Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii

Autor: Angela Vollstedt, Volker Thiemann, Thomas Schäfer, Jürgen Puls, Costanzo Bertoldo, Bodo Saake, Roland Freudl, Garabed Antranikian
Rok vydání: 2006
Předmět:
DNA
Bacterial
Starch
Staphylococcus
Molecular Sequence Data
Size-exclusion chromatography
Oligosaccharides
Protein Sorting Signals
Gram-Positive Bacteria
medicine.disease_cause
Applied Microbiology and Biotechnology
Bacteria
Anaerobic
chemistry.chemical_compound
Bacterial Proteins
1
4-alpha-Glucan Branching Enzyme
Enzymatic hydrolysis
Dextrins
Enzyme Stability
medicine
Glycogen branching enzyme
Amino Acid Sequence
Cloning
Molecular
Escherichia coli
Staphylococcus carnosus
Bacteria
Sequence Homology
Amino Acid
biology
Molecular mass
Escherichia coli Proteins
Temperature
Sequence Analysis
DNA
General Medicine
Hydrogen-Ion Concentration
biology.organism_classification
Recombinant Proteins
Molecular Weight
Biochemistry
chemistry
biology.protein
Amylose
Heterologous expression
Chromatography
Liquid
Biotechnology
Zdroj: Applied Microbiology and Biotechnology. 72:60-71
ISSN: 1432-0614
0175-7598
Popis: 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.
Databáze: OpenAIRE
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