CN113373134B - A kind of extraction method of N-acetylglucosamine deacetylase - Google Patents

Abstract

The invention discloses a method for extracting N-acetylglucosamine deacetylase and belongs to the technical field of bioengineering. The invention uses fermentation broth containing N-acetylglucosamine deacetylase as raw material, collects the concentrated microbial suspension through flocculation centrifugation or ceramic microfiltration membrane, and simultaneously removes residual nutrients during the fermentation process; and then ruptures the cell wall through high-pressure homogenization , releasing the cell contents and target enzymes, and finally dialyzing the solution rich in N-acetylglucosamine deacetylase through a ceramic microfiltration membrane or ultrafiltration membrane, while retaining and removing cell debris components. While improving the enzyme extraction recovery rate, the present invention reduces material consumption and wastewater production during the extraction process, achieving good economic benefits, environmental protection and safety effects.

Description

A kind of extraction method of N-acetylglucosamine deacetylase

Technical field

The invention relates to a method for extracting N-acetylglucosamine deacetylase and belongs to the technical field of bioengineering.

Background technique

D-glucosamine has important physiological significance in the human body and is used in medicine, food, cosmetics and other fields. Chitin is widely distributed in biological raw materials such as shrimp shells and crab shells, but the production process requires the use of strong acids and alkalis, which can easily cause serious environmental pollution. Genetic engineering technology can be used to construct recombinant microorganisms that produce N-acetylglucosamine, and large-scale production of N-acetylglucosamine can be achieved, but it is still one step away from D-glucosamine and requires N-acetylglucosamine deacetylase catalysis It can be obtained after hydrolysis.

N-acetylglucosamine deacetylase (EC 3.5.1.33) is an important deacetylase with a molecular weight of 30-60kDa, and the molecular weight of some enzyme complexes is between 160-200kDa. The catalyzed biological reaction formula is: N-acetyl-D-glucosamine + H ₂ O = D-glucosamine + acetic acid. Roseman first discovered in 1957 that this enzyme catalyzes and participates in the metabolic process of D-glucosamine. Deacetylase enzymes with similar functions in the same family also include chitosan deacetylase (EC 3.5.1.41), diacetylchitobiose deacetylase (EC 3.5.1.105) and N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25). The main microorganisms that have been found to contain N-acetylglucosamine deacetylase genes include Escherichia coli, Lactococcus lactis, Thermococcus kodaka, Thermococcus Horikoshi, etc. (J. Biol. Chem. 2004, 279, 30021-30027). Efficient expression and production can be achieved through aerobic fermentation of recombinant Escherichia coli, recombinant Bacillus subtilis and other microorganisms. Extraction of N-acetylglucosamine deacetylase is one of the key steps in the production of D-glucosamine.

The 1993 U.S. patent (US 5219749) disclosed a method for purifying chitosan deacetylase. It used ammonium sulfate precipitation method to enrich chitosan deacetylase in Mucor ruckeri fermentation broth, and then successively passed through hydrophobic chromatography, strong anion Through three units of exchange chromatography and strong cation exchange chromatography, a relatively pure chitosan deacetylase is finally obtained.

In 2002, Bouriotis and others from the University of Crete in Greece extracted chitosan deacetylase from Saccharomyces cerevisiae. The extraction steps mainly include: enriching the crude enzyme by ammonium sulfate precipitation method, and then adding it to a phenyl-Sepharose HiLoad (hydrophobic) chromatographic column. After gradient elution, it was loaded onto a Mono S cation exchange chromatography column for separation, and finally a relatively pure chitosan deacetylase was obtained with a recovery rate of 24.5%.

In 2006, Cai Jun of Wuhan University used the aerobic fermentation of Scopus brevis to obtain chitose deacetylase, enriched the crude enzyme using ammonium sulfate precipitation method, and then passed through two chromatography units of Sephadex G-25 and Sephadex G-100, and finally obtained a relatively Pure chitose deacetylase.

In 2012, the U.S. patent (US2012009627A1) and the Chinese patent (CN 105039464A) announced N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25) and N-acetylglucosamine deacetylase (EC3.5.1. 33), genetic modification, fermentation process and enzyme reaction conditions of chitin deacetylase and acyltransferase.

