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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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Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwith pH control at increased glucose concentration.L. plantarum WCFS1 harbouring the pEH9R plasmid was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 40 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by the addition of varying amounts of pheromone (ng/ml fermentation broth; see insert) at different OD600 values: immediately after inoculation (A), at OD600 of 0.3 (B), or at OD600 of 3.0 (C). All data points represent the average value from 2 independent experiments.
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Fig3: Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwith pH control at increased glucose concentration.L. plantarum WCFS1 harbouring the pEH9R plasmid was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 40 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by the addition of varying amounts of pheromone (ng/ml fermentation broth; see insert) at different OD600 values: immediately after inoculation (A), at OD600 of 0.3 (B), or at OD600 of 3.0 (C). All data points represent the average value from 2 independent experiments.

Mentions: Subsequently, we studied the effect of varying glucose concentrations on β-galactosidase production under pH-controlled conditions (pH 6.5). Figure 3 shows that an increase of the glucose concentration from 20 g/l to 40 g/l approximately doubled the maximum OD600 values, which now reached 15–18. Concomitantly, the recombinant enzyme production also increased approximately two-fold; β-galactosidase levels continuously increased during the cultivation to reach a maximum of about 35 U/ml when the stationary growth phase was reached. Maximum specific activities were only slightly higher than those obtained with 20 g/l glucose, indicating that the increased volumetric yields are primarily caused by the increased cell densities. Dose–response effects for the pheromone were tested in a limited range only (20–80 ng/ml) and were generally small, as observed in other experiments for this concentration range. Comparison of the experiments displayed in Figure 3 further shows that under these conditions it may be favourable to induce somewhat later during growth since this yielded slightly higher specific activities. Higher concentrations of glucose (80, 120 g/l) were also tested, and this did not lead to a significant increase in enzyme yield even though higher cell densities were obtained (data not shown).Figure 3


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. reuteriwith pH control at increased glucose concentration.L. plantarum WCFS1 harbouring the pEH9R plasmid was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 40 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by the addition of varying amounts of pheromone (ng/ml fermentation broth; see insert) at different OD600 values: immediately after inoculation (A), at OD600 of 0.3 (B), or at OD600 of 3.0 (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

Fig3: Time course of the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteriwith pH control at increased glucose concentration.L. plantarum WCFS1 harbouring the pEH9R plasmid was cultivated in 400-ml laboratory fermentors at 37°C using MRS medium with 40 g/l glucose and pH control at pH 6.5. Expression of β-galactosidase was induced by the addition of varying amounts of pheromone (ng/ml fermentation broth; see insert) at different OD600 values: immediately after inoculation (A), at OD600 of 0.3 (B), or at OD600 of 3.0 (C). All data points represent the average value from 2 independent experiments.
Mentions: Subsequently, we studied the effect of varying glucose concentrations on β-galactosidase production under pH-controlled conditions (pH 6.5). Figure 3 shows that an increase of the glucose concentration from 20 g/l to 40 g/l approximately doubled the maximum OD600 values, which now reached 15–18. Concomitantly, the recombinant enzyme production also increased approximately two-fold; β-galactosidase levels continuously increased during the cultivation to reach a maximum of about 35 U/ml when the stationary growth phase was reached. Maximum specific activities were only slightly higher than those obtained with 20 g/l glucose, indicating that the increased volumetric yields are primarily caused by the increased cell densities. Dose–response effects for the pheromone were tested in a limited range only (20–80 ng/ml) and were generally small, as observed in other experiments for this concentration range. Comparison of the experiments displayed in Figure 3 further shows that under these conditions it may be favourable to induce somewhat later during growth since this yielded slightly higher specific activities. Higher concentrations of glucose (80, 120 g/l) were also tested, and this did not lead to a significant increase in enzyme yield even though higher cell densities were obtained (data not shown).Figure 3

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