Dataset Information

Property Improvement of ?-Amylase from?Bacillus stearothermophilus by Deletion of Amino Acid?Residues Arginine 179 and Glycine 180.

ABSTRACT: To improve the properties of ?-amylase from Bacillus stearothermophilus (AmyS), a deletion mutant AmyS?R179-G180 was constructed by deleting arginine (Arg179) and glycine (Gly180) using site-directed mutagenesis. AmyS and AmyS?R179-G180 were expressed in Bacillus subtilis and purified by ammonium sulfate precipitation, after which the enzymatic properties were characterized and compared. By deleting amino acids Arg179 and Gly180, the thermostability of ?-amylase AmyS?R179-G180 was enhanced and the half-life at 100 °C significantly increased from 24 to 33 min. In addition, AmyS?R179-G180 exhibited greater acid resistance and lower calcium requirements to maintain ?-amylase activity. The secretory capacity of the recombinant strain was evaluated by fed-batch fermentation in a 7.5-litre fermentor in which high ?-amylase activity was obtained. The highest activity reached 3300 U/mL with a high productivity of 45.8 U/(mL·h).

SUBMITTER: Gai Y

PROVIDER: S-EPMC5956274 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Property Improvement of α-Amylase from Bacillus stearothermophilus by Deletion of Amino Acid Residues Arginine 179 and Glycine 180.

Gai Yuanming Y Chen Jingqi J Zhang Shibin S Zhu Beiwei B Zhang Dawei D

Food technology and biotechnology 20180301 1

To improve the properties of α-amylase from Bacillus stearothermophilus (AmyS), a deletion mutant AmyS∆R179-G180 was constructed by deleting arginine (Arg179) and glycine (Gly180) using site-directed mutagenesis. AmyS and AmyS∆R179-G180 were expressed in Bacillus subtilis and purified by ammonium sulfate precipitation, after which the enzymatic properties were characterized and compared. By deleting amino acids Arg179 and Gly180, the thermostability of α-amylase AmyS∆R179-G180 was ...[more]

PMID: 29795997

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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.

Project description:Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%-48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels.

Dataset Information

Property Improvement of ?-Amylase from?Bacillus stearothermophilus by Deletion of Amino Acid?Residues Arginine 179 and Glycine 180.

Publications

Property Improvement of α-Amylase from <i>Bacillus stearothermophilus</i> by Deletion of Amino Acid Residues Arginine 179 and Glycine 180.

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Dataset Information

Property Improvement of ?-Amylase from?Bacillus stearothermophilus by Deletion of Amino Acid?Residues Arginine 179 and Glycine 180.

Publications

Property Improvement of α-Amylase from <i>Bacillus stearothermophilus</i> by Deletion of Amino Acid Residues Arginine 179 and Glycine 180.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Property Improvement of α-Amylase from <i>Bacillus stearothermophilus</i> by Deletion of Amino Acid Residues Arginine 179 and Glycine 180.