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Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

Paper ID Volume ID Publish Year Pages File Format Full-Text
34738 45042 2013 7 PDF Available
Title
Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1
Abstract

•An amylopullulanase from Thermococcus (Apu-Tk) was cloned and purified to homogeneity.•The optimal temperature for Apu-Tk to hydrolyse pullulan and starch was >100 °C.•The Apu-Tk was active at a broad range of pH (4–7) with the optimum pH at 5.0–5.5.•Apu-Tk retained >30% activity in the presence of 8% SDS or 10% β-mercaptoethanol.

Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 °C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.

Keywords
Amylopullulanase; GH57 family; Thermophilic archaeon; Extremely stable; Industrial application
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Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 48, Issues 5–6, May–June 2013, Pages 878–884
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us