The efficiency of enzyme extraction depends on the composition of the impurities in the raw material, the difference in physical and chemical properties between the target substance and the impurity, the relative concentration of the target product, and other factors. At present, there are relatively few published documents on the extraction method of N-acetylglucosamine deacetylase. There are relatively many chitose deacetylase enzymes with similar properties and functions, but they are still mainly implemented on a laboratory scale. The basic steps include salt Precipitation enrichment and multi-stage chromatography purification require many separation steps, extraction-related materials are expensive, and the reagents used in the separation process are usually organic solvents, which is not conducive to green and safe production, and the recovery rate of the final product is low.

Contents of the invention

In view of the shortcomings of high material consumption and low efficiency in the existing technology, the present invention provides a new N-acetylglucosamine deacetylase extraction method, using recombinant microbial fermentation broth rich in N-acetylglucosamine deacetylase as the For raw materials, solid-liquid separation is achieved through ceramic membrane microfiltration or flocculation sedimentation, and the microbial cells are collected and extracellular impurities such as residual nutrients in the culture medium are removed at the same time; the cell wall is ruptured using a high-pressure homogenization method to release N- The cell contents of acetylglucosamine deacetylase; finally, a ceramic microfiltration membrane or ceramic ultrafiltration membrane is used to intercept cell debris, etc., so that N-acetylglucosamine deacetylase can be dialyzed out to obtain a relatively pure enzyme solution.

The first object of the present invention is to provide a method for isolating and purifying N-acetylglucosamine deacetylase, which method includes the following steps:

(1) Use microbial fermentation broth containing N-acetylglucosamine deacetylase as raw material, use ceramic microfiltration membrane concentration method or flocculation centrifugation method to achieve solid-liquid separation, and obtain microbial concentrate containing N-acetylglucosamine deacetylase. suspension;

(2) Subject the concentrated microbial suspension to high-pressure homogenization to rupture the microbial cell wall and release N-acetylglucosamine deacetylase from the cell, thereby obtaining a wall-broken suspension containing N-acetylglucosamine deacetylase;

(3) Separate the broken suspension through a ceramic microfiltration membrane or ceramic ultrafiltration membrane, and obtain a higher-purity N-acetylglucosamine deacetylase extract from the permeate.

In one embodiment, the microbial fermentation broth in step (1) is the fermentation broth obtained by microorganisms fermenting for a period of time in a culture medium containing a carbon source, a nitrogen source and an inorganic salt, and contains N-acetylglucosamine deacetylation. Enzymes, cell metabolites and incompletely consumed culture medium components; the microbial fermentation broth can be recombinant Escherichia coli fermentation broth, recombinant Bacillus subtilis fermentation broth, Lactococcus fermentation broth, or recombinant yeast fermentation broth liquid.

In one embodiment, the microfiltration membrane in step (1) is a ceramic membrane component, the average pore size of the microfiltration membrane is 1-5 μm, and the operating pressure is 0.3-0.8MPa. The operation process is The temperature is controlled at 4~30℃.

In one embodiment, the flocculant used in step (1) is a food-safe flocculant, including but not limited to organic flocculants or inorganic flocculants;

In one embodiment, the flocculant is one or more of polyacrylamide, dimethylamine-epichlorohydrin copolymer, polyferric chloride, ferric chloride, ferric sulfate, and ferrous sulfate. The combination;

In one embodiment, the flocculant is added in (a) or (b):

(a) Add a single-component flocculant at the following dosage: the amount of polyacrylamide added is 0.01 to 0.2% of the dry weight of biomass in the raw material; the amount of dimethylamine-epichlorohydrin copolymer added is 0.01% to 0.2% of the dry weight of biomass in the raw material. The added amount of polyferric chloride is 0.1-2.0% of the dry weight of biomass; the added amount of ferric chloride is 0.1-1.0% of the dry weight of biomass; the added amount of iron sulfate is 0.1-1.0% of the dry weight of biomass; the added amount of iron sulfate is 0.1-2.0% of the dry weight of biomass 0.1~1.5% of the dry weight; the added amount of ferrous sulfate is 0.2~2.0% of the dry weight of the biomass;

(b) When adding flocculants containing two or more components, the amount of each flocculant added is 20-50% of the amount of a single component as described in (a).

