Protein engineering of amylases

Svensson, Birte; Søgaard, Morten

doi:10.1042/bst0200034

Cited by 27 publications

(11 citation statements)

References 9 publications

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“…Domain B is formed by a protrusion between the third β-strand and the third α-helix of the TIM barrel. It has a rather irregular β-rich structure and varies substantially in size and structure among the α-amylases [34]. Domain B forms a large part of the substrate binding cleft and is presumed to be important for the substrate specificity differences observed between α-amylases [35].…”

Section: [15]mentioning

confidence: 99%

α-Amylase: An Ideal Representative of Thermostable Enzymes

Prakash

Jaiswal

2009

Appl Biochem Biotechnol

167

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The conditions prevailing in the industrial applications in which enzymes are used are rather extreme, especially with respect to temperature and pH. Therefore, there is a continuing demand to improve the stability of enzymes and to meet the requirements set by specific applications. In this respect, thermostable enzymes have been proposed to be industrially relevant. In this review, alpha-amylase, a well-established representative of thermostable enzymes, providing an attractive model for the investigation of the structural basis of thermostability of proteins, has been discussed.

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Section: [15]mentioning

confidence: 99%

α-Amylase: An Ideal Representative of Thermostable Enzymes

Prakash

Jaiswal

2009

Appl Biochem Biotechnol

167

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“…Even in the e-amylase family, only one residue, TAA Gly-323, appears to be conserved in a short eo/~ connecting turn for structural reasons [26,43]. Hybrids (chimera) of isozymes [29,65] or enzymes from closely related species [69], however, show changes in pH or temperature stability (for a short review see [73]) and hence highlight regions and residues having significant impact on conformational stability.…”

Section: Regions Critical For the Conformational Stabilitymentioning

confidence: 99%

“…73] and subjected to mutational structure/ function relationship investigations. Guided by known sequences and crystal structures, changes in specificity by mutation of the binding residues in a-amylases [45][46][47], cyclodextrin glucanotransferases (CGTases) [ 11,12], and neopullulanase [37] establish a basis for rational design of all family members.…”

Section: Introductionmentioning

confidence: 99%

Protein engineering in the ?-amylase family: catalytic mechanism, substrate specificity, and stability

1994

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Most starch hydrolases and related enzymes belong to the e-amylase family which contains a characteristic catalytic (fi/e)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the e-amylases, the cyclodextrin glucanotransferases, and the oligo-l,6-glucosidases. Throughout the e-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the fl--+ e loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of e-amylases, changed the distribution of e-,/3-and 7-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative e-1,4: e-1,6 dual-bond specificity of neopullulanase. Barley e-amylase isozyme hybrids and Bacillus e-amylases demonstrate the impact of a small domain B protruding from the (/3/e)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as e-amylase, starch branching and debranching enzymes, and amylomaltase.Abbreviations: CGTase, cyclodextrin glucanotransferase; SBD, starch binding domain; TAA, takaamylase A; TIM, triose-phosphate isomerase. The mutations are described with the one-letter code, i.e.

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“…The use of α‐amylases in industrial processes requires that they be adapted to the prevailing process conditions. Protein engineering techniques have been applied to the BLA to improve its thermal stability by rational protein engineering (Svensson and Sogaard 1992; Svensson 1994; Declerck et al 1995). Recently, the tolerance of the BLA toward low pH was enhanced by directed evolution (Shaw et al 1999).…”

mentioning

confidence: 99%

Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity

Bessler¹,

Schmitt²,

Maurer³

et al. 2003

Protein Science

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Abstract␣-Amylases, in particular, microbial ␣-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for ␣-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the ␣-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4-and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial ␣-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial ␣-amylases.

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Protein engineering of amylases

Cited by 27 publications

References 9 publications

α-Amylase: An Ideal Representative of Thermostable Enzymes

α-Amylase: An Ideal Representative of Thermostable Enzymes

Protein engineering in the ?-amylase family: catalytic mechanism, substrate specificity, and stability

Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity

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