Engineering of a Bacillus α-Amylase with Improved Thermostability and Calcium Independency

Ghollasi, Marzieh; Khajeh, Khosro; Naderi-Manesh, Hossein; Ghasemi, Ali

doi:10.1007/s12010-009-8879-2

Cited by 27 publications

(19 citation statements)

References 30 publications

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“…Recently, protein engineering techniques, such as site‐directed mutagenesis, were also employed to identify calcium‐binding residues or to enhance the calcium independence of α‐amylases from Bacillus sp. TS‐23, B. licheniformis MTCC 6598, and B. megaterium WHO . Although previous studies have demonstrated that the calcium‐binding residues (Asp231, Asp233, and Asp438) of BAA are critical for the thermostability and activity of BAA, little information is available with respect to enhancing its calcium independence.…”

Section: Introductionmentioning

confidence: 99%

Engineering of Bacillus amyloliquefaciens α‐Amylase with Improved Calcium Independence and Catalytic Efficiency by Error‐Prone PCR

Tian

Dong

et al. 2017

Starch Stärke

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Bacillus amyloliquefaciens α‐amylase (BAA) is one of the most important amylases and presents a wide range of applications in many processes involving starch‐liquefaction. However, its activity and thermostability largely depend on the existence of calcium ions, which is a major obstacle for its wide‐scale industrial applications. In the present study, to enhance the calcium independence of a mesophilic α‐amylase from B. amyloliquefaciens CICIM B2125, a novel calcium‐independent mutant Q264S is evolved by directed evolution using error‐prone polymerase chain reaction (PCR). Enzymatic properties and kinetic parameters of mutant Q264S were subsequently analyzed. Although the thermostability at 60°C and optimum temperature of Q264S are slightly decreased, its calcium independence and catalytic efficiency are significantly improved, with no apparent changes of specific activity and optimum pH compared with those of the wild‐type enzyme. Furthermore, three‐dimensional structure analysis showed that the improved calcium independence and catalytic efficiency of Q264S are most likely to be a subtle balance among the hydrophobic interactions, electrostatic forces, and hydrogen bonds around calcium‐binding sites and active‐site regions. To the best of our knowledge, this is the first report on increasing calcium independence of BAA by error‐prone PCR. These results could aid the understanding of mechanistic bases of calcium independence of BAA and thus facilitate the design and engineering of more efficient and robust calcium‐independent BAA variants.

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Section: Introductionmentioning

confidence: 99%

Engineering of Bacillus amyloliquefaciens α‐Amylase with Improved Calcium Independence and Catalytic Efficiency by Error‐Prone PCR

Tian

Dong

et al. 2017

Starch Stärke

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“…In most GH13 α-amylases, the conserved calcium-binding site located between domains A and B is thought to significantly contribute to the protein’s structural stability and to regulate its thermostability and enzymatic activity7832. However, the B domain of AmyP is the smallest among those of GH13 subfamilies of α-amylases, and it does not bind Ca 2+ or other metal ions.…”

Section: Resultsmentioning

confidence: 99%

“…For several α-amylases, it has been reported that the metal and substrate binding sites are located in domain B and its interface with domain A and that these regions affect the activity and stability of the enzyme45678. The specific role of domain B in the GH13_37 subfamily will be clearer when the three-dimensional structure of the novel α-amylase AmyP has been solved and described in detail.…”

mentioning

confidence: 99%

Crystal structure of a raw-starch-degrading bacterial α-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13

Liu

et al. 2017

Sci Rep

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Subfamily 37 of the glycoside hydrolase family GH13 was recently established on the basis of the discovery of a novel α-amylase, designated AmyP, from a marine metagenomic library. AmyP exhibits raw-starch-degrading activity and consists of an N-terminal catalytic domain and a C-terminal starch-binding domain. To understand this newest subfamily, we determined the crystal structure of the catalytic domain of AmyP, named AmyPΔSBD, complexed with maltose, and the crystal structure of the E221Q mutant AmyPΔSBD complexed with maltotriose. Glu221 is one of the three conserved catalytic residues, and AmyP is inactivated by the E221Q mutation. Domain B of AmyPΔSBD forms a loop that protrudes from domain A, stabilizes the conformation of the active site and increases the thermostability of the enzyme. A new calcium ion is situated adjacent to the -3 subsite binding loop and may be responsible for the increased thermostability of the enzyme after the addition of calcium. Moreover, Tyr36 participates in both stacking and hydrogen bonding interactions with the sugar motif at subsite -3. This work provides the first insights into the structure of α-amylases belonging to subfamily 37 of GH13 and may contribute to the rational design of α-amylase mutants with enhanced performance in biotechnological applications.

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“…For example, to improve the thermal stability of α-amylase from Bacillus megaterium WHO (BMW), researchers compared BMW-amylase with the most similar protein ( Halothermothrix orenii α-amylase, 67%) through bioinformatic methods and modified the protein using site-specific mutagenesis. The thermal stability was dramatically improved by H58I mutation, which corresponds to Ile50 in H. orenii α-amylase [93]. …”

Section: Amylasementioning

confidence: 99%

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

2016

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Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

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Engineering of a Bacillus α-Amylase with Improved Thermostability and Calcium Independency

Cited by 27 publications

References 30 publications

Engineering of Bacillus amyloliquefaciens α‐Amylase with Improved Calcium Independence and Catalytic Efficiency by Error‐Prone PCR

Engineering of Bacillus amyloliquefaciens α‐Amylase with Improved Calcium Independence and Catalytic Efficiency by Error‐Prone PCR

Crystal structure of a raw-starch-degrading bacterial α-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

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