CN109207535B

CN109207535B - Method for synthesizing uracil by using pseudomonas aeruginosa

Info

Publication number: CN109207535B
Application number: CN201811103549.8A
Authority: CN
Inventors: 杨西宁; 邢善涛; 李涛; 王东琨; 卓凯悦; 柳芳
Original assignee: Xinxiang Pharmaceutical Co ltd; Xinxiang Tuoxin Pharmaceutical Co ltd
Current assignee: Xinxiang Pharmaceutical Co ltd; Xinxiang Tuoxin Pharmaceutical Co ltd
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2021-06-25
Anticipated expiration: 2038-09-20
Also published as: CN109207535A

Abstract

The invention discloses a method for synthesizing uracil by using pseudomonas aeruginosa, belonging to the technical field of biological fermentation. The process takes pseudomonas aeruginosa as a strain, and utilizes wet thalli of the strain as an enzyme source to convert cytosine into uracil in one step. Compared with the uracil produced by the process reported in the literature, the uracil produced by the process has the advantages of high reaction speed, high conversion rate, substrate conversion rate of over 99 percent, simple process, low cost, easy operation and suitability for industrialization.

Description

Method for synthesizing uracil by using pseudomonas aeruginosa

Technical Field

The invention belongs to the technical field of biological fermentation in biomedicine, relates to biosynthesis of pyrimidine bases, and particularly relates to a method for synthesizing uracil by using pseudomonas aeruginosa.

Background

Pseudomonas aeruginosa (Pseudomonas aeruginosa) is called Pseudomonas aeruginosa. It is widely distributed in nature and is one of the most common bacteria present in soil. The bacteria exist in various water, air, normal skin, respiratory tract and intestinal tract. An important condition for the presence of the present bacteria is a humid environment. The bacterium is a common conditional pathogen, and belongs to non-fermentation gram-negative bacilli. The cells are elongated and vary in length, and may be arranged in a club-like or linear form, in pairs or in short chains. One end of the thallus is provided with single whip hair, and the movement and activity of bacteria can be observed under a dark field microscope or a phase contrast microscope.

The bacterium is an obligate aerobic bacterium, the growth temperature range is 25-42 ℃, the optimal growth temperature is 25-30 ℃, and particularly, the characteristic that the bacterium can not grow at 4 ℃ and can grow at 42 ℃ can be used for identifying. Can survive on common culture medium and can produce water soluble pigment, such as pyocyanin (pyocynin), fluorescence water soluble fluorescein (pyoverdin) and the like. There were clear hemolysin rings on the blood plates. The bacterium contains an O antigen (bacterial antigen) and an H antigen (flagellar antigen). The O antigen contains two components: one is its outer membrane protein, which is a protective antigen; the other is lipopolysaccharide, and has specificity.

Cytosine is an important intermediate of fine chemical engineering, pesticides and medicines, is particularly used for synthesizing anti-AIDS drugs and anti-hepatitis B drugs, namely lamivudine, anti-cancer drugs, namely gemcitabine, enocitabine, 5-fluorocytosine and the like in the field of medicines, and is very widely applied. The synthesis method is mainly a chemical synthesis method, and the direct synthesis of uracil from cytosine by adopting biosynthesis has no breakthrough progress.

Disclosure of Invention

In order to overcome the defects, the process takes the pseudomonas aeruginosa as a strain, converts cytosine into uracil by using wet thalli of the strain as an enzyme source in one step, and is simple in process, low in cost, easy to operate and suitable for industrialization.

The technical transformation scheme of the invention comprises the following steps: the preparation of the thallus comprises three stages of thallus preparation, enzymatic reaction, product refining and the like, wherein the thallus preparation comprises the following steps: activating thallus, culturing thallus and collecting thallus. The detailed process flow is shown in figure 1.

And (3) microorganism information: the Pseudomonas aeruginosa strain adopted by the invention and the Pseudomonas aeruginosa (Pseudomonas aeruginosa) used in the specific implementation mode are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 16110.

The preservation address is as follows: west road No.1, north west of chaoyang district, beijing, institute of microbiology, china academy of sciences, with a date of collection of 2018, 07 months and 16 days.

First, preparation of the cells

1.1 activation of cells and enzyme production culture

Strain: pseudomonas aeruginosa (Pseudomonas aeruginosa)

The strain number is as follows: CUR201604001

Activating a culture medium: 10g/L beef extract, 10g/L peptone, 5g/L sodium chloride and pH7.0;

the culture conditions are as follows: 35 ℃, 200rpm and 24 hours;

enzyme production culture medium: 75g/L beef extract, 15g/L sodium chloride, 60mL/L soybean meal hydrolysate, 0.6g/L magnesium sulfate heptahydrate, 0.4g/L manganese sulfate monohydrate, 0.6g/L calcium chloride, 0.05g/L zinc chloride and pH7.0;

the culture conditions are as follows: DO is more than or equal to 50% at 38 ℃ for 24 h.

