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Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase.

ABSTRACT: Inulosucrase is an enzyme that synthesizes inulin-type ?-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced the stability and activity of LrInu, but also modulated the pattern of the IFOS produced. Circular dichroism spectroscopy and molecular dynamics simulation indicated that these mutations had limited impact on the overall conformation of the enzyme. One of Ca-I residues most critical for controlling LrInu-mediated polymerization of IFOS, Asp418, was also subjected to mutagenesis, generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity of these mutants demonstrated that the IFOS chain length could be controlled by a single mutation at the Ca-I site.

SUBMITTER: Charoenwongpaiboon T

PROVIDER: S-EPMC7643167 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase.

Charoenwongpaiboon Thanapon T Punnatin Panachai P Klaewkla Methus M Pramoj Na Ayutthaya Pratchaya P Wangpaiboon Karan K Chunsrivirot Surasak S Field Robert A RA Pichyangkura Rath R

ACS omega 20201020 43

Inulosucrase is an enzyme that synthesizes inulin-type β-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced ...[more]

PMID: 33163783

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Project description:BackgroundThe 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.MethodsDenaturation 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.ResultsExpression 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.ConclusionIn 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.

Dataset Information

Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase.

Publications

Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by <i>Lactobacillus reuteri</i> 121 Inulosucrase.

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