title: Comparative analysis of heterologous expression, biochemical characterization optimal production of an alkaline α-amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris.
abstract: An alkaline α-amylase gene from alkaliphilic Alkalimonas amylolytica was synthesized based on the preferred codon usage of Escherichia coli and Pichia pastoris, respectively, and then was expressed in the according heterologous host, E. coli BL21 (DE3) and P. pastoris GS115. The alkaline α-amylase expressed in E. coli was designated AmyA, whereas that produced by P. pastoris was designated AmyB. The specific activity of AmyA and AmyB was 16.0 and 16.6 U/mg at pH 9.5 and 50°C, respectively. The optimal pH and pH stability of AmyA and AmyB were similar, whereas the optimum temperature and thermal stability of AmyB were slightly enhanced compared with those of AmyA. The AmyA and AmyB had a similar melting temperature of 64°C and the same catalytic efficiency (k(cat) /K(m) ) of 2.0 × 10(6) L/(mol min). AmyA and AmyB were slightly activated by 1 mM Co(2+) , Ca(2+) , or Na(+) , but inhibited by all other metal ions (K(+) , Mg(2+) , Fe(3+) , Fe(2+) , Zn(2+) , Mn(2+) , and Cu(2+) ). Tween 80 or Tween 60 (10% (w/v)) had little influence on the stability of AmyA and AmyB, while the 10% (w/v) sodium dodecyl sulfate caused the complete loss of AmyA and AmyB activities. The AmyA and AmyB were stable in the presence of solid detergents (washing powder), while were less stable in liquid detergents. Under the optimal conditions in 3-L bioreactor, the extracellular AmyB activity reached 600 U/mL, which was about 10 times as that of AmyA. These results indicated that P. pastoris was a preferable host for alkaline α-amylase expression and the produced alkaline α-amylase had a certain application potential in solid detergents.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Haiquan
authors.initials: H
authors.lastname: Yang
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Chen
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D013696:Temperature; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D000516:alpha-Amylases