Document Detail

Improvement of the acid stability of Bacillus licheniformis alpha amylase by error-prone PCR.
MedLine Citation:
PMID:  22681571     Owner:  NLM     Status:  Publisher    
AIMS: The purpose of this research was to obtain the mutant of Bacillus licheniformis alpha amylase (BLA) with improved acid stability and elucidate the difference in catalytic mechanism under acidic conditions between wild type and mutant BLAs. METHODS AND 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) /Km 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 improved acid stability under low pH condition as a result of the interactions of hydrogen bonding, hydrophobicity, helix propensity and electrostatic fields. SIGNIFICANCE AND IMPACT OF STUDY: It provides theoretical basis and background data for the improvement of acid stability in BLA by protein engineering. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
Yihan Liu; Bo Hu; Yanjing Xu; Jiaxin Bo; Shuai Fan; Jianling Wang; Fuping Lu
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-6-11
Journal Detail:
Title:  Journal of applied microbiology     Volume:  -     ISSN:  1365-2672     ISO Abbreviation:  -     Publication Date:  2012 Jun 
Date Detail:
Created Date:  2012-6-11     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9706280     Medline TA:  J Appl Microbiol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, 300457, People's Republic of China; National Engineering Laboratory for Industrial Enzymes, Tianjin, 300457, People's Republic of China; The College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
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