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α-Amylase from Thermococcus hydrothermalis: Re-cloning aimed at the improved expression and hydrolysis of corn starch

Paper ID Volume ID Publish Year Pages File Format Full-Text
18352 42719 2006 6 PDF Available
Title
α-Amylase from Thermococcus hydrothermalis: Re-cloning aimed at the improved expression and hydrolysis of corn starch
Abstract

A gene encoding the thermostable α-amylase from hyperthermophilic archaeon Thermococcus hydrothermalis was cloned in Escherichia coli. In an effort to achieve the improved expression, four DNA primers (three reverse and one forward) were designed to yield three recombinant variants of α-amylase. The additional aim of the re-cloning was to verify how the changes at the C-teminal end of the recombinant α-amylase introduced by the pET vector and 6-His tag may affect the resulting specific activity of the variants. Biochemical characteristics of the recombinant α-amylases, i.e. molecular weight, temperature optimum, and pH optimum, were confirmed to be the same as those of the original α-amylase: 53.6 kDa, 85 °C, and 5.5, respectively. Concerning the specific activities, the addition of two residues succeeded by six histidines (LEHHHHHH) had in fact no influence, whereas the insertion of a longer peptide between the original α-amylase C-terminus and the 6-His tag (DPNSSSVDKLAAALEHHHHHH) caused a substantial decrease (more than 40%) in comparison with the specific activity of the original protein. The recombinant α-amylases were preliminary tested for their ability to hydrolyze 1% and 10% corn starch suspensions. Two types of starch were used: Meritena 100 (amylose starch) and Meritena 300 (amylopectin starch). Concerning the α-amylase concentration sufficient to reach the industrial requirements, i.e. dextrose equivalent 6–12% in 3 h, one unit of enzyme activity at defined conditions per mg starch can be considered as optimal concentration with 10% starch suspension. It was found that even a simple and partial purification (boiling the enzyme supernatant for 5 min) is enough for improving the course of corn starch hydrolysis in order to accomplish the technological parameters in a reasonable time.

Keywords
Thermostable recombinant α-amylase; Thermococcus hydrothermalis; Re-cloning of the α-amylase gene; Improved expression; Starch liquefying
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α-Amylase from Thermococcus hydrothermalis: Re-cloning aimed at the improved expression and hydrolysis of corn starch
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 39, Issue 6, 3 October 2006, Pages 1300–1305
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