Article pubs.acs.org/JAFC
Nanosilica Sol Leads to Further Increase in Polyethylene Glycol (PEG) 1000-Enhanced Thermostability of β‑Cyclodextrin Glycosyltransferase from Bacillus circulans Caiming Li,†,‡ Min Huang,†,‡ Zhengbiao Gu,*,†,‡,§ Yan Hong,†,‡,§ Li Cheng,†,‡ and Zhaofeng Li*,†,‡,§ †
State Key Laboratory of Food Science and Technology, ‡School of Food Science and Technology, and §Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, People’s Republic of China ABSTRACT: A major disadvantage of cyclodextrin production is the limited thermostability of cyclodextrin glycosyltransferase. The ability of combinations of nanosilica sol with polyethylene glycol (PEG) 1000 to enhance the thermostability of the βcyclodextrin glycosyltransferase from Bacillus circulans was investigated. It was found that 10% PEG 1000 combined with 0.05% nanosilica sol could activate the β-cyclodextrin glycosyltransferase by 17.2%. Furthermore, 0.05% nanosilica sol leads to further increase in PEG 1000-enhanced thermostability of β-cyclodextrin glycosyltransferase. With the simultaneous addition of 10% PEG 1000 and 0.05% nanosilica into the enzyme solution, which was allowed to incubate for 60 min at 60 °C, 61.3% of βcyclodextrin-forming activity could be retained, which was much higher than that with only 10% PEG 1000 added. Atomic force microscopy, fluorescence spectroscopy, and circular dichroism analysis indicated that silica nanoparticles helped PEG 1000 further protect the tertiary and secondary structures of β-cyclodextrin glycosyltransferase. This study provides an effective approach for improving the thermostability of cyclodextrin glycosyltransferase and related enzymes. KEYWORDS: cyclodextrin, CGTase, polyethylene glycol, thermostability, silica nanoparticles
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INTRODUCTION Cyclodextrins are industrially important cyclic oligomers of glucose that form inclusion complexes with a variety of small hydrophobic guest molecules.1 Complex formation increases the aqueous solubility of the guests, altering their physical and chemical properties.2 Cyclodextrins have found numerous applications in the food, agricultural, cosmetic, pharmaceutical, and chemical industries3,4 and have the potential for more widespread use if more efficient methods of production can be devised. The industrial production of cyclodextrins is currently performed by treating starch with cyclodextrin glycosyltransferases at elevated temperatures. These elevated reaction temperatures help reduce the risk of microbial contamination and influence the solubility and bioavailability of organic compounds.5 High process temperatures also increase the diffusion coefficient and decrease the viscosity of substrates, leading to an increase in reactivity, greater process stability, and increased process yields.6 Cyclodextrin glycosyltransferases (CGTases; EC 2.4.1.19) convert starch, or starch derivatives, into cyclodextrins by intramolecular transglycosylation.7 CGTases also catalyze the hydrolysis, disproportionation, and coupling of these same substrates, but cyclization is their predominant enzymatic activity.8 All of the CGTases currently available produce mixtures of α-, β-, and γ-cyclodextrins, which contain six, seven, and eight glucose monomers, respectively. However, each CGTase produces one cyclodextrin as its predominant product during the initial stage of substrate conversion. This has allowed the classification of CGTases into three main groups (α-, β-, and γ-CGTases) on the basis of this product preference.9 Because the industrial production of cyclodextrins is carried out at elevated temperatures, the CGTases employed must have excellent stability at these temperatures. The thermo© 2014 American Chemical Society
stability of the CGTase, like any enzyme, depends on both its amino acid sequence and environmental factors, such as the presence of metal ions or additives in the buffer and the pH of the process.10,11 The thermal stability of the enzyme being used in an industrial process can be increased in several ways. The existing enzyme can be replaced by a more thermostable enzyme from a different source; its thermostability can be enhanced by chemical modification, protein engineering, or immobilization; or the reaction buffer can be altered using additives, such as polyhydroxy compounds, to increase the existing enzyme’s thermostability.12,13 Polyethylene glycol (PEG), a hydrophilic nonionic polymer, is used widely to increase the thermostability of enzymes. PEG forms directional bonds with water and exhibits complex phase behavior; it can also bind weakly to proteins.14 The mechanisms by which PEG molecules enhance protein stability are complex, but have generally been attributed to a preferential hydration of the protein resulting from the steric exclusion of polymer from the vicinity of the protein.15 However, PEG may also interact with, and possibly bind to, accessible nonpolar surface residues. Our previous studies have shown that the β-CGTase from Bacillus circulans STB01 has a half-life of
