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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
Synthesis of αGG with immobilized SP at 60 °C. The conversions were performed with 5 mg/mL CLEAs (○) or iCLEAs (●) in a 50 mM MOPS buffer pH 7 containing 800 mM sucrose and 2 M glycerol.
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f4-ijms-13-11333: Synthesis of αGG with immobilized SP at 60 °C. The conversions were performed with 5 mg/mL CLEAs (○) or iCLEAs (●) in a 50 mM MOPS buffer pH 7 containing 800 mM sucrose and 2 M glycerol.

Mentions: To further asses its applicability in carbohydrate conversions, the thermostability of the iCLEA preparation was evaluated by incubation at 60 °C and measuring the residual activity at several points in time (Figure 3). The iCLEA was found to exhibit a comparable thermostability to the non-imprinted CLEA, and thus to be drastically more stable than the soluble enzyme. Indeed, an impressive 75% of the initial activity was still present after three weeks incubation at 60 °C. Next, the efficiency of the newly formed iCLEA was demonstrated by comparing its performance with that of the reference CLEA in a batch production of αGG at 60 °C (Figure 4). This clearly showed that the maximal transfer yield of 84% [5] could be reached twice as fast with the iCLEA than with its non-imprinted counterpart. Furthermore, the newly formed iCLEA also proved its excellent operational stability by losing no activity whatsoever during five consecutive batch conversions (data not shown).


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)

Synthesis of αGG with immobilized SP at 60 °C. The conversions were performed with 5 mg/mL CLEAs (○) or iCLEAs (●) in a 50 mM MOPS buffer pH 7 containing 800 mM sucrose and 2 M glycerol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijms-13-11333: Synthesis of αGG with immobilized SP at 60 °C. The conversions were performed with 5 mg/mL CLEAs (○) or iCLEAs (●) in a 50 mM MOPS buffer pH 7 containing 800 mM sucrose and 2 M glycerol.
Mentions: To further asses its applicability in carbohydrate conversions, the thermostability of the iCLEA preparation was evaluated by incubation at 60 °C and measuring the residual activity at several points in time (Figure 3). The iCLEA was found to exhibit a comparable thermostability to the non-imprinted CLEA, and thus to be drastically more stable than the soluble enzyme. Indeed, an impressive 75% of the initial activity was still present after three weeks incubation at 60 °C. Next, the efficiency of the newly formed iCLEA was demonstrated by comparing its performance with that of the reference CLEA in a batch production of αGG at 60 °C (Figure 4). This clearly showed that the maximal transfer yield of 84% [5] could be reached twice as fast with the iCLEA than with its non-imprinted counterpart. Furthermore, the newly formed iCLEA also proved its excellent operational stability by losing no activity whatsoever during five consecutive batch conversions (data not shown).

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