2.1. Stability of T1 Lipase, Rand Protease and Maltogenic Amylase In Detergent Components
With the same protein concentrations of T1 lipase, Rand protease and Maltogenic amylase, 15, 100 and 230 U/mL enzyme activities were found and referred to as 1X, 1Y and 1Z, respectively. This unit of enzymes has been used for the optimization of all detergent components, including surfactants, bleaches, builders, dispersing agents and alkalinity agents. The stability of T1 lipase, Rand protease and Maltogenic amylase were checked via a compatibility test. The results are shown in Table 1
The results show that non-ionic surfactants were compatible for all three enzymes except SDS, a cationic surfactant, which destabilized all three enzymes. T1 lipase was most stable in the presence of G600, followed by Tween 80, PEG300. Sugar alcohol could lessen the thermal denaturation and allow an enzyme to react and release more products, which explains the over-activity of T1 lipase in the presence of G600 [3
]. T1 lipase was slightly inhibited (15%) by Tween 80 and PEG300. Rand protease was very stable in both Tween 80 and G600. Tween 80 activated Rand protease, that might be due to the unfolding of the substrate moiety by surfactant [8
]. The relative activity of Maltogenic amylase was increased by G600, followed by PEG 300. Also, a decrease of less than 7% was observed in the relative activity of Maltogenic amylase in the presence of Tween 80. Overall, G600 was the best surfactant for all three enzymes, since it stabilized them. Similarly, von Rybinski and Hill [9
] reported on the stability of lipase, protease, amylase and cellulase in the presence of alkyl polyglucoside, G600. SDS is an anionic surfactant which is well known in detergent formulation. However, SDS destabilized the T1 lipase, Rand protease and Maltogenic amylase tested in this study. The ionic interactions between enzymes and the SDS head group may cause the inactivation of globular proteins [10
Bleaching agents, sodium percarbonate, and sodium perborate destabilized all three enzymes (Table 1
). The bleaching agents released hydrogen peroxide—an oxidizing agent—when dissolved into water. This could easily lead to the oxidization of some amino acids and cause the inactivation of enzymes. Most commercial proteases have been reported to be unstable in the presence of bleaching agents [11
]. However, Maxamal is a genetically modified protease produced by mutagenesis that has shown improved stability in the presence of bleaching agents [12
]. Other examples of oxidative stable mutants enzymes include Durazym, Purafect [11
] and Lipolase®
Sodium polyacrylate was the only dispersing agent tested in this study (Table 1
). The activity of T1 lipase and Rand protease were moderately affected by polyacrylate, whereas Maltogenic amylase showed more than 70% stability compared to the control.
The builders tested in this study had a different impact on the stability of the enzymes. In the presence of sodium citrate, T1 lipase and Rand protease were moderately stable and Maltogenic amylase retained 96% of its stability. However, sodium metasilicate and sodium silicate destabilized all three enzymes. Since T1 lipase and Rand protease are Ca2+
dependent enzymes, this builder likely competes with the enzymes for the available Ca2+
ions, which can contribute to effect on the stability of enzymes [13
A compatibility test of T1 lipase, Rand protease, and Maltogenic amylase with different alkalinity agents is presented in Table 1
. Rand protease was reported to have a broad range of pH stability of between pH 6.5 to 10.0 [14
], whereas T1 lipase was reported to be stable between pH 6.0 to 11.0 [15
] and Maltogenic amylase was found to be stable in the range of pH 6.0 to 9.0 [16
]. Since they have similar ranges, it is easier to find suitable alkalinity agents for them. T1 lipase has an optimum pH of 9.0, whereas Rand protease and Maltogenic amylase have an optimum pH of 7.0. All three enzymes showed high stability in Sodium carbonate-glycine, at a pH of 9.25. However, the mixture of SC: SB at pH 9.5 drastically decreased the activity of T1 lipase and also had a moderate effect on Rand protease and Maltogenic amylase which indicated that the enzymes are affected by the component of the buffer, not only the pH.