1.2 Collection of cells

Centrifuging the enzyme-producing culture solution (centrifuging at 5000rpm for 10min), washing wet thallus with 20mmol/L potassium dihydrogen phosphate buffer solution with pH of 7.0, and freezing and storing at-20 deg.C.

Enzymatic reaction

The process utilizes pseudomonas aeruginosa as a strain and wet thalli of the strain as an enzyme source to convert cytosine to synthesize uracil in one step.

Reacting 50-600g/L cytosine and 10-200g/L wet bacteria in a purified water system at 15-75 ℃ to obtain uracil.

Further, in the technical scheme, the system is stirred at 200rpm, reacts for 24-72 hours under the water bath condition, and then is cooled for crystallization, and a crude uracil product is obtained through plate-frame or suction filtration.

Thirdly, refining the product

Directly dissolving the crude uracil product in hot water, filtering, cooling and crystallizing the collected filtrate, filtering and drying to obtain the pure uracil product.

Further, in the technical scheme, the filtration adopts suction filtration or plate-and-frame filter pressing.

Furthermore, in the technical scheme, the pure uracil is HPLC (high performance liquid chromatography) > 99.9%, and the 1HNMR and the 13CNMR are consistent with the standard samples.

Advantageous effects of the invention

1) The reaction efficiency is high

Compared with the uracil produced by the process reported in the literature, the reaction speed is high, the conversion rate is high, the substrate conversion rate reaches more than 99%, the product concentration reaches more than 500g/L, the thalli and the reaction liquid are easily separated, the product is easily extracted, and the yield is more than or equal to 95%.

2) The environmental protection pressure is small

The process utilizes purified water as a reaction system, and the uracil is efficiently converted and synthesized at high concentration in one step, so that the utilization rate of a thallus enzyme source is greatly improved, the cost is reduced, and the discharge amount of waste liquid is greatly reduced. The process uses water as a solvent, has simple extraction process and low cost, can repeatedly use the solvent, is environment-friendly and is suitable for industrialization.

3) High product quality

Compared with other processes, the target product uracil produced by the process has the advantages of easy extraction and high yield. Because the reaction system is purified water and each component in the synthetic solution is very clear, the product is easy to extract, the product quality is high, and the content of the finished product is more than or equal to 99.9 percent.

Drawings

FIG. 1 is a detailed process flow diagram of the inventive content conversion process;

FIG. 2 is the effect of 1.1 culture temperature on the growth of bacterial cells and enzyme activity in example 1;

FIG. 3 is the effect of pH 1.2 on cell growth and enzyme activity in example 1;

FIG. 4 is the fermentation enzyme production curve of the 1.3 CUR201604001 strain in example 1;

FIG. 5 shows the substrate conversion at different reaction temperatures of 2.1 in example 1;

FIG. 6 is a graph showing the effect of pH on conversion in the reaction system of 2.2 in example 1;

FIG. 7 is a graph of substrate concentration versus conversion of 2.3 in example 1;

FIG. 8 is a graph of 2.4 reaction time versus conversion for example 1;

FIG. 9 is a graph of 2.5 wet biomass versus substrate conversion in example 1.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1

Optimization of conversion reaction conditions

1. Enzyme production culture condition optimization

1.1 optimal culture temperature of CUR201604001 Strain

Inoculating the seed culture solution cultured for 12h into enzyme production culture medium at 5% inoculum size, stirring at 500rpm at 28-42 deg.C under ventilation of 600L/h for 16h, and measuring culture solution OD660 and enzyme activity (FIG. 2). The results show that: the bacterial quantity and the enzyme activity of the CUR201604001 strain increase along with the increase of the culture temperature within the range of 28-36 ℃, and the enzyme activity and the bacterial quantity are obviously reduced above 38 ℃. Therefore, the optimal enzyme production cultivation temperature range for the selection of the CUR201604001 strain was 36-38 ℃.