In one embodiment, the centrifugal equipment described in step (1) can be a continuously operating centrifuge or an intermittent operating centrifuge; the continuous centrifugal equipment can be a disc centrifuge, and the centrifugal factor is between 8000 and 15000g. ; The intermittent operating centrifuge can be an automatic discharge centrifuge with a centrifugal factor of 4000 to 8000g.

In one embodiment, the operating conditions of the high-pressure homogenization wall-breaking equipment described in step (2) are: the pressure of high-pressure homogenization is 60-120MPa, the number of homogenization times is 1-3 times, and the temperature is controlled at 4 -30℃.

In one embodiment, the ceramic membrane separation equipment described in step (3) can be a microfiltration membrane or an ultrafiltration membrane. The pore size of the microfiltration membrane is 0.1-1.0 μm, and the molecular weight cutoff of the ultrafiltration membrane is 300- 1000kDa, the operating pressure is 0.3~0.8MPa, and the temperature during the operation is controlled at 4~30℃.

In one embodiment, the amount of water added during the ceramic membrane separation process in step (3) is 1-5 times the volume of the raw material liquid. Higher purity N-acetylglucosamine deacetylase was obtained in the transluate.

In one embodiment, after step (3) is completed, steam is used to sterilize or disinfect the used production equipment and pipelines to prevent microbial contamination during the production process. The pressure of the steam is 0.2 to 0.6 MPa, sterilization time is 5 to 40 minutes.

Beneficial effects:

Compared with the prior art, the present invention has the following advantages:

(1) The present invention realizes efficient purification of N-acetylglucosamine deacetylase on an industrial production scale. The production process has the advantages of high recovery rate, low raw material consumption, and environmental protection and safety. The product recovery rate can reach up to 88%, and the specific activity of the produced N-acetylglucosamine deacetylase can reach up to 22U/g protein;

(2) The enzyme extraction conditions used in the present invention are mild, without using organic solvents and high-concentration salting out. Cell wall breaking is achieved through membrane separation and high-pressure homogenization, so that the enzyme can be extracted efficiently and the amount of pollutants discharged is small. The process is relatively environmentally friendly and safe;

(3) The present invention uses steam to sterilize and disinfect the separation equipment and pipelines, and uses pure physical methods to achieve and maintain cleanliness and hygiene in the production process, effectively solving the problems of bacterial contamination and health and safety that are prone to occur during the enzyme extraction process.

(4) The process of the present invention is suitable for the production of N-acetylglucosamine deacetylase at different scales and has wide industrial application value.

Description of the drawings

Figure 1 Extraction process route of N-acetylglucosamine deacetylase.

Detailed ways

Technical terms:

Microbial suspension: a flocculated sediment containing microbial cells in a flowing state.

Cut-off liquid: a solution that is retained by an ultrafiltration membrane and contains proteins with a molecular weight greater than the molecular weight cutoff of the ultrafiltration membrane.

Biomass (based on dry weight): refers to the weight of dry matter of microbial cells per unit volume. Place a certain volume of fermentation broth in a centrifuge tube, centrifuge at 8000r/min for 10min to remove the supernatant, and wash the precipitate with clean water 2 to 3 times. The resulting precipitate is dried at 105°C to constant weight and the microbial dry matter is determined. weight. The unit is g/L.

Enzyme activity recovery rate: Enzyme activity recovery rate is calculated as follows:

The deacetylase activity unit (U) is defined as: at 30°C, using N-acetylglucosamine as the substrate and using 50mmol/L sodium phosphate buffer (pH 7.5), it is necessary to react to obtain 1mmol of glucosamine per 1 minute. The amount of enzyme is 1U enzyme activity unit.

The purity of deacetylase is expressed in units of enzyme activity per gram of protein, recorded as U/g protein.

Analytical method:

The method for measuring deacetylase activity refers to J. Microbiol. Biotechnol. 2018, 28(11), 1850-1858; the method for measuring protein is the Bicinchoninic Acid colorimetric method.

Example 1

According to the process route shown in route A in Figure 1, the recombinant Escherichia coli fermentation broth containing N-acetylglucosamine deacetylase is used as raw material.

The recombinant Escherichia coli fermentation broth containing N-acetylglucosamine deacetylase is the recombinant Escherichia coli in a culture medium containing glucose, peptone, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, calcium carbonate and other ingredients The fermentation broth obtained through aerobic fermentation contains some unmetabolized culture medium components, recombinant microbial cells and some residual cell fragments and metabolic by-products, such as acetic acid, propionic acid, amino acids, pigments, etc.

