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An imprinted cross-linked enzyme aggregate (iCLEA) of sucrose phosphorylase: combining improved stability with altered specificity.

De Winter K, Soetaert W, Desmet T - Int J Mol Sci (2012)

Bottom Line: In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate.As a consequence, the enzyme's specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C.This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.

View Article: PubMed Central - PubMed

Affiliation: Centre of Expertise for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Biosciences Engineering, Ghent University, Coupure Links 653, Ghent B-9000, Belgium; E-Mails: karel.dewinter@ugent.be (K.D.W.); wim.soetaert@ugent.be (W.S.).

ABSTRACT
The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA). In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme's specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.

Show MeSH
General scheme for the production of imprinted cross-linked enzyme aggregates (iCLEAs).
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f1-ijms-13-11333: General scheme for the production of imprinted cross-linked enzyme aggregates (iCLEAs).

Mentions: For the preparation of the imprinted cross-linked enzyme aggregate (iCLEA), the thermostable variant LNFI of the SP from B. adolescentis has been used [12]. The enzyme was recombinantly expressed in E. coli and partially purified by means of a heat treatment, which has been shown to remove the contaminating phosphatase activity [10]. The next steps in the preparation of the iCLEA then consist of imprinting, followed by precipitation (Figure 1).


An imprinted cross-linked enzyme aggregate (iCLEA) of sucrose phosphorylase: combining improved stability with altered specificity.

De Winter K, Soetaert W, Desmet T - Int J Mol Sci (2012)

General scheme for the production of imprinted cross-linked enzyme aggregates (iCLEAs).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472748&req=5

f1-ijms-13-11333: General scheme for the production of imprinted cross-linked enzyme aggregates (iCLEAs).
Mentions: For the preparation of the imprinted cross-linked enzyme aggregate (iCLEA), the thermostable variant LNFI of the SP from B. adolescentis has been used [12]. The enzyme was recombinantly expressed in E. coli and partially purified by means of a heat treatment, which has been shown to remove the contaminating phosphatase activity [10]. The next steps in the preparation of the iCLEA then consist of imprinting, followed by precipitation (Figure 1).

Bottom Line: In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate.As a consequence, the enzyme's specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C.This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.

View Article: PubMed Central - PubMed

Affiliation: Centre of Expertise for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Biosciences Engineering, Ghent University, Coupure Links 653, Ghent B-9000, Belgium; E-Mails: karel.dewinter@ugent.be (K.D.W.); wim.soetaert@ugent.be (W.S.).

ABSTRACT
The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA). In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme's specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.

Show MeSH