Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter sp. Critical Effect of the Crosslinking Agent

Mayerlenis Rojas,Roberto Fernandez-Lafuente,Paulo Tardioli,Gisella Zanin,Raquel Giordano,Murilo Amaral-Fonseca

doi:10.3390/catal9020120

Abstract

Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration of the crosslinker) were evaluated on the production of CLEAs of CGTase. Among the seven studied precipitants, acetone with a 75% (v/v) concentration produced the aggregates of CGTase with higher activity, which retained 97% of the initial activity. Concerning the cross-linker (glutaraldehyde, starch–aldehyde, and pectin–aldehyde), starch–aldehyde produced the most active CLEAs. The use of bovine serum albumin as co-feeder decreased the expressed activity. Addition of polyethylenimine at the end of cross-linking step prevented the leakage of the enzyme and the subsequent Schiff’s bases reduction with sodium borohydride permitted to maintain 24% of the initial activity even with the large dextrin as substrate. The optimal conditions for the immobilization process required were defined as 75% (v/v) acetone as precipitation reagent for 1 h at 20 °C, 20 mM starch–aldehyde as crosslinking reagent for 2 h at 20 °C, treatment with 1 mg/mL of polyethylenimine for 5 min, reduction with 1 mg/mL of sodium borohydride. The CLEAs of CGTase were active catalyst (similarly to the free enzyme) in the production of cyclodextrins at 50 °C and pH 6.0 for 6 h reaction, maintaining intact their structures. Besides this, after five cycles of 3 h the total cyclodextrin yield was 80% of the initial value (first batch, with around 45% CD yield).

Highlights

Cyclodextrin glucosyltransferases (CGTase) (EC 2.4.1.19) are extracellular microbial enzymes capable of converting starch and their derivatives into cyclodextrins (CDs), which are non-reducingCatalysts 2019, 9, 120; doi:10.3390/catal9020120 www.mdpi.com/journal/catalystsCatalysts 2019, 9, 120 cyclic oligosaccharides formed commonly by 6, 7, or 8 glucose units linked by α(1→4) bonds, namely of α, β, and γ-CD, respectively [1,2]
We evaluated the reduction with sodium borohydride in the presence of β-CD, maltose, and maltotriose, because these compounds might protect the the reduction of Crosslinked enzyme aggregates (CLEAs) with sodium borohydride produces secondary amino bonds active site of the enzyme against deleterious effect of borohydride [49,50,51]
The results presented in this work showed that the free-carrier aggregation/crosslinking is a promising method for immobilizing cyclodextrin glucosyltransferase (CGTase) when properly performed

Summary

Introduction

Cyclodextrin glucosyltransferases (CGTase) (EC 2.4.1.19) are extracellular microbial enzymes capable of converting starch and their derivatives into cyclodextrins (CDs), which are non-reducingCatalysts 2019, 9, 120; doi:10.3390/catal9020120 www.mdpi.com/journal/catalystsCatalysts 2019, 9, 120 cyclic oligosaccharides formed commonly by 6, 7, or 8 glucose units linked by α(1→4) bonds, namely of α-, β-, and γ-CD, respectively [1,2]. CDs have a truncated cone shape with a hydrophobic inner cavity that allows encapsulation of hydrophobic organic molecules, increasing its solubility in some media and increasing its stability. In addition to the cyclization reaction (CD production), this enzyme catalyzes other two intermolecular transglycosylation reactions (coupling and disproportionation) and the hydrolysis reaction of the starch [3,5]. It is mainly used at industrial scale for the production of cyclodextrins, recent studies performed by Yu et al [6] have demonstrated that CGTase from

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