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Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

Nguyen TT, Nguyen HM, Geiger B, Mathiesen G, Eijsink VG, Peterbauer CK, Haltrich D, Nguyen TH - Microb. Cell Fact. (2015)

Bottom Line: Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium.As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism.The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

View Article: PubMed Central - PubMed

Affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria. nguyenthanh.ibft@gmail.com.

ABSTRACT

Background: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase.

Results: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase.

Conclusions: The over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

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SDS-PAGE analysis of cell free extract of crudeL. plantarumoverexpressing β-galactosidase fromL. reuterifrom the cultivations without pH control (A) and with pH control at pH 6.5 (B).L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose, and samples were taken at different time points. The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).
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Fig5: SDS-PAGE analysis of cell free extract of crudeL. plantarumoverexpressing β-galactosidase fromL. reuterifrom the cultivations without pH control (A) and with pH control at pH 6.5 (B).L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose, and samples were taken at different time points. The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).

Mentions: Maintaining the pH at a set value of 6.5 was clearly beneficial for β-galactosidase yields, both in terms of the volumetric and the specific β-galactosidase activities. This indicates that the decrease in pH during a non-controlled cultivation has a negative effect of the production of β-galactosidase. As expected the constant pH of 6.5 led to increased cell densities. However, this increase in biomass cannot solely explain the higher yields of recombinant protein, as indicated by the considerably higher specific activities that were obtained. One possible beneficial effect of the constant pH could be higher effectiveness of the induction process, as mentioned above. The difference in specific activities between pH controlled and non-controlled fermentations was further confirmed by SDS-PAGE analysis of cell free crude extract obtained from these cultivations (Figure 5), with the bands for the recombinant β-galactosidase being more prominent for the samples obtained with pH control.Figure 5


Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

Nguyen TT, Nguyen HM, Geiger B, Mathiesen G, Eijsink VG, Peterbauer CK, Haltrich D, Nguyen TH - Microb. Cell Fact. (2015)

SDS-PAGE analysis of cell free extract of crudeL. plantarumoverexpressing β-galactosidase fromL. reuterifrom the cultivations without pH control (A) and with pH control at pH 6.5 (B).L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose, and samples were taken at different time points. The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358714&req=5

Fig5: SDS-PAGE analysis of cell free extract of crudeL. plantarumoverexpressing β-galactosidase fromL. reuterifrom the cultivations without pH control (A) and with pH control at pH 6.5 (B).L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose, and samples were taken at different time points. The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).
Mentions: Maintaining the pH at a set value of 6.5 was clearly beneficial for β-galactosidase yields, both in terms of the volumetric and the specific β-galactosidase activities. This indicates that the decrease in pH during a non-controlled cultivation has a negative effect of the production of β-galactosidase. As expected the constant pH of 6.5 led to increased cell densities. However, this increase in biomass cannot solely explain the higher yields of recombinant protein, as indicated by the considerably higher specific activities that were obtained. One possible beneficial effect of the constant pH could be higher effectiveness of the induction process, as mentioned above. The difference in specific activities between pH controlled and non-controlled fermentations was further confirmed by SDS-PAGE analysis of cell free crude extract obtained from these cultivations (Figure 5), with the bands for the recombinant β-galactosidase being more prominent for the samples obtained with pH control.Figure 5

Bottom Line: Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium.As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism.The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

View Article: PubMed Central - PubMed

Affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria. nguyenthanh.ibft@gmail.com.

ABSTRACT

Background: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase.

Results: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase.

Conclusions: The over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Show MeSH
Related in: MedlinePlus