipulation- Amplification (SYMA) system that combines the copy number amplification system of YAC with a convenient YAC manipulation system. To achieve the desired split and to amplify a YAC clone-harboring plant chromosome, a pBGTK plasmid containing a conditional centromere and thymidine kinase (TK) gene was constructed as a template to amplify the splitting fragment via PCR. By splitting, new 490-kb and 100-kb split YACs containing the elements for copy number amplification were simultaneously generated from a 590-kb YAC clone. The 100-kb split YAC was then successfully amplified 14.4-fold by adding 3 ㎎/㎖ sulfanilamide and 50 ㎍/㎖ methotrexate (S3/M50) as inducing substances.