Abstract
The purification of thermo-acidic amylase enzyme from red pitaya (Hylocereus polyrhizus) peel for the first time was investigated using a novel aqueous two-phase system (ATPS) consisting of a thermo-separating copolymer and an organic solvent. The effectiveness of different parameters such as molecular weight of the thermo-separating ethylene oxide-propylene oxide (EOPO) copolymer and type and concentration of organic solvent on the partitioning behavior of amylase was investigated. In addition, the effects of phase components, volume ratio (VR), pH and crude load of purification factor and yield of amylase were evaluated to achieve the optimum partition conditions of the enzyme. In the novel ATPS method, the enzyme was satisfactorily partitioned into the polymer-rich top phase in the system composed of 30% (w/w) EOPO 2500 and 15% (w/w) 2-propanol, at a volume ratio of 1.94 and with a crude load scale of 25% (w/w) at pH 5.0. Recovery and recycling of components was also measured in each successive step of the ATPS process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 96.6% and copolymer was also recovered and recycled at a rate above 97%, making the method was more economical than the traditional ATPS method.
Highlights
An aqueous two-phase system (ATPS) method has been proposed as an ideal purification technique for the separation and recovery of biomolecules because of the system’s high productivity, simplicity, short processing time, cost effectiveness, scalability and versatility [1,2]
The feasibility of recovering thermo-acidic amylase and recycling the phase components in a novel ATPS composed of a thermo-separating polymer and organic solvent was investigated for the first time
The extracted enzyme was mixed with thermo-separating polymer and organic solvents to investigate the effect of the phase components on the amylase activity
Summary
An aqueous two-phase system (ATPS) method has been proposed as an ideal purification technique for the separation and recovery of biomolecules because of the system’s high productivity, simplicity, short processing time, cost effectiveness, scalability and versatility [1,2]. ATPS has been widely employed for the separation of biological materials such as cells, cell debris, proteins and nucleic acids [3] This method could be used to overcome the problem faced by conventional purification methods because of its speed, low interfacial tension, easy scale-up, minimized protein denaturation, non-toxicity and mild conditions [4]. Amylases represent one of the most important industrial enzymes and have a wide variety of applications in different industrial fields, such as the food, fermentation, textile, paper, detergent, pharmaceutical and sugar industries [7]. The feasibility of recovering thermo-acidic amylase and recycling the phase components in a novel ATPS composed of a thermo-separating polymer and organic solvent was investigated for the first time. The recycle recovery of the thermo-separating copolymer at each recycling step was investigated
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