Microbial Amylolytic Enzyme

Vihinen, Mauno; Mäntsälä, Pekka

doi:10.3109/10409238909082556

Cited by 393 publications

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“…They catalyze the hydrolysis of starch, glycogen, and related polysaccharides by cleaving internal a-1,Cglycosidic bonds. In addition to their biochemical interest, a-amylases have a number of important biotechnological applications in food and starch processing industries (Vihinen & Mantsala, 1989).Alteromonas haloplanctis is a gram negative bacterium collected in Antarctica and secreting a calcium-and chloride-dependent a-amylase. It grows at sub-zero temperatures in its natural environment and can be considered as an extreme psychrophile (Feller et al, 1992).…”

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Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

et al. 1998

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Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the a-amylase (AHA) secreted by this bacterium, in its native state to 2.0 8, resolution as well as in complex with Tris to 1.85 8, resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known a-amylases of various origins with a surprisingly greatest similarity to mammalian a-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate a-1,4-glycosidic bonds. The chloride binding site is situated -5 8, from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found -22 A from the active site. We found that this triad is equally present in other chloride dependent a-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted a-amylase.Keywords: allosteric activation; cold adaptation; crystal structure; glycosyl hydrolases; inhibition; psychrophilic a-Amylases a-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1 are endoglucanases widely distributed in bacteria, fungi, animals, and plants. They catalyze the hydrolysis of starch, glycogen, and related polysaccharides by cleaving internal a-1,Cglycosidic bonds. In addition to their biochemical interest, a-amylases have a number of important biotechnological applications in food and starch processing industries (Vihinen & Mantsala, 1989).Alteromonas haloplanctis is a gram negative bacterium collected in Antarctica and secreting a calcium-and chloride-dependent a-amylase. It grows at sub-zero temperatures in its natural environment and can be considered as an extreme psychrophile (Feller et al., 1992). The psychrophilic a-amylase is characterized by a high catalytic efficiency which compensates for the reduction of chemical reaction rates inherent to low temperatures. In addition, all biophysical parameters recorded suggest a flexible conformation of this heat-labile a-amylase. The two crystal structures reported here are the first three-dimensional structures of a psy- chrophilic enzyme; moreover, they provide a number of significant features related to various aspects of biochemistry and microbiology. The structure of A. haloplanctis a-amylase cornplexed with Tris allows to understand the molecular basis of glycosidase inhibition by this cation which is a widely used compound in biochemistry. This bacterial a-amylase requires a chloride ion for its activity, a property previously considered as specific to mammalian a-amylases (Brayer et al., 1995). ...

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Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

et al. 1998

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“…Many types of amylases with unique properties have been isolated and characterized for various important applications in the food and starch industry. 1,2) According to the classification system by Henrissat, 3) most of the starch hydrolyzing enzymes belong to the family 13 glycosyl hydrolases (GH13) based on amino acid sequence homology.This group of enzymes have the following features: (i) they hydrolyze glycosidic bonds of various glucans to produce anomeric mono or oligosaccharides (hydrolysis), form 1,4 or 1,6 glycosidic linkages (transglycosylation), or a combination of both activities; (ii) they possess a ( )8 barrel structure containing the catalytic site residues; (iii) they have four highly conserved regions in their primary sequence which contain the amino acids that form the catalytic site. Although these enzymes share many structural and mechanical characteristics, they can be divided into several groups according to substrate specificities, patterns of starch cleavage, transglycosylation or cyclization activities, and structural features.…”

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Function and Tertiary- and Quaternary-structure of Cyclodextrin-hydrolyzing Enzymes (CDase), a Group of Multisubstrate Specific Enzymes Belonging to the α-Amylase Family

