Abstract
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0–9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5–12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
Highlights
Anoxybacillus spp. are thermophilic bacteria that grow optimally at temperatures ranging from 50 to 65 ◦ C [1,2,3,4]
The sequenced genome of strain SK3-4 (NCBI Bioproject accession number: PRJNA174378) revealed several genes encoding glycoside hydrolases (GHs) known to degrade starch [7], and some of these enzymes have been extensively studied by our research group [8,9,10,11,12,13,14]. α-Amylases (EC 3.2.1.1) hydrolyze α-1,4-glycosidic bonds of polysaccharides [15]. α-Amylase from Anoxybacillus sp
SK3-4 (ASKA) is a novel thermostable enzyme that produces a high level of maltose upon reaction with starch, which is useful for starch saccharification [2,8,9,10,15,16]
Summary
Anoxybacillus spp. are thermophilic bacteria that grow optimally at temperatures ranging from 50 to 65 ◦ C [1,2,3,4]. The sequenced genome of strain SK3-4 (NCBI Bioproject accession number: PRJNA174378) revealed several genes encoding glycoside hydrolases (GHs) known to degrade starch [7], and some of these enzymes have been extensively studied by our research group [8,9,10,11,12,13,14]. SK3-4 (ASKA) is a novel thermostable enzyme that produces a high level of maltose upon reaction with starch, which is useful for starch saccharification [2,8,9,10,15,16]. Janecek et al [17] proposed that ASKA may be classified as a novel subfamily of glycoside hydrolase 13 (GH13).
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