TWI704008B - Separating method of protein - Google Patents

Abstract

A protein separating method of present having following steps is provided: providing at least one tubular membrane containing adsorbents therein; providing a crude extracted protein containing separated substance; feeding the crude extracted protein into a channel in the tubular membrane and through the tubular membrane longitudinally, the separated substance contained in the crude extracted protein is adsorbed by the adsorbent and a residual liquid is exhausted; detecting a concentration of the separated substance in the residual liquid and stopping feeding until the concentration of the separated substance in the residual liquid is greater than a predetermined ratio of the concentration of the separated substance in the crude extracted protein; feeding a desorption buffer into the channel and through the tubular membrane longitudinally. Thereby, the separated substance adsorbed in the adsorbent is separated by dissolving into the desorption buffer.

Description

Protein separation method

The present invention relates to protein separation, in particular to a protein separation method for separating specific proteins from a protein liquid.

In the prior art, the protein can be separated from the protein liquid by means of penetration filtration. Generally speaking, the protein liquid is pressurized to penetrate the filter membrane containing the adsorbent, and the specific protein contained in the protein liquid is adsorbed by the adsorbent when the protein liquid penetrates the filter membrane. Then put the filter membrane with the protein adsorbed into the desorption buffer, and the protein is dissolved into the desorption buffer for separation.

In order to facilitate pressurization, the filter membrane is made as a tubular tubular membrane with one end open and the other end closed. The pressurized protein solution passes through the tubular membrane and then flows out from the side of the tubular membrane. The disadvantage is that the high-pressure pipeline is not easy to set up and the cost is high, and the residual liquid to be filtered out also requires a larger container for recovery, so the equipment scale is limited. Furthermore, since the film thickness is only about 0.4mm to 0.6mm, the time for the protein solution to penetrate the tubular membrane is short, the contact time between the adsorbent and the protein solution is insufficient, and the adsorption efficiency is poor.

In view of this, the inventor of the present invention focused on the above-mentioned prior art, especially concentrated research and the application of scientific theory, and tried to solve the above-mentioned problems, which became the goal of the present inventor's improvement.

The invention provides a protein separation method for separating specific proteins from a protein liquid.

The present invention provides a protein separation method, which includes the following steps: providing at least one tubular membrane, the structure of the tubular membrane itself contains an adsorbent, the tubular membrane has at least one channel longitudinally penetrating the tubular membrane; A crude protein extract of the substance; the crude protein extract is fed into the channel and passes through the tubular membrane along the longitudinal direction of the tubular membrane, and the substance to be separated in the crude protein extract is adsorbed by the adsorbent and becomes a residual liquid; Over-detect the concentration of the substance to be separated in the residual liquid until the concentration of the substance to be separated in the residual liquid is greater than a predetermined ratio of the concentration of the substance to be separated in the crude protein extract, then stop the input of the crude protein extract to Channel; and provide a desorption buffer input channel, and pass the tubular membrane along the longitudinal direction of the tubular membrane, and the substance to be separated absorbed in the adsorbent is dissolved in the desorption buffer to become a desorption fluid.

In the protein separation method of the present invention, the adsorbent is a cation exchange resin and the pH value of the crude protein extract is lower than the isoelectric point of the substance to be separated. The substance to be separated is lysozyme. The desorption buffer contains tris (Tris) with a molar concentration of 45mM to 55mM and sodium chloride with a molar concentration of 0.5M to 1.5M. The pH of the desorption buffer is 7.5 to 8.5. The flow direction of the desorption buffer through the tubular membrane is opposite to the flow direction of the crude protein extract through the tubular membrane.

The protein separation method of the present invention further comprises a step of providing a washing buffer input channel and passing the tubular membrane along the longitudinal direction of the tubular membrane to remove the crude protein extract remaining in the tubular membrane. The washing buffer contains Mo Tris (Tris) with an ear concentration of 45mM to 55mM and sodium chloride with a molar concentration of 0.05M to 0.15M. The pH of the wash buffer is 7.5 to 8.5. The flow direction of the washing buffer through the tubular membrane is opposite to that of the crude protein extract through the tubular membrane.

