Process for purifying bromelain by chelated metal affinity membrane

A technology of bromelain and metal chelation, applied in the direction of hydrolytic enzymes, etc., can solve the problems of low product purity, high cost, short diffusion path, etc., achieve the effect of high purification multiple and enzyme activity, convenient source of raw materials, and easy extraction and purification

Inactive Publication Date: 2011-06-22
DONGHUA UNIV
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Problems solved by technology

[0003] The traditional extraction process of bromelain is adsorption method and precipitation method, but these two methods are cumbersome and the purity cannot meet the needs of the pharmaceutical industry
The article "Research on the Extraction of Highly Active Bromelain" uses nano-zinc oxide adsorption, ceramic membrane ultrafiltration concentration and freeze-drying technology to extract bromelain from pineapple stems and skins, which can effectively maintain the activity of protease. Alcohol is used as a precipitant to purify bromelain. These methods all have defects such as low product purity and low enzyme activity.
Patent CN1980165 discloses a physical extraction method of bromelain, which includes cleaning and pulverizing the raw materials for pretreatment, then squeezing, centrifuging, freezing, and then filtering, ultrafiltration, reverse osmosis and vacuum drying; patent CN1186118 provides a A method for extracting bromelain is to carry out freezing pretreatment on pineapple plants, then crush and squeeze the juice, ultrafiltration, reverse osmosis, and freeze-drying again. The purity and enzyme activity of the bromelain obtained by the physical extraction method are high, but the cost is high and the process High energy consumption
[0004] Membrane is a material with selective separation function, which can realize the separation, purification and concentration of different components of feed liquid. It is characterized by large surface area, short diffusion path, low operating pressure and large processing capacity. Separation, but the traditional membrane separation technology can only separate species with a relative molecular mass difference of more than 10 times, which cannot meet the needs of biochemical separation

Method used

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  • Process for purifying bromelain by chelated metal affinity membrane
  • Process for purifying bromelain by chelated metal affinity membrane
  • Process for purifying bromelain by chelated metal affinity membrane

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Activation of nylon membrane and modification of bonded hydroxyethyl cellulose, the specific steps are as follows:

[0028] (1) Nylon membranes (10 sheets) were first hydrolyzed in 1 mol / L HCl at 25°C for 24 hours, and then activated with formaldehyde. Ten hydrolyzed nylon membranes were immersed in 20ml of formaldehyde solution (>36.5wt.%), added with 0.2ml of phosphoric acid (85wt.%), reacted at 60°C for 7h, and washed with hot water at 40-50°C for several times.

[0029] (2) Immerse the above-mentioned formaldehyde-activated nylon membranes (10 sheets) in 100ml of 2% hydroxyethyl cellulose solution, react at room temperature for 1h, then transfer to a 90°C oven, take it out after 1h, and wash with 1% Triton La (X-100) NaOH solution (0.1mol / L) was used to wash away unreacted hydroxyethyl cellulose, and then washed in deionized water for 2 days.

Embodiment 2

[0031] The modified nylon membrane chelates metal zinc ions, and the specific steps are as follows:

[0032] (1) 100 sheets (about 10 grams) of nylon membranes after the above-mentioned modification were immersed in 4.6ml epichlorohydrin, 46ml 2mol / L sodium hydroxide and 5mmol sodium borohydride mixed solution, reacted at room temperature for 2 hours, and then directly applied to Add 23ml of epichlorohydrin and 46ml of 2mol / L sodium hydroxide to the mixed solution, react at 60°C for 12h, and wash with deionized water several times.

[0033] (2) Immerse 10 films (approximately 1 gram) of the above-mentioned combined cyclopropane into a mixed solution containing 86mmol of iminodiacetic acid (IDA), 8mmol of sodium borohydride and 114.4ml of 2mol / L sodium carbonate, and shake at a constant temperature of 60°C After reacting for 12 hours, wash with deionized water, 5vol.% acetic acid and deionized water several times.

[0034] (3) Immerse the above-mentioned film in 500ml of 100pp...

Embodiment 3

[0036] Use a metal chelate membrane to adsorb bromelain and optimize the conditions. The specific steps are as follows:

[0037] (1) Determination of reaction time. Stack 10 modified nylon membranes to form a membrane stack, put them into the membrane bridge, and send 100ml of 1.0mg / ml bromelain Tris-HCl solution (pH=8) with a peristaltic pump at a flow rate of 2.0ml / min. 10min, 20min, 30min, 40min, 50min, 60min, 80min and 100min, carry out adsorption, and measure the amount of bromelain adsorbed. Experimental results such as figure 1 , the optimal response time is selected at 60min.

[0038] (2) Determination of reaction pH. Stack 10 modified nylon membranes to form a membrane stack, put them into the membrane bridge, and send 100ml of 1.0mg / ml bromelain Tris-HCl solution with different pH values ​​into it with a peristaltic pump, and circulate the reaction for 2 hours at a flow rate of 2.0ml / min , adding bromelain in Tris-HCl solution with pH values ​​of 5.0, 6.0, 7.0, 8...

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Abstract

The invention relates to a method for purifying bromelain by means of a metal chelate affinity membrane. The method comprises the following steps that: (1) activated nylon membrane is modified through hydroxyethyl cellulose bonding; (2) the modified nylon membrane reacts with the mixed solution of epichlorohydrin, sodium hydroxide and sodium borohydride, and then the membrane is respectively dipped in the mixed solution of sodium carbonate, sodium borohydride and a coupling agent and a zinc sulfate solution, thereby generating a chelated zinc ionic metal affinity membrane; (3) the metal affinity membrane is piled up into a membrane stack, and is fed into a membrane bridge; a peristaltic pump pumps in a Tris-HCl buffer solution and an eluent and then pumps in a bromelain solution so as tocarry out elution by eluant, thereby generating bromelain; (4) enzymatic activity and protein content are tested to calculate purification multiple. The method has quickness, high efficiency, simplicity, convenience, great fractional dose and high activity of extracted enzyme, and is suitable for industrialscale production.

Description

technical field [0001] The invention relates to the field of protease purification, in particular to a method for purifying bromelain by using a metal chelating affinity membrane. Background technique [0002] Bromelain (Bromelain) referred to as bromelain, also known as bromelain or bromelain, enzymatic number EC3.4.22.32, is extracted from pineapple fruit stems, stalks, leaves, skins, refined, purified, concentrated, enzyme immobilized A pure natural plant protease obtained by melting and freeze-drying. It is a kind of sulfhydryl protease, its active center is sulfhydryl group (--SH), it can carry out various biochemical reactions such as protein hydrolysis, it can be widely used in food, medicine and biological industries, and can be used as food additive for meat tenderization , Beer clarification, also used in the production of cheese, gelatin and hydrolyzed protein. [0003] The traditional extraction process of bromelain is adsorption method and precipitation method...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50
Inventor 朱利民何智妍聂华丽
Owner DONGHUA UNIV
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