The steps are as follows:

(1) Take ^50m3 of recombinant E. coli fermentation broth containing N-acetylglucosamine deacetylase (enzyme activity of the fermentation broth is 101U/L). The biomass dry weight of each L of fermentation broth is 60g, and add it to the fermentation broth storage tank. Pump in 20L of 100g/L ferric sulfate solution and 25L of 10g/L food grade polyacrylamide solution. Stir and mix completely and then flocculate for 20 minutes;

(2) Continuously pump the coagulated liquid containing microbial cells after flocculation in step (1) into a disc centrifuge with a rotation speed of 12,000 rpm. The collected ^12m3 microbial concentrated suspension enters the temporary storage tank; the clear liquid flows into the clear liquid. In the liquid tank, it is discharged to the sewage treatment unit after testing.

(3) The microbial suspension collected in step (2) is subjected to high-pressure homogenization treatment to rupture the microbial cell wall and release N-acetylglucosamine deacetylase from the cell to obtain N-acetylglucosamine deacetylase. Wall-breaking suspension; the pressure of high-pressure homogenization is 110MPa, the feed flow rate is 3m ³ /h, and the temperature is controlled at 10°C. After the homogenization is completed, the homogenization operation is repeated once, and a total of 2 homogenizations are performed.

(4) Separate the ^12m3 wall-broken suspension obtained in step (3) through a ceramic ultrafiltration membrane. The molecular weight cutoff of the ultrafiltration membrane is 500kDa, the operating pressure is 0.5MPa, and the temperature during the operation is controlled at 4 to 30°C. When the volume of the retentate is about 10 m ³ , deionized water is added intermittently three times for a total of 36 m ³ to wash the retentate, and 38 m ³ of the translucent liquid is collected to obtain N-acetylglucosamine deacetylation with a purity of 18 U/g protein. The enzyme extraction liquid and retentate are discharged to the sewage treatment unit.

(5) Use steam to sterilize or disinfect the production equipment and pipelines used to prevent microbial contamination during the production process. The pressure of the steam is 0.3MPa, and the sterilization time is 20 minutes. The extraction results are shown in Table 1.

Example 2

According to the process route shown in route B in Figure 1, the recombinant E. coli fermentation broth containing N-acetylglucosamine deacetylase is used as raw material. The operation steps are as follows:

(1) Take 50m ³ of the recombinant E. coli fermentation broth containing N-acetylglucosamine deacetylase (enzyme activity of the fermentation broth is 111U/L), pump it into the ceramic microfiltration membrane module for solid-liquid separation, and collect the microbial suspension. , the average pore size of the microfiltration membrane is 2 μm, the operating pressure is 0.4MPa, and the temperature during the operation is controlled at 4 to 30°C. When the volume of the retained liquid is about ^20m3 , add deionized water intermittently in three times for a total of ^50m3 for concentration. ^20m3 of washed microbial suspension is collected, and the permeate is discharged to the sewage treatment unit.

(2) The microbial suspension collected in step (1) is subjected to high-pressure homogenization treatment to rupture the microbial cell wall and release N-acetylglucosamine deacetylase from the cell to obtain N-acetylglucosamine deacetylase. Wall-breaking suspension; the pressure of high-pressure homogenization is 100MPa, the feed flow rate is 3m ³ /h, and the temperature is controlled at 15°C. After the homogenization is completed, the homogenization operation is repeated once, and a total of 2 homogenizations are performed.

(3) Separate the ^20m3 wall-broken suspension obtained in step (2) through a ceramic ultrafiltration membrane. The molecular weight cutoff of the ultrafiltration membrane is 500kDa, the operating pressure is 0.4MPa, and the temperature during the operation is controlled at 4 to 30°C. When the volume of the retentate is about ^10m3 , add deionized water intermittently three times for a total of ^30m3 to wash the retentate, and collect ^40m3 of the translucent liquid to obtain a high-purity N-acetylglucosamine deacetylase extract. , the retained liquid is discharged to the sewage treatment unit.

(4) Use steam to sterilize or disinfect the production equipment and pipelines used to prevent microbial contamination during the production process. The pressure of the steam is 0.3MPa, and the sterilization time is 20 minutes. The extraction results are shown in Table 1.

