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C-TERMINAL TRUNCATIONS OF A THERMOSTABLE BACILLUS-STEAROTHERMOPHILUS ALPHA-AMYLASE

Vihinen, Mauno LU ; PELTONEN, T; IITIA, A; SUOMINEN, I and MANTSALA, P (1994) In Protein Engineering 7(10). p.1255-1259
Abstract
A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues, The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The K-m values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating... (More)
A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues, The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The K-m values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile, The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling, The liquefying amylases seem to require similar to 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
PROTEIN TRUNCATION, SUBSTRATE RECOGNITION, THERMOSTABLE ALPHA-AMYLASE
in
Protein Engineering
volume
7
issue
10
pages
1255 - 1259
publisher
Oxford University Press
external identifiers
  • wos:A1994PM11300010
  • scopus:0028091811
ISSN
1460-213X
DOI
10.1093/protein/7.10.1255
language
English
LU publication?
no
id
9f00d19a-d01f-436b-883d-3cd08bfb3541 (old id 3853351)
date added to LUP
2013-06-28 14:48:24
date last changed
2017-01-01 04:57:10
@article{9f00d19a-d01f-436b-883d-3cd08bfb3541,
  abstract     = {A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues, The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The K-m values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile, The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling, The liquefying amylases seem to require similar to 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.},
  author       = {Vihinen, Mauno and PELTONEN, T and IITIA, A and SUOMINEN, I and MANTSALA, P},
  issn         = {1460-213X},
  keyword      = {PROTEIN TRUNCATION,SUBSTRATE RECOGNITION,THERMOSTABLE ALPHA-AMYLASE},
  language     = {eng},
  number       = {10},
  pages        = {1255--1259},
  publisher    = {Oxford University Press},
  series       = {Protein Engineering},
  title        = {C-TERMINAL TRUNCATIONS OF A THERMOSTABLE BACILLUS-STEAROTHERMOPHILUS ALPHA-AMYLASE},
  url          = {http://dx.doi.org/10.1093/protein/7.10.1255},
  volume       = {7},
  year         = {1994},
}