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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

Fourier Transform Infrared (FT-IR) spectra in the amide I region of different GTFB preparations. Soluble GTFB, refolded GTFB, and ncIB GTFB proteins isolated from E. coli incubated at different temperatures (25 and 30 °C) were analyzed. (a) FT-IR spectra; (b) second derivatives of the FT-IR spectra
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Fig5: Fourier Transform Infrared (FT-IR) spectra in the amide I region of different GTFB preparations. Soluble GTFB, refolded GTFB, and ncIB GTFB proteins isolated from E. coli incubated at different temperatures (25 and 30 °C) were analyzed. (a) FT-IR spectra; (b) second derivatives of the FT-IR spectra

Mentions: The secondary structures of the soluble, refolded and ncIB GTFB (25 °C) proteins were examined by infrared spectroscopy (Fig. 5). The frequency and the shape of the amide I bands in the spectral region between 1690 and 1620 cm−1 provides information about the type of secondary structure present in proteins [21]. Because the components in amide I, resulting from different secondary structure elements, are strongly overlapping (Fig. 5a), a second derivative analysis was applied. The main band, at 1653 cm−1 in second derivative spectra (Fig. 5b) indicates the presence of α-helical structures, which may be expected for a GH70 family enzyme containing a TIM barrel fold with 8 α-helices [22]. The ncIBs GTFB show distinctive bands at 1627 and 1695 cm−1 (Fig. 5b) with higher intensity than those of soluble and refolded GTFB, indicating that the inclusion bodies have more intermolecular β-sheet structure [23]. The FI-IR spectra of the inclusion bodies also show peaks at 1633 and 1653 cm−1, suggesting they also contain some native β-sheet and native α-helix structures of the soluble GTFB enzyme, respectively [24].Fig. 5


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)

Fourier Transform Infrared (FT-IR) spectra in the amide I region of different GTFB preparations. Soluble GTFB, refolded GTFB, and ncIB GTFB proteins isolated from E. coli incubated at different temperatures (25 and 30 °C) were analyzed. (a) FT-IR spectra; (b) second derivatives of the FT-IR spectra
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Fourier Transform Infrared (FT-IR) spectra in the amide I region of different GTFB preparations. Soluble GTFB, refolded GTFB, and ncIB GTFB proteins isolated from E. coli incubated at different temperatures (25 and 30 °C) were analyzed. (a) FT-IR spectra; (b) second derivatives of the FT-IR spectra
Mentions: The secondary structures of the soluble, refolded and ncIB GTFB (25 °C) proteins were examined by infrared spectroscopy (Fig. 5). The frequency and the shape of the amide I bands in the spectral region between 1690 and 1620 cm−1 provides information about the type of secondary structure present in proteins [21]. Because the components in amide I, resulting from different secondary structure elements, are strongly overlapping (Fig. 5a), a second derivative analysis was applied. The main band, at 1653 cm−1 in second derivative spectra (Fig. 5b) indicates the presence of α-helical structures, which may be expected for a GH70 family enzyme containing a TIM barrel fold with 8 α-helices [22]. The ncIBs GTFB show distinctive bands at 1627 and 1695 cm−1 (Fig. 5b) with higher intensity than those of soluble and refolded GTFB, indicating that the inclusion bodies have more intermolecular β-sheet structure [23]. The FI-IR spectra of the inclusion bodies also show peaks at 1633 and 1653 cm−1, suggesting they also contain some native β-sheet and native α-helix structures of the soluble GTFB enzyme, respectively [24].Fig. 5

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