Alternating current (AC) magnetic field assisted three-phase partitioning system

Abstract

The present invention relates to an alternating current magnetic field assisted three-phase partitioning system developed as an alternative method in the field of extraction and purification of all biomolecules including proteins, enzymes, polysaccharides, phytochemicals, pigments, oils and antioxidants from biological substances of various microorganism, plant and animal origin, in order to realize the separation of biomolecules in a shorter time, with high efficiency and low cost with the support of AC magnetic field.

Description

[0001] ALTERNATING CURRENT (AC) MAGNETIC FIELD ASSISTED THREE-PHASE PARTITIONING SYSTEM

[0002] Field of the Invention

[0003] The present invention relates to an alternating current (AC) magnetic field assisted three-phase partitioning system developed as an alternative method in the field of extraction and purification of all biomolecules including proteins, enzymes, polysaccharides, phytochemicals, pigments, oils and antioxidants from biological substances of various microorganism, plant and animal origin, in order to realize the separation of biomolecules in a shorter time, with high efficiency and low cost with the support of magnetic field.

[0004] State of the Art

[0005] Nowadays, there is a strong demand for advanced separation techniques to purify biomolecules. This is because an effective purification process is one of the most important steps in production and accounts for a significant portion of the total cost.

[0006] Significant efforts are being made towards the development of biotechnology processes, where recombinant therapeutic proteins, food proteins and industrial enzymes are having an increasing impact on the global market. The production processes of these proteins and enzymes must be economical as well as feasible. Therefore, the process of extracting and purifying industrially important proteins in a highly efficient and economical way has long been one of the most important research topics.

[0007] Traditional purification techniques used to purify enzymes in the state of the art consist of several laborious, costly and time-consuming steps. Therefore, alternative recovery techniques such as three-phase partitioning (TPP) have been developed to solve these drawbacks. The TPP method for the separation of enzymes and other proteins from complex mixtures is a new, simple, inexpensive and effective separation technique in which an organic solvent and a macromolecular salt are mixed to form three phases. TPP is a fast procedure with all chemicals used being recycled, making the process easily inexpensive.

[0008] The efficiency of the TPP method, which has recently been very popular in the art and is considered as a new enzyme purification technique, has been shown by an increase in total enzyme activity in various cases in studies.

[0009] Again, assisted or modified TPP systems have been developed to generate faster and higher yields based on conventional TPP for efficient extraction and purification of different bioactive molecules in the known state of the art. These techniques carried out with the TPP system to minimize the time of TPP and increase its recovery and purity include ultrasonic assisted TPP, microwave assisted TPP, macro affinity ligand-facilitated TPP and ionic liquid TPP.

[0010] TPP and its modifications / variants, which have a wide range of applications from the state of the art, have been used for the extraction of different enzyme inhibitors, the extraction of recombinant green fluorescent protein, the extraction of antitumor proteins such as trichosanthine, and the dissolution and refolding of inclusion bodies.

[0011] TPP as a protein enrichment method has also been demonstrated on various other simple proteins such as meat proteins, immunoreactive proteins ovalbumin and whey proteins.

[0012] The increasing demand for proteins and enzymes due to advances in molecular biology over the last two decades has led to increased research interest in the development of economical, scalable and simple techniques for the production of these macromolecules. For this purpose, in recent years, as an alternative to chromatographic bioseparation techniques, high-throughput, rapid and low-cost non- chromatographic methods such as TPP, which can be applied directly to the crude extract, have been developed. However, the TPP technique is still a relatively new method. Therefore, modification studies by incorporating external approaches into TPP to improve the separation process have attracted much attention in recent years. In addition to these methods in the art, several modified TPP variants have been developed to increase the extraction yield and purification rate of enzymes and other proteins. These variants include ultrasonically assisted TPP, microwave-assisted TPP, macro affinity ligand-facilitated TPP and ionic liquid TPP.

[0013] When the studies in the state of the art are examined, it is obvious that although there are many methods and their modifications used today, new techniques and modification applications are needed since modified TPP applications are effective in the separation and purification of not only proteins but also other biomolecules.

