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Effect of differential processing of the native and recombinant ?-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties.

ABSTRACT: AmyJ33, an ?-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant ?-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native ?-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.

SUBMITTER: Montor-Antonio JJ

PROVIDER: S-EPMC5605471 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Effect of differential processing of the native and recombinant α-amylase from <i>Bacillus amyloliquefaciens</i> JJC33M on specificity and enzyme properties.

Montor-Antonio Juan José JJ Hernández-Heredia Sarahi S Ávila-Fernández Ángela Á Olvera Clarita C Sachman-Ruiz Bernardo B Del Moral Sandra S

3 Biotech 20170920 5

AmyJ33, an α-amylase isolated from <i>Bacillus amyloliquefaciens</i> JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed i ...[more]

PMID: 28955633

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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

Effect of differential processing of the native and recombinant ?-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties.

Publications

Effect of differential processing of the native and recombinant α-amylase from <i>Bacillus amyloliquefaciens</i> JJC33M on specificity and enzyme properties.

Similar Datasets

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