• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.585-591
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• 2001

To compare the p10 promoter activity of Bombyx mori nucleopolyhedrovirus (BmNPV)K1 and K4, recombinant viruses Bm101-LacZ and Bm104-LacZ with a lacZ gene under the control of each p10 promoter were constructed. The $\beta$-galactosidase activity due to Bm101-LacZ was about 5.5- and 1.1-fold higher than that due to Bm104-LacZ and BmK1-LacZ, respectively. expressing ${\beta}$-galactosidase under the control of a polyhedrin promoter. The recombinant virus BmK1-104LacZ with the same genome structure as Bm101-LacZ, except for a p10 promoter region, produced a similar ${\beta}$-galactosidase activity to that due to Bm104-LacZ and 5.5-fold lower than that due to Bm101-LacZ. The virus yield, expression level of polyhedrin, and polyhedra productivity for each recombinant virus was almost similar. These results suggested that the difference in the expression level of ${\beta}$-galactosidase resulted from a difference in the p10 promoter regions, and that an expression vector using the p10 promoter of BmNPV-K1 could be usefully exploited in the mass production of foreign proteins with silkworm larvae by means of oral ingestion.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.142-146
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• 2003

Three insect cell lines, Sf9, Sf21 and Tn5Bl-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was a ppropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5Bl-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3 times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammoniumion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line. respectively. The maximum specific ${\beta}$-galactosidase production rate was 4.5 fold that of the Sf9 cell line, a 3 times higher protease activity per cell.

• KSBB Journal
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• v.13 no.3
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• pp.325-330
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• 1998

The methylotrophic yeast, Pichia pastoris, is known to be a potential host to offer many advantages for production of recombinant proteins. Fermentation parameters were optimized to enhance the heterologous ${\beta}$-galactosidase productivity with P. pastoris. Optimum concentration of methanol, used as inducer, was observed to be 8 g/L and the extent of repression of AOX1 promoter by glycerol was lower than by glucose. The degradation of the gene product ${\beta}$-galactosidase by protease was inhibited as the pH increased from 5 to 8 and the yeast extract(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%). Induction method, in which methanol is just added to fermentation medium without centrifugation, was found to be as much effective as the one with centrifugation.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.550-558
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• 2021

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37℃ and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.353-356
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• 2005

The effect of silkworm hemolymph on the expression of recombinant protein in Escherichia coli was investigated. The addition of silkworm hemolymph to the culture medium in­creased the production of recombinant $\beta$-galactosidase in E. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1, 3, and $5\%$ silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.

• KSBB Journal
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• v.4 no.3
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• pp.266-270
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• 1989

Insect cell cultures were performed in laboratory-scale vessels. The batch growth of insect cells was affected by such parameters as serum content, other nutrients, seeding density, and mechanical agitation. Lactate and ammonium were not likely to be environmental factors that inhibited cell growth at the concentrations observed at the end of batch cultures. In addition, redox potential was found to be a useful index in monitoring low-level dissolved oxygen during the cultivation of insect cells. Recombinant protein production by cells infected with a genetically-modified baculovirus was also demons treated. The maximum beta-galactosidase synthesis of 2800 units per reactor volume was achieved at the dilution rate of $0.006hr^{-1}$.

• KSBB Journal
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• v.10 no.3
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• pp.317-322
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• 1995

Insect cells were grown and infected with baculovirus for the production of recombinant protein. Later infection gave the lower expression of recombinant protein. This indicates that the expression rate is lower at higher cell concentration. This phenomena provides a well-posed optimization problem with respect to the infection time. The optimal infection time was experimentally shown to exist for the maximum productivity of recombinant protein. Also, the expression increased with the addition of 5% silkworm hemolymph. This is considered to be due to the increase of intracellular viruses and the longevity of viable cells after the infection. The production of ${\beta}$-galaclosidase increased about ten-fold with the addition of yeastolate and silkworm hemolymph for high cell density and high expression, respectively.

• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1026-1031
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• 2006

The optimization of the production of a fusion protein containing the antigenic domain 1 (AD-1) is of a great importance, considering its use in diagnostic tests. The fusion protein is produced by the fermentation of a recombinant strain of Escherichia coli containing the plasmid Mbg58, which expresses the AD-1 (aa 484-650) of human cytomegalovirus glycoprotein B as a fusion protein together with aa 1-375 of ${\beta}-galactosidase$. An important characteristic of promoters (lac and derivatives) used in recombinant protein production in E. coli is their inducibility. Induction by IPTG is widely used for basic research; however, its use in large-scale production is undesirable because of its high cost and toxicity. In this work, studies using different inducers and carbon sources for the production of a fusion protein containing the AD-l were performed. The results showed that lactose could be used as an inducer in the fermentation process for the production of this protein, and that expression levels could exceed those achieved with IPTG. The use of lactose for protein expression in E. coli should be extremely useful for the inexpensive, large-scale production of heterologous proteins in E. coli. Addition of sucrose to the fermentation medium improved the yield of recombinant protein, whereas addition of fructose or trehalose decreased the yield.

• Microbiology and Biotechnology Letters
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• v.15 no.6
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• pp.396-401
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• 1987

Protein productivities of a cloned gene ($\beta$-galactosidase) and the cultural performances of two recombinant Escherichia coli strains, which use different host-vector systems, were studied. E. coli JM109/pTBG10 strain which carries Tac promoter had higher protein productivity than E. coli MH3000 (pRKc1857)/pASI(lacZ) strain which carries pL promoter. Induction of protein syn-thesis was optimum at the initial-and mid-logarithmic growth phases for both strains. Oxygen demand was observed to be very high during the cloned gene expression, and could be alleviated to some extent through pH control. The ratio of specific growth rates of plasmid-harboring to plasmidfree cell, $\mu$+ /$\mu$-, of the high productivity strain was observed to be lower than that of the low productivity one. Plasmid stability was analyzed for 20-30 generations, and it was found that the traction of plasmid-harboring cells dropped to l0% level in about 25 generations for both strains when the cloned gene expression was induced.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.4
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• pp.288-305
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• 2000

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects-the basics of yeast flocculation, the development of "new" flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer's yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous $\beta$-galactosidase production using a recombinant flocculent Saccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculating bioreactors and discussing potential new uses of these systems.e systems.