Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4.
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.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing 100101, China.
Yingying
Y
Zheng
Yanfen
Y
Xue
Yueling
Y
Zhang
Cheng
C
Zhou
Ulrich
U
Schwaneberg
Yanhe
Y
Ma
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase