CN100406557C - Process for preparing fixed cytochrome p450 BM-3 - Google Patents

Abstract

The invention discloses a preparation method of immobilized cytochrome P450 BM-3. The steps are as follows: 1) Extraction of crude enzyme solution: 3 g of wet bacteria obtained from fermentation were dissolved with 5 ml of buffer solution, suspended and ultrasonicated in an ice bath for 3 min, the cell-breaking liquid was centrifuged at 4°C, 8000 r/min for 20 min, and the supernatant was Crude enzyme solution, ultrasonic power 200W, ultrasonic time 2s, interval time 5s, working times 75; 2) Enzyme immobilization: Take 1.2ml specific activity of 39.5U/gPro crude enzyme solution and dissolve in 3.6%, 4.2% , 4.5%, 4.7%, 5.1% NaCS in 10mM, 25mM, 50mM, 75mM, pH75, pH8.0, pH8.2, pH8.5 Tris-Hcl buffer 18.8mL, mix, drop into 50ml of solution with No. 4 needle In the 6% and 6.2% PDMDAAC of the same buffer solution, continue to stir for 40 min, then wash with 50 mM Tris-HCl three times at pH 8.0, and then dry it. The invention has the advantages of simple and convenient operation and mild conditions; the immobilized enzyme has good stability; it can be used in bioreactors and has the prospect of industrial application.

Description

Preparation method of immobilized cytochrome P450 BM-3

technical field

The invention relates to the field of immobilized enzyme biotechnology, in particular to a method for preparing NaCS-PDMDAAC microcapsules immobilized cytochrome P450 BM-3.

Background technique

The application of enzymes in food, light industry, medicine, chemical industry, analysis and detection, environmental protection and scientific research has achieved remarkable results. However, due to the poor stability of the enzyme, the variability and inactivation; after the enzyme reaction, it is mixed with the substrate and the product, which is difficult to recycle, resulting in waste, and also brings difficulties to the separation of the product. Artificial immobilization of enzymes to make immobilized enzymes not only enables the enzymes to be recycled and used repeatedly or continuously, reducing costs, but also improves the stability of the enzymes and simplifies the purification process of the products. Enzyme immobilization methods can be divided into three categories: binding method, cross-linking method and embedding method. The embedding method can be divided into two types: grid type and microcapsule type. The enzyme embedded in the polymer gel fine grid is called the grid type; the enzyme embedded in the polymer semi-permeable membrane is called Microcystic. The encapsulation method generally does not require a binding reaction with the amino acid residues of the enzyme protein, rarely changes the high-level structure of the enzyme, and has a high recovery rate of enzyme activity, so it is the most widely used method.

Cytochrome P450 BM-3, also known as CYP102, is derived from Bacillus megaterium and is soluble in water. It is a long-chain fatty acid (C ₁₂ -C ₂₀ ) ω-hydroxylase, which can catalyze the terminal oxidation and Epoxidation. The molecular weight of P450 BM-3 is 117,641Da. It consists of two different regions, one part contains FAD and FMN reductase, and the other part is heme protein, which together form a polypeptide chain. The heme part can bind substrates and absorb oxygen. The flavin sites (FAD and FMN) are capable of accepting electrons from NADPH and transferring them to the heme active site.

At present, the P450 BM-3 mutants obtained through directed evolution (such as F87V, L188G and A74G) have further expanded the substrates, which can hydroxylate short-chain alkanes and alkanoic acids (C ₈ -C ₁₀ ), cycloalkanes, aromatics and heterocyclic compounds, etc.

Cytochrome P450 BM-3 acts on alkanes, fatty acids, etc., usually showing a high degree of substrate and stereoselectivity. It can introduce an oxygen atom in the C-H bond to produce optically pure compounds. In addition, it can be used to mono-oxidize stereo- and site-selective compounds to produce important biological activities, such as converting arachidonic acid to 14, 15-EET. Since the structure of the substrate is usually a hydrophobic compound and the product is more water-soluble, P450 BM-3 can be used to treat environmentally toxic substances, such as polycyclic aromatic hydrocarbons. In summary, cytochrome P450 BM-3 has broad application prospects in pharmaceutical industry, agriculture, environmental detection and protection, and chemical synthesis.

However, natural P450 BM-3 is very expensive, easily inactivated, and very unstable. Therefore, these defects have become the bottleneck of P450 BM-3 widely used in industrial production, and immobilized P450 BM-3 is undoubtedly one of the better choices, it can overcome some shortcomings of free enzymes, improve the stability of P450 BM-3, prolong the retention time of its activity. The immobilized P450 BM-3 can also be applied to bioreactors.

NaCS-PDMDAAC was first developed by Dr. Dautzenberg of the Institute of Polymer Chemistry of the Academy of Sciences of the GDR in the early 1980s. The microcapsule is prepared by reacting two water-soluble polymer electrolytes such as sodium cellulose sulfate (NaCS) and polydimethyldiallylammonium chloride (PDMDAAC). The capsule prepared by the reaction of NaCS and PDMDAAC is a hollow microcapsule surrounded by a porous membrane with a thickness of about 20-100 μm. Capsules have strong mechanical strength and can withstand a positive pressure of 3-6 Newtons. Moreover, the physical and chemical properties of the porous membrane are stable, not affected by light and heat, do not change in the pH range of 2 to 10, and are insoluble in most organic solvents. In addition, the film has good biocompatibility and has no obvious toxicity to microorganisms, animal and plant cells.

十程需在4℃冷藏至少3天，然后再需冻干48h以上。整个制备过程比较复杂，周期也很长，而且此过程酶也很容易失活。It is known from European patent EP1196603 that P450 BM-3 is immobilized on the following media Glasperlen 100-200 μm (Schueller), Amberlite XAD16, Dowex 50 WX4 and EP-100, and the residual enzyme activity is 3.1%, 15.2%, 9.1% and 18.2%. The recovery of enzyme activity in this immobilization method is very low, and with the help of physical adsorption immobilization, the enzyme is easy to fall off from the medium. In 2003, Maurer Steffen et al. co-immobilized P450 BM-3 and NADPH-dependent formate dehydrogenase in tetraethyl orthosilicate sol-gel. The relative activity of the immobilized enzyme was 52.4%, the half-life at room temperature was 29 days, and there was no loss of activity at 4°C for 36 days. But this immobilized

The ten-course should be refrigerated at 4°C for at least 3 days, and then freeze-dried for more than 48 hours. The whole preparation process is relatively complicated and the cycle is very long, and the enzymes in this process are also easily inactivated.

Escherichia coli (Escherichia coli) CGMCC No: 1185 was deposited on July 6, 2004 in the General Microorganism Center of China Committee for the Collection of Microorganisms.

Contents of the invention

The purpose of the invention is to provide a preparation method of NaCS-PDMDAAC microcapsule immobilized P450 BM-3.

The steps of preparation method are as follows:

1) Extraction of crude enzyme solution

3g of wet bacteria obtained from fermentation was dissolved in 5ml of buffer solution, suspended and ultrasonicated in an ice bath for 3 minutes, and the cell-breaking solution was centrifuged at 4°C and 8000r/min for 20 minutes. The supernatant was the crude enzyme solution. The ultrasonic power was 200W, and the ultrasonic time was 2s, gap time 5s, working times 75;

2) Immobilization of enzymes

Take 1.2ml of crude enzyme solution with a specific activity of 39.5U/gPro and dissolve it in 10mM, 25mM, 50mM, 75mM containing 3.6%, 4.2%, 4.5%, 4.7%, 5.1% NaCS, pH 7.5, pH 8.0, pH 8.2 , pH8.5 Tris-Hcl buffer 18.8mL, mix, use 4# needle to drip into 50ml of 6%, 6.2% PDMDAAC dissolved in the same condition buffer, and continue to stir for 40min, and then use it at pH8.0 Wash with 50mM Tris-HCl three times, then blot dry.