Water hardness can be reduced, as most alkalinity agents tend to bind to cations. Phosphate pH 7.0 and Sodium bicarbonate pH 8.6 reduced the relative activity of T1 lipase. This might be due to the binding of phosphate and carbonate to Ca2+
, producing Ca3
], while Ca2+
is an essential ion for T1 lipase structural stability [15
]. Rand protease was found to be stable in Phosphate and Tris-HCl buffers, pH 7.0. However, Tris-HCl buffer at a pH of 7.0 decreased the activity of T1 lipase and Maltogenic amylase. Sodium bicarbonate, pH 8.6, increased the activity of Maltogenic amylase much higher than did the other buffers tested in this study, indicating that the CaCO3
precipitates could help stabilize the enzyme. Sulong [16
] reported a drastic inhibition of the activity of Maltogenic amylase in the presence of Ca2+
ions in the reaction mixture.
In summary, Glucopon UP 600 as surfactant, sodium polyacrylate as the dispersing agent, sodium citrate as builder, and Sodium carbonate–glycine (30:70), pH 9.25, as buffer were used in a new ADD formulation. None of the bleaching agents were added to the formulation, which makes the new ADD more environmentally friendly. Bleaching agents are usually used to break down stains and kill bacteria, both of which can be done using enzymes. Moreover, using the new ADD, washing will be performed at high temperatures, as hot water can kill most bacteria.
2.3. Efficiency of Individual Enzyme Concentration
In the next experiment, the soil removal performance of the formulated detergent with the addition of individual free enzymes at different concentration was investigated to determine the best amount of lipase, protease and amylase required in the formulation (Figures S1 and S2
A different amount of each enzyme was individually added into the soft and hard water at 60 °C. The results showed that the high concentration of T1 lipase, Rand protease and Maltogenic amylase did not substantially improve washing. Based on post-hoc analysis, there was no signification difference in the lowest amount of enzyme compared to other amounts in soft water (Table S2
). However, in hard water, post-hoc analysis showed significant differences between 1% and the higher amounts of T1 lipase, between 3% and 6% and the higher amount of Rand protease and between 1% and the higher amounts of Maltogenic amylase (Table S3
). Therefore, 3% of T1 lipase, 9% of Rand protease and 1.5% of Maltogenic amylase were incorporated together into the same detergent.
The lowest possible enzyme level with good efficiency used in the detergent formulation is considered economical for future application. Moreover, a study by Hemachander and Puvanakrishnan [18
] showed that the addition of lipase into protease-containing detergents improved the washing efficiency and removal of fatty stains, demonstrating that combinations of more than two enzymes may be a better option.
2.4. Enzymes Encapsulation Performance
Encapsulation of T1 lipase using additives, gum arabic (GA) and maltodextrin (MD) resulted in activity retained compared to the free enzyme (Table 2
). The activity loss was greater in the powder form of the enzyme; the spray dried supernatant containing 3% (w
) T1 lipase without any wall material, compared to the encapsulated or free enzyme—which is the control—before spray drying. The loss of activity in the powder form of the enzyme may be due to the thermal denaturation of the enzyme during the spray drying process.
However, the addition of additives had a positive effect on the enzyme and led to an increase in the total activity. This suggests that the additives protect the enzymes from direct exposure to the spray dryer heat. In addition, it also reduces the stickiness and hygroscopicity of the powder [19
]. Hygroscopicity refers to the ability to absorb moisture from the air.
The blend, consisting of GA/MD/T1 at a ratio of 6:12:3, produced products that were mostly spherical, with a smooth surface and few dents (Figure 2
a). Moreover, the products from this blend also yielded a two times higher residual enzymatic activity than did the free enzyme (Table 2
). The addition of wall materials also made spray-dried products more soluble compared with the free enzyme. This is because GA and MD are hydrophilic and soluble in water—however, GA is highly viscous upon dissolution in water, especially at concentrations exceeding 10% (w
Although the spray-dried product from the blend of GA/MD/T1 yielded decent powder morphology and characteristics as well as satisfactory enzymatic activity retention, the size distribution of the powder was still wide, and many of the powder sizes were still less than 100 μm. At very low particle sizes, the enzyme powder may become a health risk. Therefore, agglomeration or granulation is typically preferable to spray-drying alone.
