title: Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii.
abstract: The gene encoding the branching enzyme (BE) from the thermoalkaliphilic, anaerobic bacterium Anaerobranca gottschalkii was fused with a twin arginine translocation protein secretory-pathway-dependent signal sequence from Escherichia coli and expressed in Staphylococcus carnosus. The secreted BE was purified using hydrophobic interaction and gel filtration chromatography. The monomeric enzyme (72 kDa) shows maximal activity at 50 degrees C and pH 7.0. With amylose the BE displays high transglycosylation and extremely low hydrolytic activity. The conversion of amylose and linear dextrins was analysed by applying high-performance anion exchange chromatography and quantitative size-exclusion chromatography. Amylose (10(4)-4 x 10(7) g/mol) was converted to a major extent to products displaying molecular masses of 10(4)-4 x 10(5) g/mol, indicating that the enzyme could be applicable for the production of starch or dextrins with narrow molecular mass distributions. The majority of the transferred oligosaccharides, determined after enzymatic hydrolysis of the newly synthesized alpha-1,6 linkages, ranged between 10(3) and 10(4) g/mol, which corresponds to a degree of polymerisation (DP) of 6-60. The minimal donor chain length is DP 16. Furthermore, the obtained results support the hypotheses of a random endocleavage mechanism of BE and the occurrence of interchain branching.
authors.affiliation: Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Thiemann
authors.affiliation: Institute for Wood Chemistry and Chemical Technology of Wood, Federal Research Centre for Forestry and Forest Products, 21002, Hamburg, Germany.
authors.forename: Bodo
authors.initials: B
authors.lastname: Saake
authors.affiliation: Biotechnology I, Research Centre Jülich GmbH, 52425, Jülich, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Vollstedt
authors.affiliation: Microbial Discovery and Molecular Biotechnology, Novozymes, 2880, Bagsvaerd, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schäfer
authors.affiliation: Institute for Wood Chemistry and Chemical Technology of Wood, Federal Research Centre for Forestry and Forest Products, 21002, Hamburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Puls
authors.affiliation: Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany.
authors.forename: Costanzo
authors.initials: C
authors.lastname: Bertoldo
authors.affiliation: Biotechnology I, Research Centre Jülich GmbH, 52425, Jülich, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Freudl
authors.affiliation: Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany. antranikian@tuhh.de.
authors.forename: Garabed
authors.initials: G
authors.lastname: Antranikian
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000688:Amylose / Q000378:metabolism; D001419:Bacteria; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D003912:Dextrins / Q000378:metabolism; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D003912:Dextrins; D029968:Escherichia coli Proteins; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000688:Amylose; D015061:1,4-alpha-Glucan Branching Enzyme