The protein separation method of the present invention further includes a step of drying the desorption liquid to obtain a solid substance to be separated.

In the protein separation method of the present invention, the tubular membranes are plural, and the tubular membranes are arranged parallel to each other and arranged in bundles to form an adsorption module. In the tubular membrane, a plurality of channels are formed longitudinally through the tubular membrane.

The protein separation method of the present invention separates the substances to be separated by adsorption, it does not need to drive the crude protein extract to penetrate the tubular membrane by high pressure, and the pipeline pressure loss is low, so the installation cost is lower and the installation is easier The implementation of low-pressure pipeline equipment. Furthermore, in the protein separation method of the present invention, the liquid for various operations is inputted at a single point from one end of the tubular membrane, and recovered at a single point from the other end of the tubular membrane. Therefore, the tubular membrane of the present invention can be arbitrarily arranged or connected in series between the input and output points, so it is easy to expand the processing capacity and the adsorption capacity.

100: Adsorption module

110: Shell

120: Tubular membrane

121: Channel

210: crude protein extract

220: residual liquid

310: Washing buffer

320: cleaning fluid

410: Desorption buffer

420: Desorption Liquid

a~g: steps

Figure 1 is a flow chart of the steps of the protein separation method of the preferred embodiment of the present invention.

Figure 2 is a schematic diagram of a single-channel tubular membrane provided by a protein separation method according to a preferred embodiment of the present invention.

Figure 3 is a longitudinal sectional view of the tubular membrane shown in Figure 2.

Figure 4 is a schematic diagram of a multiple channel tubular membrane provided by a protein separation method according to a preferred embodiment of the present invention.

Fig. 5 is a longitudinal sectional view of the tubular membrane shown in Fig. 4.

Figure 6 is a schematic diagram of the pipeline configuration of the protein separation method of the preferred embodiment of the present invention.

1 to 5, a preferred embodiment of the present invention provides a protein separation method provided by the present invention, which includes the following steps: in step a, first provide at least one tubular membrane 120, the structure of the tubular membrane 120 contains The adsorbent, and in this embodiment, the adsorbent is preferably a cation exchange resin. The tubular membrane 120 is open at both ends, and a channel 121 longitudinally penetrating the tubular membrane 120 is formed in the tubular membrane 120. The present invention does not limit the number of channels 121 in the tubular film 120, and each channel 121 respectively extends along the longitudinal direction of the tubular film 120 and penetrates the tubular film 120. In the simplest embodiment, the channel 121 is the working interface of the tubular membrane 120. Therefore, as shown in FIGS. 2 and 3, the tubular membrane 120 with a single channel 121 can implement the present invention. 4 and 5, in this embodiment, a tubular membrane 120 including a plurality of channels 121 is preferably configured. Each tubular mold 120 is formed with a single or multiple channels 121 longitudinally penetrating and parallel to each other, and the tubular membranes 120 are parallel to each other and arranged in bundles to form the adsorption module 100. Specifically, in the adsorption module 100, the tubular membranes 120 are installed in a tubular housing 110 and are bound and fixed by the housing 110.

Referring to Figures 1, 2 and 4 to 6, in step b, following step a, a crude protein extract 210 is provided. The crude protein extract 210 is extracted from egg whites, and the crude protein extract 210 includes a The substance to be separated, specifically, the substance to be separated is a specific protein (protein) dissolved in the crude protein extract 210, and in this example, the substance to be separated is preferably lysozyme. The pH value of the solution where the protein is located is different, and the concentration of the positively charged hydrogen ions and the negatively charged hydroxide ions in the solution change the charge of the protein accordingly. In a solution with a specific pH value, the positive and negative charges of the protein are balanced and not charged, then the pH value is the isoelectric point of the protein, and different proteins have different isoelectric points. The pH of the crude protein extract 210 is generally between 6.0 and 7.7, and the isoelectric point of lysozyme is generally between 11.0 and 11.2. In this embodiment, the pH value of the crude protein extract 210 is less than the isoelectric point of the substance to be separated, so the lysozyme is in the protein The crude protein extract 210 is positively charged; the isoelectric point of the general protein is usually less than pH 5, so other proteins are negatively charged in the crude protein extract 210.