Example 3

The specific implementation is the same as in Example 1, except that the 12 m ³ broken suspension obtained in step (3) is separated through a ceramic microfiltration membrane. The pore size of the microfiltration membrane is 0.2μm, the operating pressure is 0.4MPa, and the temperature during the operation is controlled at 4 to 10°C. When the volume of the retentate is about 8 m ³ , add deionized water intermittently three times for a total of 24 m ³ to wash the retentate, and collect 24 m ³ of the translucent liquid to obtain a high-purity N-acetylglucosamine deacetylase extract. , the retained liquid is discharged to the sewage treatment unit. The extraction results are shown in Table 1.

Example 4

The specific implementation is the same as in Example 2, except that the 20 m ³ broken suspension obtained in step (2) is separated through a ceramic microfiltration membrane. The pore size of the microfiltration membrane is 0.1μm, the operating pressure is 0.5MPa, and the temperature during the operation is controlled at 4 to 30°C. When the volume of the retentate is about 10 m ³ , add deionized water intermittently three times for a total of 25 m ³ to wash the retentate, and collect 25 m ³ of the translucent liquid to obtain a high-purity N-acetylglucosamine deacetylase extract. , the retained liquid is discharged to the sewage treatment unit. The extraction results are shown in Table 1.

Table 1 Extraction effects of N-acetylglucosamine deacetylase using different processes

Example 5

The specific implementation is the same as Example 1, except that one or a combination of two or more of polyacrylamide, dimethylamine-epichlorohydrin copolymer, polyferric chloride, ferric chloride, and ferrous sulfate is used instead. The flocculant ferric sulfate in Example 1;

Among them, the adding method of flocculant is (a) or (b):

(a) Add a single-component flocculant at the following dosage: the amount of polyacrylamide added is 0.01 to 0.2% of the dry weight of biomass in the raw material; the amount of dimethylamine-epichlorohydrin copolymer added is 0.01% to 0.2% of the dry weight of biomass in the raw material. The added amount of polyferric chloride is 0.1-2.0% of the dry weight of the biomass; the added amount of ferric chloride is 0.1-1.0% of the dry weight of the biomass; the added amount of iron sulfate is 0.1-1.0% of the dry weight of the biomass; 0.1~1.5% of the dry weight; the added amount of ferrous sulfate is 0.2~2.0% of the dry weight of the biomass;

(b) When adding flocculants containing two or more components, the amount of each flocculant added is 20-50% of the amount of a single component as described in (a).

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (2)

1. The method for separating and purifying the N-acetylglucosamine deacetylase is characterized by comprising the following steps of:

(1) Taking microbial fermentation liquor containing N-acetylglucosamine deacetylase as a raw material, and adopting a flocculation centrifugation method to realize solid-liquid separation to obtain microbial concentrated suspension containing the N-acetylglucosamine deacetylase;

(2) Homogenizing the microorganism concentrated suspension collected in the step (1) under high pressure, and collecting wall-broken suspension containing N-acetylglucosamine deacetylase;

(3) Separating the wall-broken suspension obtained in the step (2) through a ceramic microfiltration membrane or a ceramic ultrafiltration membrane, and collecting N-acetylglucosamine deacetylase extracting solution in the permeate; the aperture of the ceramic microfiltration membrane is 0.1-1.0 mu m, the molecular weight cut-off of the ceramic ultrafiltration membrane is 300-1000kDa, the operating pressure is 0.3-0.8 MPa, and the operating process control temperature is 4-30 ℃;

the microbial fermentation broth in the step (1) is recombinant escherichia coli fermentation broth;

the flocculating agent of the step (1) is polyacrylamide and ferric sulfate;

the centrifugation in the step (1) adopts a continuously operated centrifuge or a intermittently operated centrifuge; the continuous centrifugal equipment is a disc type centrifugal machine, and the centrifugal factor is 8000-15000 g; the intermittently operated centrifugal machine is an automatic discharging centrifugal machine, and the centrifugal factor is 4000-8000 g;

the high-pressure homogenization of step (2) is operated under the following conditions: the pressure is 60-120MPa, the temperature is 4-30 ℃, and the homogenization times are 1-3.

2. The method according to claim 1, wherein after the step (3) is completed, the used production equipment and pipelines are sterilized or disinfected by steam, the pressure of the steam is 0.2-0.6 mpa, and the sterilization time is 5-40 min.