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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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Effect of pH control on the growth (A) and enzyme production (B) ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by adding 20 ng/ml pheromone at different OD600: immediately after inoculation, at OD600 of 0.3 or at OD600 of 3.0. All data points represent the average value from 2 independent experiments.
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Fig2: Effect of pH control on the growth (A) and enzyme production (B) ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by adding 20 ng/ml pheromone at different OD600: immediately after inoculation, at OD600 of 0.3 or at OD600 of 3.0. All data points represent the average value from 2 independent experiments.

Mentions: In order to study the effect of the pH value on recombinant protein production when using the pSIP system, a series of cultivations was carried out where the pH was maintained at 6.5 by adding sodium hydroxide. Induction was performed using a non-saturating pheromone concentration of 20 ng/ml. The results, depicted in Figure 2A, B, show that culture pH had a strong positive effect on both growth and protein expression, and that the time of induction (immediately after inoculation, at OD600 of 0.3, or at OD600 of 3.0) hardly affected the outcome of the cultivations. OD600 values around 7 were reached after 10 hours of cultivation regardless of the induction time (Figure 2A) as compared to an OD600 of 4.5-5.0 obtained for growth without pH control (Figure 1). Accordingly, recombinant protein production was improved: β-galactosidase levels increased until the cells reached the early stationary phase to yield final volumetric activities of 15–19 U/ml, which is a 2.5–3 fold increase compared to the cultivations without pH control. Interestingly, specific β-galactosidase activities also increased about two-fold, reaching values of around 90–100 U/mg. This indicates that the improved performance of pH-controlled cultivations is not just a matter of increased cell densities.Figure 2


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)

Effect of pH control on the growth (A) and enzyme production (B) ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by adding 20 ng/ml pheromone at different OD600: immediately after inoculation, at OD600 of 0.3 or at OD600 of 3.0. All data points represent the average value from 2 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Effect of pH control on the growth (A) and enzyme production (B) ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.L. plantarum WCFS1 harbouring pEH9R was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 20 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by adding 20 ng/ml pheromone at different OD600: immediately after inoculation, at OD600 of 0.3 or at OD600 of 3.0. All data points represent the average value from 2 independent experiments.
Mentions: In order to study the effect of the pH value on recombinant protein production when using the pSIP system, a series of cultivations was carried out where the pH was maintained at 6.5 by adding sodium hydroxide. Induction was performed using a non-saturating pheromone concentration of 20 ng/ml. The results, depicted in Figure 2A, B, show that culture pH had a strong positive effect on both growth and protein expression, and that the time of induction (immediately after inoculation, at OD600 of 0.3, or at OD600 of 3.0) hardly affected the outcome of the cultivations. OD600 values around 7 were reached after 10 hours of cultivation regardless of the induction time (Figure 2A) as compared to an OD600 of 4.5-5.0 obtained for growth without pH control (Figure 1). Accordingly, recombinant protein production was improved: β-galactosidase levels increased until the cells reached the early stationary phase to yield final volumetric activities of 15–19 U/ml, which is a 2.5–3 fold increase compared to the cultivations without pH control. Interestingly, specific β-galactosidase activities also increased about two-fold, reaching values of around 90–100 U/mg. This indicates that the improved performance of pH-controlled cultivations is not just a matter of increased cell densities.Figure 2

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