Park

2006

J. Appl. Glycosci.

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Enzymes have a long history of use in industrial applications to produce a broad range of commercial products. Many types of amylases with unique properties have been isolated and characterized for various important applications in the food and starch industry. 1,2) According to the classification system by Henrissat, 3) most of the starch hydrolyzing enzymes belong to the family 13 glycosyl hydrolases (GH13) based on amino acid sequence homology.This group of enzymes have the following features: (i) they hydrolyze glycosidic bonds of various glucans to produce anomeric mono or oligosaccharides (hydrolysis), form 1,4 or 1,6 glycosidic linkages (transglycosylation), or a combination of both activities; (ii) they possess a ( )8 barrel structure containing the catalytic site residues; (iii) they have four highly conserved regions in their primary sequence which contain the amino acids that form the catalytic site. Although these enzymes share many structural and mechanical characteristics, they can be divided into several groups according to substrate specificities, patterns of starch cleavage, transglycosylation or cyclization activities, and structural features.Jespersen et al . 4) used sequence alignments and structure prediction models to forecast the presence of amylase type ( )8 barrel domains and the positions of the strand and helices found in various amylolytic and related enzymes. An evolutionary distance tree constructed from 37 residues representing the four most highly conserved strands of these related enzymes revealed several clusters of enzymes harboring similar reaction specificities. The correlation strongly suggests that the conserved strands may determine substrate and or product specificity.Typical amylases (EC 3.2.1.1; 1,4 D glucan glucanohydrolase) catalyze the hydrolysis of 1,4 glucosidic linkages in starch and give rise to oligosaccharides with certain lengths of glucose as the major product depending on the specific enzyme.

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“…These enzymes possess an 8 (α/β) or TIM barrel structure containing the catalytic site residues and consisted of four highly conserved regions in their primary sugar syrups, to produce cyclodextrins for the pharmaceutical industry (19). The properties of α-amylases such as thermo-stability and pH profile should match the application (20,21).…”

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Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

Afzaljavan

Mobini-Dehkordi

2013

Jundishapur J Microbiol

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Background: Alpha-amylases are digestive enzymes which hydrolyze starch glycosidic bonds to glucose, maltose, maltotriose and dextrin which have diverse applications in a wide range of industries such as food, textile, paper, detergents representing approximately 30% of the world enzyme production. Objectives: In this study, the gene encoding the alpha-amylase enzyme of native isolated Bacillus subtilis was amplified with specific primers containing of NotI and AscI restriction sites by PCR and then sequenced. Purified PCR product and shuttle episomal vector p316TDH3 were cut by restriction enzymes and cloned into Escherichia coli and yeast hosts. Materials and Methods:The haploid auxotroph (ura3-) strain of Saccharomyces cerevisiae and p316TDH3 were used as the host and vector for cloning and expression of the alpha amylase gene, respectively. In native Bacillus sp. the amyE gene without signal sequence was amplified with specific primers that introduced AscI and NotI restriction sites. After constructing the recombinant plasmid, it was transformed into E. coli competent cells. Then, colonies selection and confirmation were performed and the extracted plasmid was introduced to competent yeast cells using carrier sperm DNA. Recombinant yeast cells could grow on minimal media and produce extracellular enzyme. Results:The presence of alpha-amylase gene in recombinant bacteria was certificated by colony-PCR method. After extraction of recombinant vector from E. coli, the competent S. cerevisiae cells were transformed using polyethylene glycol and carrier sperm DNA. The recombinant yeast strains were screened by URA3 auxotrophic marker and analyzed for alpha-amylase gene existence. In the other hand, the amylase gene length of native B. subtilis was 1887 base pairs (bp) with an approximately 93.65% similarity with standard bacterial strain. Conclusions: Based on this similarity and our bioinformatics evaluations, this mentioned alpha-amylase gene can be expressed in S. cerevisiae as extracellular enzyme.

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Microbial Amylolytic Enzyme

Cited by 393 publications

References 539 publications

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

Function and Tertiary- and Quaternary-structure of Cyclodextrin-hydrolyzing Enzymes (CDase), a Group of Multisubstrate Specific Enzymes Belonging to the α-Amylase Family

Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

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Microbial Amylolytic Enzyme

Cited by 393 publications

References 539 publications

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

Function and Tertiary- and Quaternary-structure of Cyclodextrin-hydrolyzing Enzymes (CDase), a Group of Multisubstrate Specific Enzymes Belonging to the &alpha;-Amylase Family

Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

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Function and Tertiary- and Quaternary-structure of Cyclodextrin-hydrolyzing Enzymes (CDase), a Group of Multisubstrate Specific Enzymes Belonging to the α-Amylase Family