To prepare the powder form or to encapsulate the Rand protease and Maltogenic amylase, spray drying was performed because both enzymes are thermostable and able to survive at high temperatures. But, due to exposure of the enzymes to high temperatures of 85 °C, their residual activity was very low and almost denatured. Therefore, in the next attempt, the enzymes were freeze dried. The advantages of the enzyme freeze drying include high recoveries of the end products of organic analytes regardless of water content and the prevention of the volatilization of temperature-sensitive analytes. However, in the freeze drying process, using low temperatures to dry an enzyme can induce several stresses that may cause denaturation [20
Based on Table 2
, the enzyme activity of the powder forms of the Rand protease and Maltogenic amylase were lower than the free enzymes, but the results were better than spray drying the enzymes. Even though low temperatures in freeze-drying were generated for the drying process to take place, this can still deactivate and destabilize the enzymes due to dehydration stress [19
]. Hence, stabilizers must be used when freeze-drying in order to decrease the deactivation and destabilization of the enzyme.
After the addition of additives to encapsulate the enzymes, the results showed that both encapsulated Rand protease and Maltogenic amylase produced higher activities compared to the powder form and free enzymes, at 93% and 95% of activity retained, respectively.
This proves that addition of additives to encapsulate the enzymes showed positive effects on both enzymes. A study explained that gum arabic and maltodextrin can act as effective coats during freeze drying for serine proteases [19
]. Gum arabic decreases the hydrophobicity of the product and increases water mobility in the enzyme, whereas maltodextrin provides good oxidative stability to encapsulated enzymes. The dry matter of the powder form of Rand protease without the addition of gum arabic and maltodextrin was too low and sticky. This stickiness was due to residues of the nutrient medium [21
]. A fluent powder was produced after the addition of gum arabic and maltodextrin.
The immobilization of protease (Figure 2
b) and Maltogenic amylase (Figure 2
c) was analyzed using scanning electron microscopy (SEM). The protease was immobilized with gum arabic and maltodextrin additives, whereas the amylase was immobilized with only gum arabic. The scan of the freeze-dried powders under the microscope showed a glass-like structure with a smooth surface. It was reported that during the dehydration process, an amorphous glassy structure was produced to protect the entrapped molecules from opening due to exposure to oxygen and heat [22
2.5. Comparison of Detergent with Free and Encapsulated Enzymes
In the following experiment, the dishwashing efficiency of the formulated detergent after the addition of free and encapsulated enzymes was tested at different water temperatures (40, 50 and 60 °C) and hardness (Figure 3
). Dishwashing temperatures were used. The water hardness used for dishwashing was 0 ppm CaCO3
for soft water (Figure 3
a) and 350 ppm CaCO3
for hard water (Figure 3
b). The detergent containing free enzymes was labeled Detergent A and the detergent containing encapsulated enzymes was labeled Detergent B.
As expected, dishwashing performance improved as the temperature increased for both formulated ADDs with enzymes. Based on Figure 3
a, detergent without enzymes showed around 80% dishwashing performance from 40 to 60 °C in soft water. Meanwhile, both formulated detergents A and B showed no visible difference in dishwashing performance after the addition of enzymes at 21 °C in both soft and hard water. This proves that enzymes are not active at the lower temperature, hence no washing improvement was observed. There are studies that explain that some fats inhibit enzymes reaction at low temperature since they exhibit higher melting points [23
]. In addition, T1 lipase relative activity was reported to decrease up to 75% as the temperature decreases from 70 to 40 °C, which explains the low dishwashing performance at 21 °C. A similar pattern was shown by native Maltogenic amylase, with relative activity decreasing at temperatures lower than 55 °C [16
Based on post hoc studies, in soft water at 50 and 60 °C, the addition of the encapsulated enzyme caused a significant difference in detergent performance compared to the free enzyme. It can be concluded that in soft water encapsulated enzyme detergent may work better in high temperatures.
