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Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

Bai Y, van der Kaaij RM, Woortman AJ, Jin Z, Dijkhuizen L - BMC Biotechnol. (2015)

Bottom Line: Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein.FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs.The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.

View Article: PubMed Central - PubMed

Affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. y.bai@rug.nl.

ABSTRACT

Background: The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme.

Methods: Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC.

Results: Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.

Conclusion: In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

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Related in: MedlinePlus

SEC chromatograms of AVEBE MD20 and its products following incubation with GTFB preparations. Different GTFB preparations with equal hydrolysis activity (25.0 μg/ml soluble GTFB, 38.7 μg/ml refolded GTFB and 249.4 μg/ml 25 °C ncIB GTFB) were incubated with 5 % AVEBE MD20 for 72 h at 37 °C and pH 4.7. Elution volumes of the pullulan standards corresponding to 366, 200, 113, 48.8, 21.7, 10, 6.2, 1.32 and 0.342 kDa, are plotted above the x-axis
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Fig4: SEC chromatograms of AVEBE MD20 and its products following incubation with GTFB preparations. Different GTFB preparations with equal hydrolysis activity (25.0 μg/ml soluble GTFB, 38.7 μg/ml refolded GTFB and 249.4 μg/ml 25 °C ncIB GTFB) were incubated with 5 % AVEBE MD20 for 72 h at 37 °C and pH 4.7. Elution volumes of the pullulan standards corresponding to 366, 200, 113, 48.8, 21.7, 10, 6.2, 1.32 and 0.342 kDa, are plotted above the x-axis

Mentions: The Size Exclusion Chromatograms (SEC) of AVEBE MD20 before and after incubation with soluble, refolded and ncIB GTFB proteins are shown in Fig. 4. The elution volume in SEC is directly related to the hydrodynamic volume of the linear and branched molecules [20]. The elution volumes of some pullulan standards are plotted at the x-axis. Since AVEBE MD20 is mainly composed of short oligosaccharides (average degree of polymerization 6), its major peak is seen at a high elution volume (around 32.5 ml). After incubation with GTFB preparations with equivalent hydrolysis activity, the peak at 32.5 ml decreased and shifted to a bi-modal peak at elution volumes of approximately 30 and 32 ml, demonstrating that the short oligosaccharides in MD20 substrate were converted to products with higher molecular weight. The distributions of the products of soluble, ncIB and refolded GTFB were highly similar. The relatively high ratios of hydrolysis versus transferase activity (Fig. 1b and c) of ncIB and refolded GTFB compared to soluble GTFB may initially result in enhanced synthesis of smaller products. This increased availability of shorter acceptor substrates resulted in reduced average sizes of their product molecules.Fig. 4


Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

Bai Y, van der Kaaij RM, Woortman AJ, Jin Z, Dijkhuizen L - BMC Biotechnol. (2015)

SEC chromatograms of AVEBE MD20 and its products following incubation with GTFB preparations. Different GTFB preparations with equal hydrolysis activity (25.0 μg/ml soluble GTFB, 38.7 μg/ml refolded GTFB and 249.4 μg/ml 25 °C ncIB GTFB) were incubated with 5 % AVEBE MD20 for 72 h at 37 °C and pH 4.7. Elution volumes of the pullulan standards corresponding to 366, 200, 113, 48.8, 21.7, 10, 6.2, 1.32 and 0.342 kDa, are plotted above the x-axis
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4459449&req=5

Fig4: SEC chromatograms of AVEBE MD20 and its products following incubation with GTFB preparations. Different GTFB preparations with equal hydrolysis activity (25.0 μg/ml soluble GTFB, 38.7 μg/ml refolded GTFB and 249.4 μg/ml 25 °C ncIB GTFB) were incubated with 5 % AVEBE MD20 for 72 h at 37 °C and pH 4.7. Elution volumes of the pullulan standards corresponding to 366, 200, 113, 48.8, 21.7, 10, 6.2, 1.32 and 0.342 kDa, are plotted above the x-axis
Mentions: The Size Exclusion Chromatograms (SEC) of AVEBE MD20 before and after incubation with soluble, refolded and ncIB GTFB proteins are shown in Fig. 4. The elution volume in SEC is directly related to the hydrodynamic volume of the linear and branched molecules [20]. The elution volumes of some pullulan standards are plotted at the x-axis. Since AVEBE MD20 is mainly composed of short oligosaccharides (average degree of polymerization 6), its major peak is seen at a high elution volume (around 32.5 ml). After incubation with GTFB preparations with equivalent hydrolysis activity, the peak at 32.5 ml decreased and shifted to a bi-modal peak at elution volumes of approximately 30 and 32 ml, demonstrating that the short oligosaccharides in MD20 substrate were converted to products with higher molecular weight. The distributions of the products of soluble, ncIB and refolded GTFB were highly similar. The relatively high ratios of hydrolysis versus transferase activity (Fig. 1b and c) of ncIB and refolded GTFB compared to soluble GTFB may initially result in enhanced synthesis of smaller products. This increased availability of shorter acceptor substrates resulted in reduced average sizes of their product molecules.Fig. 4

Bottom Line: Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein.FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs.The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.

View Article: PubMed Central - PubMed

Affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. y.bai@rug.nl.

ABSTRACT

Background: The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme.

Methods: Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC.

Results: Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.

Conclusion: In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Show MeSH
Related in: MedlinePlus