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Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38.

ABSTRACT: A novel alpha-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60 degrees C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H2O2 (1.8 M).

SUBMITTER: Hagihara H

PROVIDER: S-EPMC92793 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38.

Hagihara H H Igarashi K K Hayashi Y Y Endo K K Ikawa-Kitayama K K Ozaki K K Kawai S S Ito S S

Applied and environmental microbiology 20010401 4

A novel alpha-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60 degrees C. The apparent molecular mass was app ...[more]

PMID: 11282629

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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:Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction. Secretion stress was applied by overproducing the well-secreted AmyQ α-amylase (pKTH10 vector) from B. amyloliquefaciens. Besides examining secretion stress in wild-type cells, we compared transcriptome profiles of a cssS mutant strain under conditions of high-level AmyQ production. Samples for transcriptome analyses were collected at the late exponential growth stage (one hour before the transition point) and 3 hours upon entry in the stationary growth phase. Three independent cultures of each strain were used and cells were sampled for macroarray experiments. Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel.

Dataset Information

Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38.

Publications

Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38.

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