In the adsorption step c, following step b, the crude protein extract 210 is fed into the channel 121 and passes through the tubular membrane 120 along the longitudinal direction of the tubular membrane 120. Lysozyme and other proteins are charged differently in the crude protein extract 210, and therefore can be separated from the crude protein extract 210 by adsorbing positively charged lysozyme by the cation exchange tree. The substance to be separated contained in the crude protein extract 210 is adsorbed by the adsorbent and becomes a residual liquid 220.

In step d, continue with step c, repeatedly detecting the concentration of the substance to be separated in the residual liquid 220, until the concentration of the substance to be separated in the residual liquid 220 is greater than the concentration of the substance to be separated in the crude protein extract 210 A predetermined ratio (ie, lysozyme breaking; breakthrough), that is to say, the ability of the tubular membrane 120 to adsorb the substance to be separated drops to an allowable lower limit, and the input of the crude protein extract 210 to the channel 121 is stopped. In this embodiment, the aforementioned predetermined ratio is preferably between 1% and 5%.

In the desorption step e, following step d, a desorption buffer solution 410 is provided to enter the channel 121 and passes through the tubular membrane 120 along the longitudinal direction of the tubular membrane 120, and the substances to be separated adsorbed in the adsorbent are dissolved into the desorption Buffer 410. The flow direction of the desorption buffer 410 through the tubular membrane 120 is preferably opposite to the flow direction of the crude protein extract 210 through the tubular membrane 120. The pH of the desorption buffer 410 is 7.5 to 8.5, and the desorption buffer 410 contains tris (Tris) with a molar concentration of 45 mM to 55 mM and sodium chloride with a molar concentration of 0.5 M to 1.5 M. Tris soluble in water is alkaline to adjust the pH value of the desorption buffer 410. A higher concentration of sodium chloride contains a higher concentration of sodium ions, and the substance to be separated is released and dissolved in the desorption buffer 410 to become the desorption solution 420 by replacing the substance to be separated adsorbed in the adsorbent by sodium ions. Preferably, the protein separation method of the present invention can also be used to separate and purify the protein in the desorption liquid 420 again.

The protein separation method of the present invention may optionally further include a cleaning step f between step d and step e. In step f, a cleaning buffer 310 is provided to enter the channel 121 and pass through the tubular membrane 120 along the longitudinal direction. 120 to remove the crude protein extract 210 remaining in the tubular membrane 120, and the washing buffer 310 is washed and discharged as a washing liquid 320. The flow direction of the washing buffer 310 through the tubular membrane 120 is preferably opposite to the flow direction of the crude protein extract 210 through the tubular membrane 120. The pH of the washing buffer 310 is 7.5 to 8.5, and the washing buffer 310 contains Tris with a molar concentration of 45 mM to 55 mM and sodium chloride with a molar concentration of 0.05 M to 0.15 M. Tris is dissolved in water to be alkaline to adjust the pH value of the cleaning buffer 310. Low-concentration sodium chloride contains low-concentration sodium ions to avoid the release of substances to be separated adsorbed in the adsorbent.

The protein separation method of the present invention may optionally further include a drying step g, followed by step e, drying the desorption liquid 420 passing through the tubular membrane 120 to obtain a solid substance to be separated. In this embodiment, the desorption solution 420 is dried by a drying step to obtain a solid lysozyme powder.

Since the adsorbent must contact the crude protein extract 210 to adsorb the substances to be separated, the longer the contact time between the crude protein extract 210 and the adsorbent, the better the adsorption efficiency. In the existing penetrating filtration method, the crude protein extract 210 only contacts the adsorbent during the process of penetrating the tubular membrane; in the protein separation method of the present invention, the crude protein extract 210 can be contacted during the passage of the tubular membrane 120 Adsorbent, so the adsorption efficiency is better.

Compared with the existing penetration filtration method, the protein separation method of the present invention separates the substances to be separated by adsorption, which does not require high pressure to drive the crude protein extract 210 to penetrate the tubular membrane 120, and the pipeline pressure loss is low. Therefore, it can be implemented by low-pressure pipeline equipment with lower installation cost and easier installation.

