Preparation and characterization of inclusion complexes containing fixolide, a synthetic musk fragrance and cyclodextrins

AHTN (7-Acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene), commercially known as fixolide or tonalide, is a synthetic fragrance widely used in replace of natural musk odor which is more expensive. It is a popular fragrance material added in the manufacturing of personal care and household products, such as perfumes, soaps, shampoos, detergents, and fabric softeners. AHTN is semivolatile and is degraded under light exposure and high temperature. This work focuses on the complexation of AHTN with cyclodextrins in the effort to stabilize the fragrance material. AHTN was complexed with β-cyclodextrin, methyl (MβCD), and hydroxypropyl (HPβCD) derivatives in the mole ratio 1:1, 1:2, and 1:3 guest:host, and the complexes formed by physical mixing, co-precipitation, kneading, and freeze-drying were analyzed by DSC and FTIR. Percent AHTN included in the complex was also determined by hexane extraction and GC analysis. It was found that no inclusion complex was formed in the physical mixture. When co-precipitation method was performed, only βCD could form inclusion complex with AHTN, while the other two derivatives could not. Using 1:2 AHTN:βCD, no free AHTN was left in the complex as evidenced by DSC and FTIR spectrum. In kneading and freeze-drying methods, complexes could be formed with all CDs tested. However, co-precipitation method with 1:2 AHTN:βCD and kneading method with 1:2 AHTN:MβCD provided the highest complex yield with highest amount of AHTN included in the complex. AHTN in the complex form was more stable against high temperature and UV exposure than its free form.     

Biochemical properties and cyclodextrin production profiles of isoforms of cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to cyclodextrin (CD), an important host molecule for the study of host?Cguest interactions. CGTase from Paenibacillus sp. RB01 and its recombinant form showed the same isoform pattern. The three isoforms, two major (isoforms I and II) and one minor (isoform III), all had a different net charge but the same molecular mass. The aim of this work was to characterize the three isoforms, and especially to compare their CD production profiles. Isoforms I and II were separated on a FPLC Mono Q column and showed the same optimum pH (pH 5 for dextrinizing and pH 6?C7 for cyclization activity) and optimum temperature (65?C70 °C for both activities). However, the two isoforms differed in their catalytic efficiency of the coupling reaction with variable concentrations of the ??-CD donor in the presence of a fixed amount of cellobiose acceptor, with k_cat/K_m values of 3.46 × 10^?3 and 2.20 × 10^?3 mM^?1 min^?1, for isoforms I and II, respectively. Both isoforms I and II were found to have ??-CGTase activity and gave a similar CD6:CD7:CD8 product ratio of 0.2:1.0:0.6, with an increase in the ratio of the small-ring to the large-ring CDs from 1.0:0.5 to 1.0:0.3 from a 6 to 24 h reaction time. However, in terms of maximal CD yields, the two isoforms differed in their optimal reaction temperature and time required, the optimal conditions being at 40 °C for 6 h for isoform I and at 60 °C for 24 h for isoform II.     

A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products

Amylomaltase catalyzes the formation of large-ring cyclodextrins (LR-CDs) from starch. This study aims to construct the recombinant amylomaltase from Corynebacterium glutamicum and to characterize the purified enzyme with the emphasis on the profile of LR-CDs production. A novel amylomaltase from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21 (DE3) using the expression vector pET-19b. The open reading frame of amylomaltase gene of 2,121 bp (encoding the polypeptide of 706 amino acid residues) was obtained with the N-terminal His-tag fragment of 69 bp attached before the start codon of the amylomaltase gene. The deduced amino acid sequence showed a low sequence identity (20?C25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 °C for 2 h after induction with 0.4 mM isopropyl thio-??-D-galactoside (IPTG). The enzyme was 11-fold purified with a yield of 30% by a HiTrap affinity column. The purified amylomaltase showed a single band of 84 kDa on a 7.5% SDS-PAGE. When the enzyme acted on pea starch, it catalyzed an intramolecular transglucosylation (cyclization) reaction that produced LR-CDs or cycloamyloses (CA). The product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. At 4 h incubation, the product was composed of a mixture of LR-CDs in the range of CD19?CCD50, with CD27?C28 as products with highest amount. It is noted that CD19 was the smallest product in all conditions tested. The enzyme also catalyzes intermolecular transglucosylation on various malto-oligosaccharides, with maltose as the smallest substrate.     

Production of Large-Ring Cyclodextrins by Amylomaltases

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60–70 °C for thermostable amylomaltases and 30–45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0–7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases.     

Formation of Inclusion Complexes between Cyclodextrins and Carbaryl and Characterization of the Complexes

The solubility of carbaryl increased with increasing concentrations of-CD, G₂--CD, and M--CD. The result suggests theformation of soluble inclusion complex. Solubility increase was highestin M--CD-carbaryl, being 18.4 fold higher than that of carbaryl when 100 mM M--CD was used. The apparent formation constant for the complex calculated from phase solubility diagram was 223.18 M^-1. The preparation of the complex in solid form for characterization was successful by kneading andfreeze-drying. The DSC curves for kneading and freeze-drying mixture didnot show the endothermic peak characteristic of carbaryl, but a small new endothermic peak was observed. FTIR analysis showed a shift of the major peak of carbonyl group in carbaryl molecule from 1717 to 1744 and 1734 cm^-1 in kneading and freeze-dried mixtures, respectively. M--CD-carbaryl complex demonstrated higher dissolution rate, higher thermal and UV stability but lower toxicity than its parent carbaryl compound.