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A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

Amid M, Murshid FS, Manap MY, Hussin M - Biomed Res Int (2015)

Bottom Line: Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied.The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0.The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.

ABSTRACT
A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Show MeSH
Effect of low concentration (20% w/w) (a) and high concentration (60% w/w) (b) of sorbitol on structure and morphology of pectinase enzyme.
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Related In: Results  -  Collection


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fig1: Effect of low concentration (20% w/w) (a) and high concentration (60% w/w) (b) of sorbitol on structure and morphology of pectinase enzyme.

Mentions: Preliminary studies revealed that the enzyme was stable in Triton X-100, Tween 20, SDS, and sorbitol, which indicated that pectinase was suitable for the novel surfactant/sorbitol-based ATPS. Another study proved that surfactants promote the availability of reaction sites and increase the hydrolysis rate [17]. To determine the effects of each phase composition on the pectinase activity, crude feedstock pectinase was mixed with various compounds. The surfactant also induces enzyme activity and eventually reduces the rate of enzyme denaturation during hydrolysis [18] and thus functions as an inducer of enzyme activity by affecting the enzyme-substrate interaction. This process can prevent the inactivation of adsorbed enzymes, which directly facilitates the desorption of enzymes from the substrate [18–20]. High concentrations [60%] of Triton X-100 and Tween-80 slightly increased pectinase activity, whereas SDS partially inactivated this activity (Table 1). This latter effect was due to the binding of SDS, an ionic surfactant, to the proteins, which disrupted the majority of the globular proteins original structures. Moreover, the pectinase was mixed into sorbitol at different concentrations to determine its activity. In the presence of sorbitol, pectinase also exhibited high enzyme stability. The probable reason for this observation is that sorbitol helped to maintain the enzyme's open conformation by exposing the active site crevice surface and thus stimulating pectinase activity [18]. As shown in Table 1, a higher concentration of sorbitol (60%, w/w) inhibited the pectinase activity compared with the pectinase activity in 20% (w/w) sorbitol solution. Morphological observations revealed that, in 60% (w/w) sorbitol and pectinase, severe agglutination and rough surfaces within the interactions were observed, whereas 20% (w/w) sorbitol resulted in clear and smooth surfaces as shown in Figure 1. This observation might have resulted from the high concentration of sorbitol (60%, w/w) causing conformational changes in the pectinase structure that caused enzyme denaturation. Hence, this phenomenon decreased the pectinase activity and resulted in an incompatibility in the denatured enzyme-substrate interaction. Therefore, the authors speculate that the high concentrations of sorbitol denatured the pectinase and decreased its activity compared with the low concentration of sorbitol.


A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

Amid M, Murshid FS, Manap MY, Hussin M - Biomed Res Int (2015)

Effect of low concentration (20% w/w) (a) and high concentration (60% w/w) (b) of sorbitol on structure and morphology of pectinase enzyme.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4338374&req=5

fig1: Effect of low concentration (20% w/w) (a) and high concentration (60% w/w) (b) of sorbitol on structure and morphology of pectinase enzyme.
Mentions: Preliminary studies revealed that the enzyme was stable in Triton X-100, Tween 20, SDS, and sorbitol, which indicated that pectinase was suitable for the novel surfactant/sorbitol-based ATPS. Another study proved that surfactants promote the availability of reaction sites and increase the hydrolysis rate [17]. To determine the effects of each phase composition on the pectinase activity, crude feedstock pectinase was mixed with various compounds. The surfactant also induces enzyme activity and eventually reduces the rate of enzyme denaturation during hydrolysis [18] and thus functions as an inducer of enzyme activity by affecting the enzyme-substrate interaction. This process can prevent the inactivation of adsorbed enzymes, which directly facilitates the desorption of enzymes from the substrate [18–20]. High concentrations [60%] of Triton X-100 and Tween-80 slightly increased pectinase activity, whereas SDS partially inactivated this activity (Table 1). This latter effect was due to the binding of SDS, an ionic surfactant, to the proteins, which disrupted the majority of the globular proteins original structures. Moreover, the pectinase was mixed into sorbitol at different concentrations to determine its activity. In the presence of sorbitol, pectinase also exhibited high enzyme stability. The probable reason for this observation is that sorbitol helped to maintain the enzyme's open conformation by exposing the active site crevice surface and thus stimulating pectinase activity [18]. As shown in Table 1, a higher concentration of sorbitol (60%, w/w) inhibited the pectinase activity compared with the pectinase activity in 20% (w/w) sorbitol solution. Morphological observations revealed that, in 60% (w/w) sorbitol and pectinase, severe agglutination and rough surfaces within the interactions were observed, whereas 20% (w/w) sorbitol resulted in clear and smooth surfaces as shown in Figure 1. This observation might have resulted from the high concentration of sorbitol (60%, w/w) causing conformational changes in the pectinase structure that caused enzyme denaturation. Hence, this phenomenon decreased the pectinase activity and resulted in an incompatibility in the denatured enzyme-substrate interaction. Therefore, the authors speculate that the high concentrations of sorbitol denatured the pectinase and decreased its activity compared with the low concentration of sorbitol.

Bottom Line: Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied.The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0.The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.

ABSTRACT
A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Show MeSH