Furthermore, the existing penetrating filtration method needs to recover the liquid of various operations from the side of the tubular membrane. Therefore, the entire tubular membrane needs to be contained in a container. The larger the adsorption area, the larger the container and the equipment. The cost of setup has also increased dramatically. In the protein separation method of the present invention, the liquid for various operations is single-point input from one end of the adsorption module 100, and single-point recovery from the other end of the adsorption module 100. Therefore, in the protein separation method of the present invention, the adsorption module 100 can be connected in parallel between the input and output points to increase the operating flow, or the adsorption module 100 can be connected in series to increase the operating adsorption capacity, so it is easy to expand the processing capacity and adsorption capacity of the equipment.

The foregoing descriptions are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Other equivalent changes using the patent spirit of the present invention should all belong to the patent scope of the present invention.

100: Adsorption module

120: Tubular membrane

210: crude protein extract

220: residual liquid

310: Washing buffer

320: cleaning fluid

410: Desorption buffer

420: Desorption Liquid

Claims (11)

A protein separation method, comprising the steps: a) providing at least one tubular membrane, the structure of the tubular membrane itself containing an adsorbent, and at least one channel longitudinally penetrating the tubular membrane; b) providing a A crude protein extract of the substance to be separated; c) input the crude protein extract into the channel, and pass the tubular membrane along the longitudinal direction of the tubular membrane, and the substance to be separated contained in the crude protein extract is The adsorbent adsorbs and becomes a residual liquid; d) repeatedly detecting the concentration of the substance to be separated in the residual liquid until the concentration of the substance to be separated in the residual liquid is greater than the concentration of the substance to be separated in the crude protein extract If the concentration of the substance to be separated is a predetermined ratio, stop the input of the crude protein extract into the channel; f) following step d, provide a cleaning buffer to be input into the channel, and pass through the tubular membrane along the longitudinal direction of the tubular membrane To remove the crude protein extract remaining in the tubular membrane, the washing buffer contains tris (Tris) with a molar concentration of 45mM to 55mM and a chlorinated solution with a molar concentration of 0.05M to 0.15M. Sodium; and e) following step f, providing a desorption buffer into the channel, and passing through the tubular membrane along the longitudinal direction of the tubular membrane, and the substance to be separated adsorbed in the adsorbent is dissolved into the desorption The desorption buffer solution contains tris (Tris) with a molar concentration of 45 mM to 55 mM and sodium chloride with a molar concentration of 0.5 M to 1.5 M. The protein separation method according to claim 1, wherein the adsorbent is a cation exchange resin and the pH value of the crude protein extract is less than the isoelectric point of the substance to be separated. The protein separation method according to claim 2, wherein the substance to be separated is lysozyme. The protein separation method according to claim 3, wherein the pH of the desorption buffer is 7.5 to 8.5. The protein separation method according to claim 3, further comprising a step f: providing a washing buffer solution into the channel, and passing the tubular membrane along the longitudinal direction of the tubular membrane to remove the remaining in the tubular membrane Crude protein extract, the pH of the washing buffer is 7.5 to 8.5. The protein separation method according to claim 1, further comprising a step f of providing a washing buffer solution to the channel, and passing the tubular membrane along the longitudinal direction of the tubular membrane to remove the remaining in the tubular membrane Crude protein extract. The protein separation method according to claim 6, wherein the flow direction of the washing buffer through the tubular membrane is opposite to the flow direction of the crude protein extract through the tubular membrane. The protein separation method according to claim 1, wherein the flow direction of the desorption buffer through the tubular membrane is opposite to the flow direction of the crude protein extract through the tubular membrane. The protein separation method according to claim 1, further comprising a step g of drying the desorption liquid to obtain the solid substance to be separated. The protein separation method according to claim 1, wherein the tubular membranes are plural and the tubular membranes are arranged parallel to each other and arranged in a bundle. The protein separation method according to claim 1, wherein the tubular membrane has a plurality of channels extending longitudinally through the tubular membrane.

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