Biotechnologically Relevant Enzymes and Proteins

Applied Microbiology and Biotechnology

, Volume 87, Issue 1, pp 225-233

First online:

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

  • Yingying ZhengAffiliated withState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of SciencesThe Graduate School, Chinese Academy of Sciences
  • , Yanfen XueAffiliated withState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences
  • , Yueling ZhangAffiliated withTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences
  • , Cheng ZhouAffiliated withState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences
  • , Ulrich SchwanebergAffiliated withLehrstuhl für Biotechnologie, RWTH Aachen University Email author 
  • , Yanhe MaAffiliated withState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences Email author 

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Abstract

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 °C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 °C for 6 h. TtcGA can hydrolyze both α-1, 4- and α-1, 6-glycosidic linkages in various α-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.

Keywords

Glucoamylase Thermoanaerobacter tengcongensis Recombinant expression Thermostable