This invention makes full use of the good mechanical strength and biocompatibility of NaCS-PDMDAAC microcapsules. The physical and chemical properties of the porous membrane are stable, not affected by light and heat, and do not change in the pH range of 2 to 10, and are insoluble in most of organic solvents. The invention has the characteristics of simple and convenient operation and mild conditions; the immobilized enzyme has good stability; it can be used in bioreactors and has the prospect of industrial application.

Detailed ways

Properties of the invented immobilized P450 BM-3:

The optimal pH of immobilized P450 BM-3 was 7.8, slightly lower than the pH 8.0 of free P450 BM-3.

The optimum temperature of immobilized P450BM-3 was 42℃, which was higher than that of free P450BM-3 which was 37℃.

The enzyme activity of immobilized P450 BM-3 is basically unchanged in the range of 0-25 °C, and its thermal stability is greatly improved compared with free P450 BM-3.

The activity of immobilized P450 BM-3 was stable between pH 7.0 and 8.0, which was consistent with that of free P450 BM-3.

When the immobilized P450 BM-3 was stored at 4°C for 30 days, the enzyme activity lost 22.3%, while the free enzyme activity decreased to 12.3% under the same conditions.

Example 1:

1. Extraction of crude enzyme solution

The fermented cells were dissolved with 5ml buffer solution/(3g wet cells), suspended and ultrasonicated in an ice bath for about 3 minutes (200W, ultrasonic time 2s, interval time 5s, working times 75). The broken cells were centrifuged at 4°C, 8000r/min for 20min, and the supernatant was the crude enzyme solution.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (the specific activity is 39.5U/gPro) and mix it with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, drop 50ml of NaCS dissolved in the same condition buffer with a 4# needle In 6% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity is 19.7%.

Example 2:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 39.5U/gPro) mixed with 18.8mL of 4.5% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 6# needle In 6.2% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 60 mM Tris-HCl (pH 7.5) three times, and suck dry. The relative enzyme activity is 19.4%.

Example 3:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (the specific activity is 39.5U/gPro) and mix it with 18.8mL of 3.6% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, drop 40ml of NaCS dissolved in the same condition buffer with a 7# needle In 6% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-HCl (pH 7.8) three times, and suck dry. The relative enzyme activity is 17.3%.

Example 4:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 62.5U/gPro) mixed with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 4# needle In 6% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity is 26.8%.

Example 5:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (the specific activity is 62.5U/gPro) and mix it with 18.8mL of 4.2% NaCS dissolved in 50mM pH7.5 Tris-Hcl buffer solution, and drop 50ml of NaCS dissolved in the same condition buffer solution with a 4# needle In 6% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity was 22.0%.

Embodiment 6:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (the specific activity is 62.5U/gPro) and mix it with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.5 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 4# needle In 6% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity is 20.1%.

Embodiment 7:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (the specific activity is 62.5U/gPro) and mix it with 18.8mL of 4.2% NaCS dissolved in 25mM pH8.2 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 4# needle In 6% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity is 23.7%.

Embodiment 8:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 119U/gPro) and mix with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop into 50ml of 6NaCS dissolved in the same condition buffer with a 6# needle. % of PDMDAAC (molecular weight 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-Hcl (pH 8.0) three times, and suck dry. The relative enzyme activity is 28.2%.

Embodiment 9:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 119U/gPro) mixed with 18.8mL of 4.2% NaCS dissolved in 10mM pH8.0 Tris-Hcl buffer, and drop into 50ml of NaCS dissolved in the same condition buffer with a 6# needle. % of PDMDAAC (molecular weight 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-Hcl (pH 8.0) three times, and suck dry. The relative enzyme activity was 14.0%.

Example 10:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 119U/gPro) mixed with 18.8mL of 4.2% NaCS dissolved in 75mM pH8.0 Tris-Hcl buffer, and drop into 50ml of NaCS dissolved in the same condition buffer with a 4# needle. % of PDMDAAC (molecular weight 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-Hcl (pH 8.0) three times, and suck dry. The relative enzyme activity is 23.1%.

