Enhancement of enzymatic catalysis of Bacillus amyloliquefaciens α‐amylase by nonionic surfactant micelles

Abstract

Some enzymes are considerably more stable when formulated with nonionic surfactants than when formulated with anionic surfactants. The effect of a nonionic surfactant, polyoxyethylene mono-N-dodecyl ether (Brij 35; number of units of ethylene oxide moieties, 23), on the kinetic behavior of hydrolysis of amylopectin with Bacillus amyloliquefaciens α-amylase (BAA) was studied at a temperature of 25°C and a pH of 7.0. The hydrolytic rate was accelerated by the addition of the nonionic surfactant above its critical micelle concentration. Lineweaver-Burk plots for the enzymatic hydrolysis in the absence and presence of the nonionic surfactant at 0.5 to 2.5% (wt/vol) had linear relationships, and the kinetic parameters, K m and k cat , were obtained. The value of k cat was increased with an increased concentration of Brij 35, whereas the K m value was approximately constant. Therefore, the increase in k cat contributed to the acceleration of the apparent hydrolytic rate. The interaction of amylopectin with the surfactant was examined by a surface tension measurement, and the result confirmed the corresponding binding between the substrate and the surfactant. A fluorescence analysis due to tryptophan in BAA suggested that BAA bound to the nonionic micelles. The increase in k cat suggested that hydrolytic catalysis at the micellar pseudophase was more efficient than that at the aqueous pseudophase. The enhancement of the catalytic rate contributed to the effective removal of food stains containing starch when BAA was added with Brij 35 in a laundry detergent washing test.Amphiphilic media such as micelles and microemulsion droplets can influence enzyme structure and activity (1,2). An investigation of the interaction between synthetic surfactants and enzymes in aqueous solutions has shown that there is a range of possible surfactant effects on the activity of enzymes (3,4). The micelle-forming anionic surfactants are known to alter protein and substrate conformations because of the existence of strong electrostatic and hydrophobic interactions between the micelles and the proteins (5,6). Conversely, nonionic surfactants do not alter protein conformation in comparison with anionic surfactants since they are free of any electrostatic interaction (7). Moreover, some nonionic surfactants reportedly increase the catalytic activity of enzymes. For example, polyethyleneglycol mono-N-dodecyl ether (Brij 35; number of units of ethylene oxide moieties, 23) stimulates cytoplasmic glycerol-3-phosphate dehydrogenase 1.5-fold (8).α-Amylase (EC 3.2.1.1; 1,4-α-D-glucan glucanohydrolase) hydrolyzes starch by cleaving the internal α-1,4-glucosidic bonds (9-12). This enzyme, produced from genus Bacillus, has been studied extensively for industrial applications in the sugar, brewing, alcohol, desizing, and detergent industries (13,14). The nucleotide sequence of the α-amylase genes from Bacillus amyloliquefaciens has been determined (15). The kinetic properties of this enzyme with use of linear maltooligosaccharides have been studied in det...

Modification of the activity of an a-amylase from Bacillus licheniformis by several surfactants

“…All these surfactants were assayed at concentrations above their corresponding CMC, resulting in a slight increase in the enzymatic activity in all cases, in agreement with the work by Hoshino and Tanaka (2003). On the other hand, these results are the opposite of the observations with the fatty alcohol ethoxylates assayed.…”

“…The mean AER is slightly higher at concentrations below the CMC, when matched to the AER at concentrations above the CMC, the reverse situation to that described by other authors, who reported an increase in the reaction rate at concentrations above the CMC for non-ionic surfactants (Hoshino and Tanaka, 2003). Nevertheless, it should be pointed out that the differences between the mean AER values at concentrations above and below the CMC are very small, these values also being very close to 100%; it can therefore be concluded that the fatty alcohol ethoxylate Findet 1214N/23 does not significantly affect the enzymatic activity.…”

“…With respect to the substrate, in the case of starch, it is known that amylose, mainly, and to a lesser extent amylopectin can interact with surfactants, giving rise to inclusion complexes (Svensson et al 1996;Lundqvist et al 2002a;Lundqvist et al 2002b;Lundqvist et al 2002c;Tanaka and Hoshino, 2002;Hoshino and Tanaka, 2003). The formation of these complexes between the surfactants and starch can hamper their enzymatic hydrolysis with amylases; thus, it has been confirmed that the formation of surfactant complexes with amylose hinders enzymatic hydrolysis with β-amylase, being only partially hydrolysable (Kim and Robinson, 1979).…”

“…On the contrary, non-ionic surfactants do not alter the conformation of the protein, since they do not cause any electrostatic interaction (Russell and Britton, 2002) and, moreover, some non-ionic surfactants increase the catalytic activity of enzymes. On studying the effect of the non-ionic surfactant polyoxyethylene mono-N-dodecyl ether (Brij 35) on the enzymatic hydrolysis of amylopectin from potato with an α-amylase from Bacillus amyloliquefaciens, it was found that the reaction rate raised with an increasing concentration of the surfactant above its critical micelle concentration while the non-aggregated surfactant molecules did not boost the reaction rate (Hoshino and Tanaka, 2003). It has been shown that amylopectin binds to surfactant and that enzyme binds to the micelles of the surfactant in such a way that the catalysis appears to be more efficient in the micellar pseudophase than in the aqueous pseudophase.…”

Surfactant enhanced lipase containing films characterized by confocal laser scanning microscopy

“…This observation is consistent with previous studies indicating increased enzymatic activity in surfactant containing emulsions. [4,19] For example, surfactants such as Triton X-100 and Tween-80, enhanced activity and stability in solutions of enzymes. [4,20] In this study, the maximum amount of surface accumulated lipase was observed with the addition of Teric 475 (1.8wt/wt%, Figure 1 …”