Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4. 2010

Yingying Zheng, and Yanfen Xue, and Yueling Zhang, and Cheng Zhou, and Ulrich Schwaneberg, and Yanhe Ma
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing 100101, China.

A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 degrees C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 degrees C for 6 h. TtcGA can hydrolyze both alpha-1, 4- and alpha-1, 6-glycosidic linkages in various alpha-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca2+, Mn2+, Co2+, Mg2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn2+, Pb2+, Cu2+, and EDTA.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008968 Molecular Conformation The characteristic three-dimensional shape of a molecule. Molecular Configuration,3D Molecular Structure,Configuration, Molecular,Molecular Structure, Three Dimensional,Three Dimensional Molecular Structure,3D Molecular Structures,Configurations, Molecular,Conformation, Molecular,Conformations, Molecular,Molecular Configurations,Molecular Conformations,Molecular Structure, 3D,Molecular Structures, 3D,Structure, 3D Molecular,Structures, 3D Molecular
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D005087 Glucan 1,4-alpha-Glucosidase An enzyme that catalyzes the hydrolysis of terminal 1,4-linked alpha-D-glucose residues successively from non-reducing ends of polysaccharide chains with the release of beta-glucose. It is also able to hydrolyze 1,6-alpha-glucosidic bonds when the next bond in sequence is 1,4. 1,4-alpha-Glucosidase, Exo,Amyloglucosidase,Exo-1,4-alpha-Glucosidase,Glucoamylase,gamma-Amylase,Glucoamylase G1,Glucoamylase G2,1,4-alpha-Glucosidase, Glucan,Exo 1,4 alpha Glucosidase,Glucan 1,4 alpha Glucosidase,gamma Amylase
D006358 Hot Temperature Presence of warmth or heat or a temperature notably higher than an accustomed norm. Heat,Hot Temperatures,Temperature, Hot,Temperatures, Hot
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein

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