Galactosidase Gal EC is an enzyme exoglycosidase and the
α-Galactosidase (α-Gal, EC 18.104.22.168) is an enzyme exoglycosidase, and the main role of this enzyme is the enzymolysis of α-galactoside. After this hydrolysis, galactosylation of cyclodextrins by the α-galactosidase occurs, thus forming α-galactosyl-cyclodextrin. This branched CD is easy to dissolve in water, and has several applications. Some previous studies have found that alphathe α-galactopyranosyl-cyclodextrin (α-galactopyranosyl-CD) can be used as a tool in the delivery of some important healthy compounds. For instance, some authors found that α-galactopyranosyl-CD increased significantly the solubility of retinoic Pirinixic Acid mg (RA) in liver cells. And other works showed the inhibitory effect of α-galactopyranosyl-CD on the combination of lactose-carrying styrene homopolymer (PVLA) and liver cancer cells in vitro, thus showing the ability of this complex to be used as drug target for liver cells to the carrier (Abe et al., 2002, Connolly et al., 1982, Oda et al., 2008, Seo et al., 2004, Shinoda et al., 1998).
Some previous studies have used the α-Gal from green coffee beans to synthesize the α-galactopyranosyl-cyclodextrin (α-galactopyranosyl-CD), being the enzymatic synthesis the only existing method. However, the yield of the α- galactopyranosyl--CD was low (about 24%) (Hara et al., 1994, Kitahata et al., 1992, Koizumi et al., 1995, Okada et al., 1996).
In this line, some research results showed that CDs may change the process of enzymatic reaction and form complexes with special groups of the enzymes, thus activating or inhibiting the enzyme activity. Moreover, they also affected both the reaction rate and equilibrium (Hamilton, 2000, Pinotsis et al., 2003, Thoma and Koshland, 1960).
In previous studies, the interaction between glucoamylase and CDs was studied. It was observed the potential of glucoamylase to induce proteolytic removal on substrate specificity and its subsequent inhibition by β-CD (Monma et al., 1989, Fagerström, 1994). In addition, some other authors, found biomimetic reactions catalyzed by CDs and their derivatives (Breslow & Dong, 1998).
Overall, it has been shown that CDs increased the utility of enzymes in organic synthesis (Harper, Easton, & Lincoln, 2000). For instance, some previous results showed the ability of CDs to limit substrate inhibition and alter substrate selectivity displayed by enzymes (Easton, Harper, Head, Lee, & Lincoln, 2001). And, other authors investigated the inhibition mechanism of β-CD on pullulanase (Yu, Tian, Yang, Xu, & Jin, 2011), thus concluding that CDs and derivatives decreased the activity of this enzyme.
Therefore, this work will focus on understanding the inhibition induced by α-, β- and γ-CD on α-Gal. Moreover, the theoretical foundation for reducing the inhibitory effect of CDs on α-Gal and improving the synthesis efficiency of α-galactopyranosyl-CD complex will be established. For this purpose, the activity of α-Gal will be determined by measuring the concentration of the substrate p-nitrophenyl-α-d-galactopyranoside (PNGP). Moreover, the α-Gal enzyme activities will be evaluated and compared without addition of CDs and after adding CDs. The best inhibitory effect will be selected by comparing the ability of α-, β- and γ-CD to inhibit the α-Gal enzyme at different conditions (temperature, pH, concentration, time, etc.). For this purpose, circular dichroism and nuclear magnetic resonance techniques will be used.
Materials and methods
Results and discussion
Conclusions This work successfully investigated the effects of the different influential factors and hydrophobic cavities of CDs on α-Gal by exploring α-Gal activity. The highest inhibitory conditions on α-Gal were obtained when the concentration of CDs was 10mM, reaction time 90min, temperature 30°C, and pH 6.0. α-CD, β-CD and γ-CD can change the secondary structure of α-Gal in different degrees, observing different levels of α-Gal activity inhibition according to the type of CDs. α-Gal activity inhibition decreased in the following row β-CD (81%)>γ-CD (40%)>α-CD (36%). Therefore, the results established the stronger ability of β-CD to interact with α-Gal compared to α-CD and γ-CD, due to its appropriate geometric dimensions and the potential use of CDs to manipulate the concentrations of α-Gal substrates.