Journal
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
Volume 116, Issue -, Pages 70-77Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.molcatb.2015.03.001
Keywords
High-throughput screening; Maltotriose; Psychrophiles; Pullulan; Type-I pullulanase
Funding
- KAAD (Katholischer Akademischer Auslander-Dienst)
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An activity-based screening approach led to the identification of a novel glycoside hydrolase family-13 pullulanase gene (pul13A) from a psychrophilic bacterium. The recombinant enzyme exhibited a deduced peptide sequence of 1440 amino acid residues and was produced in a heterologous host in Escherichia coli. Purification from inclusion bodies was achieved by a six-step dialysis protocol enabling mild refolding of the urea-denaturated protein followed by affinity- and size-exclusion chromatography under native conditions. Pul13A is a type-I pullulanase, which is capable of hydrolysing alpha-1,6-glycosidic bonds in pullulan to produce maltotriose, while maltose and intermediate oligosaccharides are produced from soluble starch and amylopectin. The recombinant enzyme exhibited typical properties of cold-adapted proteins including low thermostability at elevated temperatures. It showed a temperature optimum at 35 degrees C, while at 10 degrees C residual activity (25%) remained. The optimal pH was in the range of 6.0-7.0, with Pul13A being stable at neutral and basic pH, but not in the acidic range. Catalytic activity was increased in the presence of divalent cations calcium and cobalt and both metal ions were also able to restore catalytic activity of EDTA-chelated enzyme samples. Pul13A represents the first type-I pullulanase from a psychrophile that has been produced in recombinant form. Moreover, its favourable enzymatic properties make this enzyme a potential candidate for industrial applications such as starch degradation for ethanol based biofuel production. (C) 2015 Elsevier B.V. All rights reserved.
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