Biomedical subjects

Byong H Lee

Publications and source records attributed to Byong H Lee.

7 recordsLinked to original sources

PubMed2005

Expression of a cold-adapted fish trypsin in Pichia pastoris.

Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.

Animals↗

PubMed2005

Cloning and characterization of a bile salt hydrolase (bsh) from Bifidobacterium adolescentis.

A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P(bsh)) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.

Amidohydrolases↗

PubMed2004

Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96.

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S. tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.

Amino Acid Sequence↗

PubMed2004

Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.

Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.

Amidohydrolases↗

PubMed2003

Growth of Lactobacillus paracasei ssp. paracasei on tofu whey.

The liquid by-product of the soybean product tofu, tofu whey (TW), was used as a growth medium for the production of Lactobacillus paracasei ssp. paracasei LG3 cultures. The TW used in this study contained stachyose, raffinose, sucrose, fructose and glucose, but the strain used could only utilize the three latter. The lactobacilli population obtained in MRS broth was three times higher than that in TW alone, and supplementation of TW was thus examined. Of 19 mixtures of yeast extracts (YE), peptones and potato extracts examined, the best nitrogen sources were YE and tryptone. The addition of YE, salts (phosphates, citrates, Mg and Mn), glucose as well as Tween to TW tripled the populations to 2.9 x 10(9) cfu/ml, which was as high as that obtained in MRS broth. Growth of L. paracasei LG3 in cow rehydrated skim milk was inferior to that in TW.

Carbohydrate Metabolism↗

PubMed2003

Induction of Oenococcus oeni H+-ATPase activity and mRNA transcription under acidic conditions.

The profiles of Oenococcus oeni IOB84.13 H(+)-ATPase activity under various conditions of growth were studied. Cells growing at low pH 3.5 had a 1.6-fold higher H(+)-ATPase activity compared to control cells grown at pH 5.3. While the pH of the growth medium was shown to be stable in the presence of malic acid, a drastic decrease in pH from 5.3 down to 3.9 during growth in the absence of malic acid induced an increase in H(+)-ATPase activity by 1.5-fold. This induction was even greater when the initial pH was 3.5. Partial cloning of the genes encoding the beta-subunit and the epsilon-subunit of the H(+)-ATPase suggested a typical F(1)F(0)-ATPase genetic organization in O. oeni. The atp mRNA was detected by slot blots. Cells shocked at acidic pH were shown to contain higher levels of atp mRNA compared to the control cells grown at pH 5.3. Taken together, these results indicate that the H(+)-ATPase of O. oeni is induced at low pH and that regulation seems to occur at the level of transcription. This agrees with the role of this enzyme in the regulation of the cytoplasmic pH and in the acid tolerance of O. oeni.

Enzyme Activation↗

PubMed2002

Positive selection vectors.

This review describes information concerning positive selection vectors on their mechanism, classification, property, and limitation. A total of 72 positive selection vectors collected were discussed. Positive selection vectors can reduce background and directly screen transformants containing cloned DNA fragments. The mechanisms to perform positive selection include insertional inactivation and the replacement of functional genes of the vectors. In general, the former is much more convenient than the latter. The functional genes are controlled either by their promoters or by heterologous promoters introduced. On the basis of the structures, positive selection vectors could be classified into five groups. The positive selection vectors are commonly based on the mechanisms of lethal genes and the sensitivity of compounds. The vectors, with molecular weights ranging from 2.6 to 17.0 kb, have diverse genetic markers and wide host ranges, including Escherichia coli, Bacillus, Streptomyces, lactic acid bacteria, yeasts, and mammalian cells. Although some limitations exist for using some positive selection vectors, they are useful in recombinant DNA experiments.

Biotechnology↗