[0014] Description of the Invention

[0015] In this detailed description, the AC magnetic field assisted three-phase partitioning system is described only for clarifying the subject matter in a manner such that no limiting effect is created.

[0016] The structural and characteristic features of the present invention will be understood clearly by the following detailed description and therefore the evaluation shall be made by taking the detailed description into consideration.

[0017] The present invention relates to an AC magnetic field-assisted three-phase partitioning system that fulfills the above-mentioned requirements, eliminating all disadvantages and introducing some additional advantages.

[0018] The preferred embodiment of the invention is a three-phase partitioning system for the extraction and purification of all biomolecules including proteins, enzymes, polysaccharides, phytochemicals, pigments, lipids and antioxidants from biological materials of various microorganism, plant and animal origin. More preferably, the invention is an AC magnetic field-assisted three-phase partitioning system for bioseparation of biomolecules.

[0019] The invention in its most preferred form is an AC magnetic field assisted three-phase partitioning system.

[0020] TPP is a mass transfer induced technique and hence improvement in mass transfer rate increases partitioning and target product purity. This process improvement in the mass transfer rate can also be achieved by combining it with a magnetic field.

[0021] The primary object of the present invention was developed as an alternative method to enable the separation of biomolecules in a shorter time, with high efficiency and low cost with the support of AC magnetic field compared to other methods mentioned.

[0022] A further object of the present invention is that the AC magnetic field applied to the conventional TPP method increases the mass transfer, thereby increasing the separation rate of the phases and the interphase diffusion of the target biomolecule; to provide the following advantages;

[0023] • Higher efficiency,

[0024] • High purity product,

[0025] • Time saving.

[0026] Along with these, this method is;

[0027] • Cheap.

[0028] • Applicable directly to the culture liquid.

[0029] • Used for small and large volumes. Therefore, it has both application areas in the laboratory and can be easily scaled to industrial production.

[0030] The process steps related to the invention are as follows; i. Preparation of the biomolecule source (separation of culture liquid or cell lysis or tissue extraction), ii. Mixing the prepared biomolecule source with organic solvent (t- butanol / isopropanol) and salt (ammonium sulfate, etc.), iii. Keeping the mixture tube under an AC magnetic field.

[0031] Alternating Current Magnetic field-assisted three-phase partitioning (AC- MFTPP) is realized by modifying the TPP technique in a manner suitable for magnetic field. in step ii. AC-MFTPP is carried out using ammonium sulfate saturation, culture liquid / t-butanol ratio and pH, which are determined as a result of the optimization made in traditional TPP. 4 mL of culture liquid is brought to 30% w / v ammonium sulfate saturation and (1.0: 1.5) t-butanol is added. The installed system is exposed to a magnetic field for a certain period of time. After the three layers are formed, the intermediate phase and the lower aqueous phase are separated and enzyme activity and protein determination are made in both phases.

[0032] After testing the success of the system in operations carried out at room temperature, optimum conditions are determined as follows;

[0033] • AC Magnetic field application time: 2-10 min.,

[0034] • Duty cycle: 30%, 50%, 70%, 90%, 100%,

[0035] • Power: 40-100 W

[0036] With the mentioned invention, a new type of modification of the TPP method, the use of 'alternating current (AC) magnetic field', is realized to be used effectively in the bioseparation of biomolecules, especially enzymes and other proteins, with higher yield and purification rate in a shorter process time compared to conventional TPP.

Claims

CLAIMS1. AC Magnetic field-assisted three-phase partitioning (AC-MFTPP) system for the extraction and purification of all biomolecules including proteins, enzymes, polysaccharides, phytochemicals, pigments, oils and antioxidants from various microorganisms, plants, animal-derived biological substances, characterized in that; it enables the separation of biomolecules in a shorter time, high efficiency and low cost with the support of magnetic field.

2. AC-MFTPP system according to claim 1 , characterized in that; alternating 3. current magnetic field is applied in three-phase partitioning (TPP).

2. AC-MFTPP system according to claim 1 and claim 2, characterized in that; the optimized condition for alternating current magnetic field application time is 2-10 minutes.