Improvement of the acid stability of Bacillus licheniformis alpha amylase by error-prone PCR.
OBJECTIVE
The purpose of this research was to obtain the mutant of Bacillus licheniformis alpha amylase (BLA) with an improved acid stability and elucidate the difference in catalytic mechanism under acidic conditions between wild-type and mutant BLAs.


RESULTS
The stability of BLA under acid condition was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, T353I and H400R, were obtained in BLA. To identify the mutation of amino acids in Thr353Ile/His400Arg related to its acid stability, single mutants Thr353Ile and His400Arg were obtained via site-directed mutagenesis. Among the resulting mutant enzymes, the k(cat)/K(m) values of the mutants Thr353Ile, His400Arg and Thr353Ile/His400Arg under pH 4.5 were 3.5-, 6.0- and 11.3-fold higher, respectively, than that of the wild-type. Thr353Ile/His400Arg exhibited stronger tolerance towards a lower pH without obvious difference in thermostability when compared with wild-type.


CONCLUSIONS
The results combined with three-dimensional structure analysis of mutant BLAs demonstrated that Thr353Ile/His400Arg showed an improved acid stability under low pH condition as a result of the interactions of hydrogen bonding, hydrophobicity, helix propensity and electrostatic fields.


CONCLUSIONS
It provides theoretical basis and background data for the improvement of acid stability in BLA by protein engineering.
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, China.
Y H
YH
Liu
B
B
Hu
Y J
YJ
Xu
J X
JX
Bo
S
S
Fan
J L
JL
Wang
F P
FP
Lu
D000143:Acids / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics
D000143:Acids; D000516:alpha-Amylases; C501919:alpha-amylase, Bacillus licheniformis