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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.

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Transglucosylation/phosphorolysis ratio for different CLEA formulations. a Cross-linking was initiated upon addition of the imprinting molecules; b Cross-linking was initiated after 30 min incubation; c 200 mM α-glucosyl glycerol (αGG) was used; d 1 M αGG was used.
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f2-ijms-13-11333: Transglucosylation/phosphorolysis ratio for different CLEA formulations. a Cross-linking was initiated upon addition of the imprinting molecules; b Cross-linking was initiated after 30 min incubation; c 200 mM α-glucosyl glycerol (αGG) was used; d 1 M αGG was used.

Mentions: The specificity of the obtained formulations was then examined and compared with that of a non-imprinted CLEA. More specifically, their activity towards phosphate (phosphorolysis) and glycerol (transglucosylation) as acceptor was determined. Imprinting with sucrose, glycerol or a combination of both was found to have no influence on the specificity of the enzyme preparation (Figure 2). However, initiating aggregation and cross-linking after 30 min incubation with sucrose and glycerol did significantly increase the transglucosylation activity. This must mean that the presence of glycosylated product is required to modify the enzyme’s acceptor specificity. Indeed, imprinting with 200 mM αGG almost doubled the specific activity on glycerol, from 2.22 U/mg for the non-imprinted CLEA to 4.12 U/mg for the iCLEA. Increasing the αGG concentration to 1 M during imprinting. However, it did not significantly improve the acceptor specificity much further.


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)

Transglucosylation/phosphorolysis ratio for different CLEA formulations. a Cross-linking was initiated upon addition of the imprinting molecules; b Cross-linking was initiated after 30 min incubation; c 200 mM α-glucosyl glycerol (αGG) was used; d 1 M αGG was used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijms-13-11333: Transglucosylation/phosphorolysis ratio for different CLEA formulations. a Cross-linking was initiated upon addition of the imprinting molecules; b Cross-linking was initiated after 30 min incubation; c 200 mM α-glucosyl glycerol (αGG) was used; d 1 M αGG was used.
Mentions: The specificity of the obtained formulations was then examined and compared with that of a non-imprinted CLEA. More specifically, their activity towards phosphate (phosphorolysis) and glycerol (transglucosylation) as acceptor was determined. Imprinting with sucrose, glycerol or a combination of both was found to have no influence on the specificity of the enzyme preparation (Figure 2). However, initiating aggregation and cross-linking after 30 min incubation with sucrose and glycerol did significantly increase the transglucosylation activity. This must mean that the presence of glycosylated product is required to modify the enzyme’s acceptor specificity. Indeed, imprinting with 200 mM αGG almost doubled the specific activity on glycerol, from 2.22 U/mg for the non-imprinted CLEA to 4.12 U/mg for the iCLEA. Increasing the αGG concentration to 1 M during imprinting. However, it did not significantly improve the acceptor specificity much further.

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