Example 11:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Mix 3ml of crude enzyme solution (specific activity 39.5U/gPro) with 17mL of 5.1% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop into 50ml of 6.2% NaCS dissolved in the same condition buffer with a 4# needle In PDMDAAC (molecular weight 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-Hcl (pH 8.0) three times, and suck dry. The relative enzyme activity is 13.5%.

Example 12:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 2ml of crude enzyme solution (specific activity is 39.5U/gPro) and mix with 18mL of 4.5% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop into 50ml of 6.0% NaCS dissolved in the same condition buffer with a 4# needle In PDMDAAC (molecular weight 200,000-350,000), continue to stir for 40 min, then wash with 50 mM Tris-Hcl (pH 8.0) three times, and suck dry. The relative enzyme activity is 15.6%.

Example 13:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 33.8U/gPro) mixed with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 6# needle In 6.0% PDMDAAC (molecular weight<200,000), continue to stir for 60 min, then wash with 50 mM Tris-Hcl (pH 8.0) three times, and suck dry. The relative enzyme activity is 23.2%.

Example 14:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 33.8U/gPro) mixed with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 6# needle In 6.0% PDMDAAC (molecular weight: 200,000-350,000), continue to stir for 60 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity is 25.1%.

Example 15:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 33.8U/gPro) mixed with 18.8mL of 4.2% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 6# needle In 6.0% PDMDAAC (molecular weight: 350,000-450,000), continue to stir for 60 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity was 37.0%.

Example 16:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 94.8U/gPro) mixed with 18.8mL of 4.7% NaCS dissolved in 50mM pH8.0 Tris-Hcl buffer, and drop 50ml of NaCS dissolved in the same condition buffer with a 6# needle In 6.0% PDMDAAC (molecular weight: 350,000-450,000), continue to stir for 60 min, then wash with 50 mM Tris-HCl (pH 8.0) three times, and suck dry. The relative enzyme activity is 32.5%.

Example 17:

1. Extraction of crude enzyme solution

With embodiment 1.

2. Enzyme Immobilization

Take 1.2ml of crude enzyme solution (specific activity is 39.5U/gPro) and mix with 18.8mL of 4.5% NaCS dissolved in 50mM pH8.0 phosphate buffer, and drop into 50ml of 6.2% NaCS dissolved in the same condition buffer with a 6# needle. % of PDMDAAC (molecular weight: 350,000-450,000), continue to stir for 40 min, then wash with 60 mM Tris-Hcl (pH 7.5) three times, and suck dry. The relative enzyme activity is 34.7%.

Claims (2)

1. a preparation method of NaCS-PDMDAAC microcapsule immobilized cytochrome P450 BM-3, is characterized in that, the steps are as follows: 1) Extraction of crude enzyme solution 3g of wet bacteria obtained from fermentation was dissolved in 5ml of buffer solution, suspended and ultrasonicated in an ice bath for 3 minutes, and the cell-breaking solution was centrifuged at 4°C and 8000r/min for 20 minutes. The supernatant was the crude enzyme solution. The ultrasonic power was 200W, and the ultrasonic time was 2s, interval time 5s, working times 75; 2) Immobilization of enzymes Take 1.2ml of crude enzyme solution with a specific activity of 39.5U/gPro and dissolve it in 10 mM, 25 mM, 50 mM or 75mM containing 3.6%, 4.2%, 4.5%, 4.7% or 5.1% NaCS, pH 7.5, pH 8.0 , pH 8.2 or pH 8.5 Tris-HCl buffer solution 18.8mL, mix, drop into 50ml of 6% or 6.2% PDMDAAC dissolved in the same condition buffer solution with a No. Wash three times with 50mM Tris-HCl at 0°C, then blot dry. 2. the preparation method of a kind of NaCS-PDMDAAC microcapsule immobilized cytochrome P450BM 3 according to claim 1 is characterized in that said wet thalline is CGMCC No.1185 recombinant Escherichia coli cell.