Dataset Information

Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 ?-amylase on Chitosan-magnetic nanoparticles.

ABSTRACT: Bacillus subtilis strain-MK1 ?-amylase was successfully immobilized on Chitosan-magnetic nanoparticles (Ch-MNP) that had been modified with polyethyleneimine (PEI) and glutaraldehyde (GA). Optimization of Ch-MNP/PEI/GA beads modification by Central Composite design enhanced the immobilization yield (IY %) by 1.5-fold. Ch-MNP/PEI/GA was characterized before and after modification and immobilization by FTIR and SEM. Ch-MNP/PEI/GA/Enzyme showed the same pH optima of free enzyme, while an elevation 10?°C in temperature optima was observed after its immobilization. Ch-MNP/PEI/GA/Enzyme displayed higher Km and Vmax values (2.1 and 1.2-fold) and lower Vmax/Km ratio (1.7-fold), respectively than the free enzyme. Compared to the free enzyme, Ch-MNP/PEI/GA/Enzyme exhibited lower activation energy, lower deactivation constant rate, higher D-values, higher half-life, and higher energy for denaturation. Immobilization of ?-amylase increased enthalpy (4.2-fold), free energy (1.1-fold) and decreased entropy (4.6-fold) of thermal inactivation. A significant increase in pH stability of Ch-MNP/PEI/GA/Enzyme was observed especially at alkaline pH values. In addition, Ch-MNP/PEI/GA/Enzyme preserved 83.2 % of its initial activity after 15 consecutive cycles. When storing Ch-MNP/PEI/GA/Enzyme at 4?°C the residual activity was 100 and 86 %, respectively after 21 and 40 days. Finally, immobilization process improved the catalytic properties and stabilities, thus raising the suitability for industrial processes with lower cost and time.

SUBMITTER: Ahmed SA

PROVIDER: S-EPMC7056624 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Catalytic, kinetic and thermal properties of free andimmobilized <i>Bacillus subtilis</i> -MK1 α-amylase on Chitosan-magnetic nanoparticles.

Ahmed Samia A SA Abdella Mohamed A A MAA El-Sherbiny Gamal M GM Ibrahim Atef M AM El-Shamy Aliaa R AR Atalla Sherien M M SMM Hassan Mohamed E ME

Biotechnology reports (Amsterdam, Netherlands) 20200226

<i>Bacillus subtilis</i> strain-MK1 α-amylase was successfully immobilized on Chitosan-magnetic nanoparticles (Ch-MNP) that had been modified with polyethyleneimine (PEI) and glutaraldehyde (GA). Optimization of Ch-MNP/PEI/GA beads modification by Central Composite design enhanced the immobilization yield (IY %) by 1.5-fold. Ch-MNP/PEI/GA was characterized before and after modification and immobilization by FTIR and SEM. Ch-MNP/PEI/GA/Enzyme showed the same pH optima of free enzyme, while an ele ...[more]

PMID: 32154128

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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

Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 ?-amylase on Chitosan-magnetic nanoparticles.

Publications

Catalytic, kinetic and thermal properties of free andimmobilized <i>Bacillus subtilis</i> -MK1 α-amylase on Chitosan-magnetic nanoparticles.

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