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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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Related in: MedlinePlus

Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwithout pH control.L. plantarum WCFS1 harbouring the pEH9R plasmid was grown in 50-ml cultures using MRS medium with 20 g/l glucose, at 37°C. Recombinant protein expression was induced by the addition of varying amounts of the inducing pheromone IP (ng/ml fermentation broth; see inset) at different phases of the cultivation, i.e., different OD600 values: immediately after inoculation of the culture (A), at OD600 of 0.4-0.5 (B), or at OD600 of 1.5 (C). All data points represent the average value from 2 independent experiments.
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Fig1: Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwithout pH control.L. plantarum WCFS1 harbouring the pEH9R plasmid was grown in 50-ml cultures using MRS medium with 20 g/l glucose, at 37°C. Recombinant protein expression was induced by the addition of varying amounts of the inducing pheromone IP (ng/ml fermentation broth; see inset) at different phases of the cultivation, i.e., different OD600 values: immediately after inoculation of the culture (A), at OD600 of 0.4-0.5 (B), or at OD600 of 1.5 (C). All data points represent the average value from 2 independent experiments.

Mentions: Cultivations were performed without pH control at 37°C using MRS medium containing 20 g/l glucose. Despite the varying induction conditions, growth of the organism was in all cases very similar and reached an OD600 of ~4.5-5.0 after 12 h of cultivation (Figure 1). The volumetric activities of β-galactosidase (U per ml of fermentation broth) in induced cultures varied between 2 U/ml and 8 U/ml, and the specific activities ranged from about 20 U/mg to 50 U/mg, depending on the conditions employed. These production levels were generally reached at OD600 2.0–3.0, regardless of the time of induction (immediately after inoculation, at OD600 of 0.4–0.5, or at OD600 of 1.5; Figure 1). The results show clear dose–response effects for the pheromone concentration, which level off at about 40 ng/ml. Maximum β-galactosidase levels were quite similar for cultures induced immediately after inoculation (Figure 1A) or at an OD600 of 0.4–0.5 (Figure 1B), but volumetric activities were clearly lower (2–4 U/ml rather than 4 – 8 U/ml) for cultures induced at OD600 of 1.5 (Figure 1C). These data also indicate that more pheromone is needed when induction takes place at a later growth phase. For example, induction with 20 ng/ml at OD600 of 0.4–0.5 maximally yielded 6 U/ml and 44 U/mg, whereas induction with 20 ng/ml at OD600 of 1.5 maximally yielded 2.6 U/ml and 34 U/mg. In the non-induced cultures very low enzyme activity was measured with approximately 0.2 U/ml of fermentation broth or 1.3 U/mg protein (Figure 1A). The average pH value of the fermentation media dropped from 6.5 to approximately 5.2 or 4.3 after 7 h (OD600 ~ 1.8-2.1) or 12 h (OD600 ~ 4.5-5.0) of growth, respectively.Figure 1


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)

Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwithout pH control.L. plantarum WCFS1 harbouring the pEH9R plasmid was grown in 50-ml cultures using MRS medium with 20 g/l glucose, at 37°C. Recombinant protein expression was induced by the addition of varying amounts of the inducing pheromone IP (ng/ml fermentation broth; see inset) at different phases of the cultivation, i.e., different OD600 values: immediately after inoculation of the culture (A), at OD600 of 0.4-0.5 (B), or at OD600 of 1.5 (C). 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

Fig1: Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwithout pH control.L. plantarum WCFS1 harbouring the pEH9R plasmid was grown in 50-ml cultures using MRS medium with 20 g/l glucose, at 37°C. Recombinant protein expression was induced by the addition of varying amounts of the inducing pheromone IP (ng/ml fermentation broth; see inset) at different phases of the cultivation, i.e., different OD600 values: immediately after inoculation of the culture (A), at OD600 of 0.4-0.5 (B), or at OD600 of 1.5 (C). All data points represent the average value from 2 independent experiments.
Mentions: Cultivations were performed without pH control at 37°C using MRS medium containing 20 g/l glucose. Despite the varying induction conditions, growth of the organism was in all cases very similar and reached an OD600 of ~4.5-5.0 after 12 h of cultivation (Figure 1). The volumetric activities of β-galactosidase (U per ml of fermentation broth) in induced cultures varied between 2 U/ml and 8 U/ml, and the specific activities ranged from about 20 U/mg to 50 U/mg, depending on the conditions employed. These production levels were generally reached at OD600 2.0–3.0, regardless of the time of induction (immediately after inoculation, at OD600 of 0.4–0.5, or at OD600 of 1.5; Figure 1). The results show clear dose–response effects for the pheromone concentration, which level off at about 40 ng/ml. Maximum β-galactosidase levels were quite similar for cultures induced immediately after inoculation (Figure 1A) or at an OD600 of 0.4–0.5 (Figure 1B), but volumetric activities were clearly lower (2–4 U/ml rather than 4 – 8 U/ml) for cultures induced at OD600 of 1.5 (Figure 1C). These data also indicate that more pheromone is needed when induction takes place at a later growth phase. For example, induction with 20 ng/ml at OD600 of 0.4–0.5 maximally yielded 6 U/ml and 44 U/mg, whereas induction with 20 ng/ml at OD600 of 1.5 maximally yielded 2.6 U/ml and 34 U/mg. In the non-induced cultures very low enzyme activity was measured with approximately 0.2 U/ml of fermentation broth or 1.3 U/mg protein (Figure 1A). The average pH value of the fermentation media dropped from 6.5 to approximately 5.2 or 4.3 after 7 h (OD600 ~ 1.8-2.1) or 12 h (OD600 ~ 4.5-5.0) of growth, respectively.Figure 1

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