Improvement of the acid stability of Bacillus licheniformis alpha amylase by error-prone PCR.
Summary of "Improvement of the acid stability of Bacillus licheniformis alpha amylase by error-prone PCR."
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.
Affiliation
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,
Journal Details
This article was published in the following journal.
Name: Journal of applied microbiology
ISSN: 1365-2672
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/22681571
- DOI: http://dx.doi.org/10.1111/j.1365-2672.2012.05359.x
Medical and Biotech [MESH] Definitions
Salivary Alpha-amylases
A subclass of alpha-amylase ISOENZYMES that are secreted into SALIVA.
Prostaglandins F
(9 alpha,11 alpha,13E,15S)-9,11,15-Trihydroxyprost-13-en-1-oic acid (PGF(1 alpha)); (5Z,9 alpha,11,alpha,13E,15S)-9,11,15-trihydroxyprosta-5,13-dien-1-oic acid (PGF(2 alpha)); (5Z,9 alpha,11 alpha,13E,15S,17Z)-9,11,15-trihydroxyprosta-5,13,17-trien-1-oic acid (PGF(3 alpha)). A family of prostaglandins that includes three of the six naturally occurring prostaglandins. All naturally occurring PGF have an alpha configuration at the 9-carbon position. They stimulate uterine and bronchial smooth muscle and are often used as oxytocics.
Pancreatic Alpha-amylases
A subclass of alpha-amylase ISOENZYMES that are secreted into PANCREATIC JUICE.
Regulatory Sequences, Ribonucleic Acid
Sequences within RNA that regulate the processing, stability (RNA STABILITY) or translation (TRANSLATION, GENETIC) of RNA.
Bacillus Phages
Viruses whose host is Bacillus. Frequently encountered Bacillus phages include bacteriophage phi 29 and bacteriophage phi 105.
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