title: Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. y.bai@rug.nl.|The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. y.bai@rug.nl.
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. r.m.van.der.kaaij@rug.nl.
authors.forename: Rachel Maria
authors.initials: RM
authors.lastname: van der Kaaij
authors.affiliation: Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. a.j.j.woortman@rug.nl.
authors.forename: Albert Jan Jacob
authors.initials: AJ
authors.lastname: Woortman
authors.affiliation: The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. zjin@jiangnan.edu.cn.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. l.dijkhuizen@rug.nl.
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000737:chemistry / Q000201:enzymology*; D052200:Limosilactobacillus reuteri / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D011489:Protein Denaturation; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D006004:Glycogen Debranching Enzyme System; C020569:4 alpha-glucanotransferase