Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii.
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.
Thiemann
Volker
V
Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany.
Saake
Bodo
B
Institute for Wood Chemistry and Chemical Technology of Wood, Federal Research Centre for Forestry and Forest Products, 21002, Hamburg, Germany.
Vollstedt
Angela
A
Biotechnology I, Research Centre Jülich GmbH, 52425, Jülich, Germany.
Schäfer
Thomas
T
Microbial Discovery and Molecular Biotechnology, Novozymes, 2880, Bagsvaerd, Denmark.
Puls
Jürgen
J
Institute for Wood Chemistry and Chemical Technology of Wood, Federal Research Centre for Forestry and Forest Products, 21002, Hamburg, Germany.
Bertoldo
Costanzo
C
Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany.
Freudl
Roland
R
Biotechnology I, Research Centre Jülich GmbH, 52425, Jülich, Germany.
Antranikian
Garabed
G
Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany. antranikian@tuhh.de.
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
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