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1.
Food Chem ; 354: 129475, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-33744660

RESUMO

The α-amylases are the most widely used industrial enzymes, and are particularly useful as liquifying enzymes in industrial processes based upon starch. Since starch liquefication is carried out at evaluated temperatures, typically above 60 °C, there is substantial demand for thermostable α -amylases. Most naturally occurring α -amylases exhibit moderate thermostability, so substantial effort has been invested in attempts to increase their thermostability. One structural feature that has the potential to increase protein thermostability is the introduction of salt bridges. However, not every salt bridge contributes to protein thermostability. The salt bridges in amylases have their characteristics in terms of distribution, configuration and location. The summary of these features helps to introduce new salt bridges based on the characteristics. This review focuses on salt bridges of α-amylases, both naturally present and introduced using mutagenesis. Its aim is to provide a bird's eye view of distribution, configuration, location of desirable salt bridges.


Assuntos
Sais/química , alfa-Amilases/metabolismo , Bacillus licheniformis/enzimologia , Sítios de Ligação , Estabilidade Enzimática , Metais/química , Simulação de Dinâmica Molecular , Temperatura , alfa-Amilases/química
2.
Food Chem ; 316: 126348, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32044699

RESUMO

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02 (GtGBE, EC 2.4.1.18) does not possess the thermostability required by modified starch industry. To increase its thermostability, a rational design strategy was used to introduce additional salt bridges into GtGBE. The strategy involved in mutation of individual residues to form "local" two-residue salt bridges. Accordingly, five of local salt bridges (Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and I571D-R569 mutants) were separately introduced into GtGBE. The half-times of these mutants at 60 °C were 17% to 51% longer than that of wild-type. Subsequently, these two-residue salt bridges were extended to form salt bridge networks (Q231R/K-D227-D131H, T339D/E-K335-I291H, and I571D-R569-R617H mutants). Among these mutants, except I571D-R569-R617H, the half-times of Q231R/K-D227-D131H, T339D/E-K335-I291H mutants at 60 °C were 15%, 17%, 21% and 17% longer than those of the corresponding two-residue salt bridges, respectively. The results showed that design and introduction of salt bridges improves enzyme thermostability in GtGBE.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Geobacillus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Estabilidade Enzimática/efeitos dos fármacos , Geobacillus/genética , Mutação , Cloreto de Sódio/farmacologia , Temperatura
3.
Crit Rev Biotechnol ; 40(3): 380-396, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31996051

RESUMO

The 1,4-α-glucan branching enzymes (GBEs) are ubiquitously distributed in animals, microorganisms and plants. These enzymes modify the structure of both starch and glycogen; changing the frequency and position of branches by forming new α-1,6-glucosidic linkages. In organisms, controlling the number and distribution of branches is an irreplaceable process that maintains the physiological state of starch and glycogen in the cell. The process is also the foundation for the industrial applications of GBEs. So far, a number of GBEs have been identified in eukaryotes and prokaryotes as researchers searched for GBEs with optimal properties. Among them, bacterial GBEs have received particular attention due to the convenience of heterologous expression and industrial applications of GBEs from bacteria than GBEs from other sources. The advantages of bacterial GBEs in potential applications stimulated the investigations of bacterial GBEs in terms of their structure and properties. However, full exploitation of GBEs in commercial applications is still in its infancy because of the disadvantages of currently available enzymes and of limited imagination with respect to future possibilities. Thus, in this review, we present an overview of the bacterial GBEs including their structure, biochemical properties and commercial applications in order to depict the whole picture of bacterial GBEs.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bactérias/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Animais , Bactérias/genética , Proteínas de Bactérias , Biotecnologia , Glucanos , Humanos , Modelos Moleculares , Amido/biossíntese
4.
Comput Struct Biotechnol J ; 17: 895-903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31333816

RESUMO

The contribution of newly designed salt bridges to protein stabilization remains controversial even today. In order to solve this problem, we investigated salt bridges from two aspects: spatial distribution and evolutionary characteristics of salt bridges. Firstly, we analyzed spatial distribution of salt bridges in proteins, elucidating the basic requirements of forming salt bridges. Then, from an evolutionary point of view, the evolutionary characteristics of salt bridges as well as their neighboring residues were investigated in our study. The results demonstrate that charged residues appear more frequently than other neutral residues at certain positions of sequence even under evolutionary pressure, which are able to form electrostatic interactions that could increase the evolutionary stability of corresponding amino acid regions, enhancing their importance to stability of proteins. As a corollary, we conjectured that the newly designed salt bridges with more contribution to proteins, not only, are qualified spatial distribution of salt bridges, but also, are needed to further increase the evolutionary stability of corresponding amino acid regions. Based on analysis, the 8 mutations were accordingly constructed in the 1,4-α-glucan branching enzyme (EC 2.4.1.18, GBE) from Geobacillus thermoglucosidans STB02, of which 7 mutations improved thermostability of GBE. The enhanced thermostability of 7 mutations might be a result of additional salt bridges on residue positions that at least one of amino acids positions is conservative, improving their contribution of stabilization to proteins.

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