Amylases represent one of the most important enzyme groups within the field of biotechnology. These enzymes are present in numerous biotechnological and industrial applications, including their use in detergent formulations (Tanaka and Hoshino, 1999). The use of enzymes in detergents offers multiple advantages from an environmental standpoint: energy savings by using lower washing temperatures, replacement or reduction of other more environmentally harmful components, lack of negative effects on the wastewater-treatment systems, and absence of risk for aquatic wildlife.

One of the main problems affecting the action of these enzymes in detergent formulations is the possible influence of surfactants on enzymatic hydrolysis, due to feasible interactions of the surfactant both with the reaction substrate as well as with the enzyme. With respect to the substrate, it is known that amylose and amylopectin are able to form complexes with surfactants (Svensson et al. 1996; Lundqvist et al. 2002b; Tanaka and Hoshino, 2002; Hoshino and Tanaka, 2003) which can hamper their enzymatic hydrolysis by amylases (Kim and Robinson, 1979). With regard to the enzyme, many enzymes are unstable in solutions of anionic surfactants (Tanaka and Hoshino, 1999; Tanaka and Hoshino, 2002), and the formation of micelles can also affect enzyme kinetics (Tanaka and Hoshino, 2002). Furthermore, some enzymes are more stable in the presence of non-ionic surfactants than in the presence of anionic surfactants. 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 such as α-amylases (Hoshino and Tanaka, 2003). Non-ionic surfactants such as fatty alcohol ethoxylates have also been verified to stabilize proteases in the presence of anionic surfactants (Russell and Britton, 2002), and others such as alkyl polyglycosides are capable of increasing enzyme stability in liquid-detergent formulations (von Rybinski and Hill, 1998).

In the present work, the influence of several non-ionic surfactants, alkyl polyglycosides, fatty alcohol ethoxylates, and nonyl phenol ethoxylate is studied in relation to the activity of a commercial α-amylase, comparing them with a classical anionic surfactant, linear alkyl benzene sulfonate (LAS).

An α-amylase of bacterial origin (Bacillus licheniformis) from Novozymes A/S, Termamyl^® 300 L Type DX was tested. As non-ionic surfactants, alkyl polyglycosides Glucopon^® 215 CS UP, Glucopon^® 600 CS UP, and Glucopon^® 650 EC (from Cognis Deutschland GmbH&Co) were used, as well as fatty alcohol ethoxylates, Findet 10/15 and Findet 1214N/23 (from Kao Corporation S.A.), and nonyl phenol (with 9.5 moles of ethylene oxide) under the commercial name of Findet 9Q/21.5NF (from Kao Corporation S.A.). In addition, an anionic surfactant was assayed, linear alkyl benzene sulfonate, LAS, from Kao Corporation S.A.

A standard method (Novozymes A/S, 2001b), based on the kit Phadebas^® Amylase Test from PHARMACIA Diagnostics AB, was used to determine the activity of the α-amylase in the presence of the surfactants. The relative enzymatic activity, AER, was defined as the quotient between the activity in the presence of the surfactant, AE_T, and the activity in its absence, AE. For each surfactant, the relative activity of the enzyme was measured at two different concentrations. Duplicate runs, at least, were performed

Findet 1214N/23 showed AER slightly higher at concentrations below the critical micelle concentration, CMC, when matched to the AER at concentrations above the CMC, the reverse situation to that described by other authors (Hoshino and Tanaka, 2003). Nevertheless, it should be pointed out that the differences were very small, these values also being very close to 100%. Therefore the fatty alcohol ethoxylate Findet 1214N/23 did not significantly affect the enzymatic activity. Regarding Findet 10/15 very significant losses in enzymatic activity were noted. This loss in activity increased with the surfactant concentration up to an AER value of 40.1% for 1.0 g/L. This situation was not foreseeable, since this is a non-ionic surfactant. However, Findet 10/15 presents the lowest hydrophilic-lipophilic balance, HLB of all non-ionic surfactants assayed, being slightly lower than 10; thus its higher hydrophobic character may determine a greater interaction with the enzyme and hence a reduction in the enzymatic activity. The last non-ionic ethoxylate surfactant studied was Findet 9Q/21.5F, showing a very slight loss of enzymatic activity in the presence of the surfactant, which furthermore did not depend on the surfactant concentration.

In the case of alkyl polyglycosides, Glucopon® 215, Glucopon® 600, and Glucopon® 650, experiments showed a slight increase in the enzymatic activity in all cases, in agreement with the work by Hoshino and Tanaka (2003). In any case, given that the increase in enzymatic activity that is observed in the presence of these alkyl polyglycosides is very small, it can be concluded that they do not significantly affect the activity of the α-amylase tested.

The anionic surfactant LAS was found to significantly decrease the activity of the α-amylase studied, even at concentrations lower than its CMC. An AER value of 62.0% for 0.2 g/L was observed, as opposed to the results previously published with another anionic surfactant, SDS (Tanaka and Hoshino, 2002). This noteworthy loss in enzymatic activity is most likely due to the electrostatic interactions inherent to the anionic character of LAS.

Finally the relationship between AER and HLB was studied. For alkyl polyglycosides, the AER remains approximately constant and independent of the HLB value. On the contrary, considering the ethoxylate surfactants, the enzymatic activity declined on decreasing the HLB, in such a way that the less hydrophilic character of Findet 9Q/21.5F with respect to Findet 1214N/23 could be the cause of the small loss in enzymatic activity detected in the assays made with Findet 9Q/21.5F. For HLB values lower than 10, as in the case of Findet 10/15, where the hydrophobic groups rules out in the molecule considered, there is a very significant loss of enzymatic activity.

From the results, it can be deduced that when α-amylase from Bacillus licheniformis is to be used in detergent formulations, and from the standpoint of the possible alteration of its enzymatic activity, the most suitable surfactants to be included in these formulations would be alkyl polyglycosides, as they even tend to increase the activity somewhat. In the case of the ethoxylate surfactants studied a loss of enzymatic activity is observed, being more significant the lower the surfactant HLB value. This loss is proved to be significant also in the case of the classical anionic surfactant LAS.

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