In hard water, by increasing the temperature, the dishwashing performance after the addition of encapsulated and free enzymes showed significant gains in efficiency compared to the detergent without enzyme. However, the performance of formulated detergents decreases in hard water compared to soft water in the same conditions. A few studies explain that the performance of surfactant can be severely affected by the presence of cations Ca2+
. These cations form highly charged structures with the surfactant, which prevent the removal of soil [24
]. The results showed that incorporation of three encapsulated enzymes into the formulation increased the dishwashing performances when increasing the temperature up to 60 °C. The improvements of the dishwashing performance in both soft and hard water were more dramatic with the addition of encapsulated enzymes at 50 and 60 °C. This may be because both Maltogenic amylase and crude Rand protease work at an optimum temperature of 50 °C. Also, the majority of the detergents have high efficiency in soft water but fail to perform in hard water [25
]. Hence, formulated detergent containing encapsulated enzymes with high efficiency in hard water could fulfill industrial demand.
In hard water, soil removal by Detergent A at 40 °C is lower compared to the higher temperature while this performance is almost same for Detergent B, which contained encapsulated enzymes. The better performance of Detergent A at a higher temperature could be due to the carbonate hardness of water , which can be reduced at higher temperatures [26
]. The carbonate hardness has easier access to the free enzymes compared to the encapsulated enzymes.
Post hoc tests revealed that there is no significant difference between the dishwashing performance of detergents from 50 to 60 °C for both the encapsulated and free enzymes in hard water. Therefore, dishwashing performance is nearly the same. It can be concluded that optimum washing temperature for this detergent is in the range 50 to 60 °C.
2.8. Comparison of Formulated Detergents Containing Enzymes with Commercial
After evaluation of both formulated detergents in both soft and hard water at different temperatures, the formulated detergents with enzymes were compared with a commercial ADD, Finish®
, as shown in Figure 5
Detergent A showed comparable performances with Finish®
at 50 °C in 0 ppm CaCO3
, soft water (Figure 5
a). Detergent B showed a better washing efficiency in soft water at a higher temperature with significant differences (Figure 3
a). Dishwashing performance using Detergent B at 50 and 60 °C in soft water showed no significant improvements—however, these performances are comparable to Finish®
. This suggests that dishwashing using Detergent B can be done at a lower temperature to save electric current and the amount of water used in washing (http://www.indesitservice.co.uk/help-and-advice/dishwasher/
The dishwashing efficiency of formulated detergent with a commercial detergent in hard water was compared and is presented in Figure 5
b. The efficiency of Detergent B is better than Detergent A and Finish®
at 40 °C in hard water. In hard water, both formulated detergents were as efficient as Finish®
at 50 °C, with no significant difference in washing. At 60 °C, in hard water, post hoc tests revealed significant differences between Detergent A, B and Finish®
showed better dishwashing performance, followed by Detergent B and Detergent A. Dishwashing at 60 °C in 350 ppm of CaCO3
with Detergent A, B and Finish®
resulted in 42.5%, 50% and 66% soil removal, respectively. Also, among the other tested temperatures in this study, Finish®
showed superior soil removal performance at 60 °C, which has been reported as the optimal operating temperature for commercial dishwashing.
The formulated detergent containing triple enzymes showed better soil removal than the detergent containing only T1 lipase. Rahman et al. [3
] reported that the formulated detergent containing only T1 lipase showed the maximum washing of 30% at 60 °C in the same condition. This explained that low dishwashing performance was due to the nature of the soil used, which contained fat, proteins, and carbohydrates. Hence, incorporation of three different thermostable hydrolysis enzymes into the formulation increased dishwashing performance.
It is concluded that Detergents A and B showed the best performance in both soft and hard water at temperatures of 50 and 60 °C. However, this would not really be of benefit in most situations, since the same performance can be achieved at a lower temperature to reduce electricity usage and the amount of water used in washing. Thus, the best energy-saving working temperature of these ADDs is 50 °C.