Enzymatic Hydrolysis of Soluble Starch with an α-Amylase from Bacillus licheniformis

Rodríguez, V. Bravo; Alameda, Encarnación Jurado; Martínez-Gallegos, Juan F.; Requena, A. Reyes; López, Antonio

doi:10.1021/bp060057a

Cited by 37 publications

(17 citation statements)

References 22 publications

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“…α-Amylase (Amy) is one of the most widely used technological enzymes, with broad applications in food processing and other industries that use starch liquefaction [ 13 , 14 ]. Glucokinase (GlcK) plays an important role in glucose homeostasis.…”

Section: Resultsmentioning

confidence: 99%

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

et al. 2015

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Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

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Section: Resultsmentioning

confidence: 99%

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

et al. 2015

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“…PHA beads have been used to immobilise a variety of enzymes that can be used for a range of applications from food production to the synthesis of fine chemicals. The successful display of a thermostable α-amylase (BLA) could be used in a wide variety of industries that require starch liquefaction including food processing, detergent manufacture, paper processing, and textiles [39,40,41]. Immobilised enzymes have a potential use in the bioremediation of toxic chemicals which would otherwise persist in the environment.…”

Section: Current Applicationsmentioning

confidence: 99%

Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

Hooks

Venning-Slater

et al. 2014

Molecules

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Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

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“…A moderately thermostable amylase from Bacillus licheniformis warrants mention due to extensive use in industry (Bravo Rodriguez et al, 2006). A number of halophilic enzymes tolerant of high salt and solvent concentrations have been reported (Li et al, 2002;Amoozegar et al, 2003;Tan et al, 2003;Antranikian and Egorova, 2007).…”

Section: Other Polymeric Biomass Resourcesmentioning

confidence: 99%

Extremophiles and their Application to Biofuel Research

Taylor¹,

Bauer²,

Mackay³

et al. 2012

Extremophiles

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Enzymatic Hydrolysis of Soluble Starch with an α-Amylase from Bacillus licheniformis

Cited by 37 publications

References 22 publications

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

Extremophiles and their Application to Biofuel Research

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