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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

Variation of the plasmid copy number during the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.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, pH control at pH 6.5 and the cells were induced at OD ~ 3 with 80 ng/ml peptide pheromone. All data points represent the average value from 2 independent experiments.
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Fig4: Variation of the plasmid copy number during the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.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, pH control at pH 6.5 and the cells were induced at OD ~ 3 with 80 ng/ml peptide pheromone. All data points represent the average value from 2 independent experiments.

Mentions: Figure 4 shows more detailed cultivation data for an experiment run under optimal conditions. The data show that all glucose was consumed and that glucose depletion coincides with reaching maximum levels of β-galactosidase and lactic acid. To check whether the gene dose was constant during the cultivation, the plasmid copy number (PCN) was determined. The PCN was found to be at a constant level of ~4 throughout the whole exponential and stationary phase, with a slight dip in the late exponential phase.Figure 4


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)

Variation of the plasmid copy number during the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.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, pH control at pH 6.5 and the cells were induced at OD ~ 3 with 80 ng/ml peptide pheromone. 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

Fig4: Variation of the plasmid copy number during the cultivations ofL. plantarumoverexpressing β-galactosidase fromL. reuteri.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, pH control at pH 6.5 and the cells were induced at OD ~ 3 with 80 ng/ml peptide pheromone. All data points represent the average value from 2 independent experiments.
Mentions: Figure 4 shows more detailed cultivation data for an experiment run under optimal conditions. The data show that all glucose was consumed and that glucose depletion coincides with reaching maximum levels of β-galactosidase and lactic acid. To check whether the gene dose was constant during the cultivation, the plasmid copy number (PCN) was determined. The PCN was found to be at a constant level of ~4 throughout the whole exponential and stationary phase, with a slight dip in the late exponential phase.Figure 4

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