Dataset Information

Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus ?-amylase at 1.9?A resolution.

ABSTRACT: The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed ?-amylases and then exo-acting ?-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial ?-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl ?-amylase variant based upon the parent Geobacillus stearothermophilus ?-amylase is presented. The structure has been solved at 1.9?Å resolution, revealing the classical three-domain fold stabilized by Ca2+ and a Ca2+-Na+-Ca2+ triad. As expected, the structure is similar to the G. stearothermophilus ?-amylase but with main-chain deviations of up to 3?Å in some regions, reflecting both the mutations and differing crystal-packing environments.

SUBMITTER: Offen WA

PROVIDER: S-EPMC4304751 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus α-amylase at 1.9 Å resolution.

Offen Wendy A WA Viksoe-Nielsen Anders A Borchert Torben V TV Wilson Keith S KS Davies Gideon J GJ

Acta crystallographica. Section F, Structural biology communications 20150101 Pt 1

The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolve ...[more]

PMID: 25615972

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Project description:BackgroundOur laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low.ResultsIn this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date.ConclusionsThis is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.

Dataset Information

Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus ?-amylase at 1.9?A resolution.

Publications

Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus α-amylase at 1.9 Å resolution.

Similar Datasets

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