• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Microbiology and Biotechnology Letters
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• v.17 no.1
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• pp.1-7
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• 1989

To enhance the capability of starch fermentation of the transformant TSD-14, the heat treated protoplasts of TSD-14 were fused with the protoplasts of C. tropicalis (lys$^-$) in the presence of 30% (w/ v) PEG and 20 mM CaC1$_2$. Fusants were selected by nutritional complementation on minium medium and the fusion frequency was 4.4$\times$10$^{-5}$. All fusants tested were possessed of complemented traits concerning carbon compound assimilation, and the cell volumes of the fusants were approximately 1.5 times larger than the parental strains. The fusants were genetically very stable, and were able to hydrolyze alpha 1,4-glucosidic linkage as well as alpha 1,6-linkage of starch contrary to one of parents TSD-14, The most promising fusant FSC-14-75 produced 8.7% (v/v) of ethanol from 15% liquefied potato starch medium, but the result was enhanced to 9.3% (v/v) by addition of 0.3% peptone. The corresponding fermentation efficiency was 86.0%.

• Molecules and Cells
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• v.28 no.4
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• pp.369-373
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• 2009

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and ${\beta}$-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an ${\alpha}$-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and ${\beta}$-glucosidase was able to produce ethanol from ${\beta}$-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.

• KSBB Journal
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• v.16 no.1
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• pp.1-10
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• 2001

Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.

• YAKHAK HOEJI
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• v.38 no.6
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• pp.696-702
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• 1994

An interspecific fusant strain, MS1, was obtained by protoplast fusion between S. peucetius subsp. caesius and S. platensis. We studied on the microbiological and cultural characteristics of the fusant MS1. In liquid culture, the viscosity of culture broth of S. peucetius increased during incubation However the fusant MS1 formed pellet like S. platensis without viscosity change. On agar medium the colony morphology of MSI resembled S. platensis but the color was similar to S. peucetius. The fermentation products of the fusant MS1 was identical with S. peucetius.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.365-369
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• 1986

The activity of glucoamylase and pullulanase, properties of glucoamylase and ethanol productivities of fusants were studied. Glucoamylase and pullulanase activity of fusants were higher than parents. The optimal pH and temperature of glucoamylase of fusants were very similar to the those produced by S. diastaticus. In alcohol fermentation. fermenting ability and fermentation rate of fusants were higher and faster than either of its parental strain. The maximum of alcohol yield in 15% of liquefied potato starch was 7.8% (v/v)

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.274-280
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• 1991

Effects of IPTG induction on cell growth and production of ${\beta}$-galactosidase-preS2 fusion protein (${\beta}$gal-preS2) were studied in a defined medium using a recombinant Escherichia coli JM109/pCMHB30. IPTG was added (0.2 mM) to induce the cloned-gene expression in the early-, mid-, and late-log growth phases. The most serious decreases in growth rate and plasmid stability were observed for the induction in the early-log growth phase. The expression level of ${\beta}$gal-preS2 attained by the induction in the mid-log phase was about 0.51 mg fusion protein/mg total cellular protein, which was 2- and 5-fold improvement over the levels obtained with the inductions in the early- and late-log phases. Formation of acidic byproducts including acetate and pyruvate showed different profiles during the fermentation period for each cases of induction; pyruvate was the major byproduct for the induction in the early-log phase while acetate production became more significant for the cases of inductions in the mid- and late-log phases.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.359-363
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• 1986

S. diastaticus hydrolysised $\alpha$-1.4 linkage of the starch and was known fermenting yeast strain, but poorly hydrolized $\alpha$-1.6 linkage of the starch. To improve the starch fermentation ability of yeast, we tried that protoplast fusion between S. diastaticus and C. tropicalis and finally two starins of fusant (FPDC42, FPDC43) were obtained. C. tropicalis well hydrolysis both $\alpha$-1.4 and $\alpha$-1.6 linkanges in the starch. The protoplast of parental auxotrophic cells were fused in the presence of 10mM CaCl$_2$ and 35% of polyethylene glycol (M. W. 4,000). The fusion frequency was 10$^{-5}$ to 10$^{-6}$. Properties of the fusants(genetic stability, assimilation of carbon sources, random spore formation, copper resistance, NaCl tolerance, DNA content, cell size and growth rate) were investigated.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.174-179
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• 1991

Protoplast fusion was carried out between Brevibacterium flavum and Corynebacterium glutamicum. For the protoplast fusion, various mutants were isolated from Brevibacterium flavum ATCC 21493 and Corynebacteriurn glutamicum ATCC 21831. The optimum conditions for protoplast fusion of these mutants were examined. In the present work, the authors obtained a fusant, MWF 9031, by the intergeneric protoplast fusion between Brevibacterium flavum 108-125 and Corynebacterium glutamicum 41-214A, which was excellent in L-arginine fermentation. Fusant MWF 9031 was found to accumulate a large amount of L-arginine reached 32.5 mg/ml with a medium containing 10% glucose. The fusant possessed intermediate characteristics between the parental strains and the stability was found to retain for 60 days.

• Korean Journal of Microbiology
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• v.19 no.4
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• pp.186-191
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• 1981

Protoplast production from Trichoderma koningii ATCC 26113 was investigated for the purpose of doing basic and applied researches by protoplast fusion of the cellulolytic filamentous fungus. High yields of protoplast were obtained by using the 18hr. old mycelia treated with the lytic enzyme Driselase of Kyowa Fermentation Co., Japan, in 0.6M $MgSO_4\;or\;(NH_4)_2SO_4$ as osmotic stabilizers. The optimum temeprature of mycelial digestion was about $28^{\circ}C$ and the number of protoplast increased rapidly after 3hr. digestion. Protoplasts produced at different periods showed distinct morphological heterogeneities in the whole size and the size of vacuole.