An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half‐life of 10 min at 70°C. It is a Ca2+ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α‐amylases, AmyKS presents a high affinity toward soluble starch with a Km value of 0.252 mg ml−1. Coupled with the size‐exclusion chromatography data, MALDI–TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non‐maltogenic α‐amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.
Immobilization
of enzymes has been extensively required in a wide
variety of industrial applications as a way to ensure functionality
and the potential of enzyme recycling after use. In particular, enzyme
immobilization on magnetic nanoparticles (MNPs) could offer reusability
by means of magnetic recovery and concentration, along with increased
stability and robust activity of the enzyme under different physicochemical
conditions. In the present work, microbial α-amylase (AmyKS)
and xylanase (XAn11) were both immobilized on different types of MNPs
[MamC-mediated biomimetic MNPs (BMNPs) and inorganic MNPs] by using
two different strategies (electrostatic interaction and covalent bond).
AmyKS immobilization was successful using electrostatic interaction
with BMNPs. Instead, the best strategy to immobilize XAn11 was using
MNPs through the hetero-crosslinker 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) and
N
-hydroxysuccinimide (NHS).
The immobilization protocols were optimized by varying glutaraldehyde
(GA) concentration, enzyme quantity, and reaction time. Under optimal
conditions, 92% of AmyKS and 87% of XAn11 were immobilized on BMNPs
and MNPs–E/N, respectively (here referred as AmyKS–BMNPs
and XAn11–MNPs nanoassemblies). The results show that the immobilization
of the enzymes did not extensively alter their functionality and increased
enzyme stability compared to that of the free enzyme upon storage
at 4 and 20 °C. Interestingly, the immobilized amylase and xylanase
were reused for 15 and 8 cycles, respectively, without significant
loss of activity upon magnetic recovery of the nanoassemblies. The
results suggest the great potential of these nanoassemblies in bioindustry
applications.
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