1.2 optimum culture pH value of CUR201604001 Strain

Inoculating the seed culture solution cultured for 12h into enzyme production culture medium at 5% inoculum size, ventilating amount of 600L/h, stirring speed of 500rpm, culture temperature of 38 deg.C, and measuring OD660 and enzyme activity of culture solution cultured for 16h under different pH conditions (FIG. 3). The results show that: the pH range of the thallus growth is 6.5-8.0, and the thallus growth amount is obviously reduced when the pH range is lower than or higher than the pH range; in the acidic pH range, the enzyme activity of unit wet thalli increases along with the increase of pH, reaches the maximum value at the pH of about 7.0, and the change of the enzyme activity is not obvious when the pH is higher than 7.0. Considering the factors of thallus growth, enzyme activity, total enzyme amount and the like, the optimum enzyme production culture pH range of the CUR201604001 strain is 6.5-8.0.

1.3 fermentation enzyme production curve of CUR201604001 Strain

Inoculating the CUR201604001 strain with an inoculum size of 5% in an enzyme production fermentation medium, culturing at 37 deg.C and pH7.0, ventilating at 600L/h under 500r/min, periodically sampling to determine the fermentation broth OD660nm and enzyme activity during the culture process, and drawing a growth curve and an enzyme production curve (FIG. 4). The results show that: the concentration of the fermentation liquid thallus is rapidly increased within the range of less than 14h, and the change is not obvious after the fermentation liquid thallus is cultured for 16 h; the enzyme activity is rapidly improved along with the prolonging of the culture time in the initial 14h of fermentation, which shows that the enzyme activity is parallel to the growth of thalli, the enzyme activity of unit wet thalli reaches the maximum value after the culture time reaches 16h, and the enzyme activity curve shows a plateau period. Therefore, the suitable enzyme-producing culture time is more than 16 h.

2. Reaction condition optimization

The optimization test of the reaction condition for synthesizing uracil is carried out on the basis of an initial reaction system, and comprises the optimization of the conditions such as wet bacterial quantity, substrate concentration, pH value, temperature, reaction time and the like, so as to achieve the aims of improving the substrate conversion rate and reducing the cost.

2.1 Effect of temperature on the rotating reaction

The initial reaction system was used to perform enzymatic conversion reaction at different temperatures for 36h, and the conversion was calculated by measuring the content of the desired product (FIG. 5). The results show that the reaction system can convert the substrate into uracil within a wider temperature range, the conversion efficiency is continuously improved along with the temperature rise within the temperature range of 30-50 ℃, the conversion reaction speed can be improved by increasing the temperature, and the conversion rate reaches the maximum value at 55 ℃; when the temperature is further increased, the conversion rate is reduced, and the conversion rate is rapidly reduced when the temperature is higher than 60 ℃, which indicates that the enzyme activity is seriously inactivated under the condition of overhigh temperature, and the conversion reaction is not facilitated. The results show that the optimum conversion reaction temperature is around 55 ℃.

2.2 Effect of pH on the conversion reaction

The pH value of the solution is adjusted by utilizing the reaction system, the conversion reaction is carried out for 36h, the content of the target product is measured, and the substrate conversion rate under different pH conditions is calculated (figure 6). The results show that the substrate conversion reaches a maximum in the pH range of 7.0 to 8.0, and that the conversion decreases rapidly both at too high and too low a pH. Therefore, the optimum pH value of the immobilized enzyme-catalyzed reaction is about 7.5.

2.3 determination of optimal substrate concentration

In the above reaction system, the concentration of the substrate was adjusted, the conversion reaction was carried out for 36 hours, the content of the objective product was measured, the conversion rate of the substrate under different conditions was calculated, and the conversion rate curve under different concentration conditions was plotted (fig. 7). The results show that: when the substrate concentration is less than 400g/L, the conversion does not change significantly, whereas after more than 400g/L, the conversion starts to decrease. According to the above results, the substrate concentration is increased without changing the substrate conversion rate, and the production cost is reduced by making full use of the catalytic potential of wet cells and increasing the productivity, so that the optimum reaction substrate concentration is about 400 g/L.

2.4 determination of optimum reaction time

Under the optimized reaction conditions, the concentration of the product at different times of the conversion reaction was measured and the substrate conversion was calculated (FIG. 8). The results show that: the product is accumulated continuously with the prolonging of the reaction time, the substrate conversion rate is increased rapidly with the increasing of the reaction time, and the change of the product concentration is not obvious after the reaction time reaches 48 hours. Thus, a suitable time for harvesting the product may be selected to be 48 hours later.

2.5 determination of the optimum Wet microbial cell amount

Under the above optimum reaction conditions, the conversion reaction was carried out while changing the amount of the bacterial cells in the range of 4 to 18% while keeping the other conditions constant, and the substrate conversion rate was calculated by measuring the product concentration of the conversion reaction for 48 hours (FIG. 9). The results show that: in the range of 4-12% of wet cell mass, the conversion rate rapidly increased with the increase of cell mass, and the change was not obvious thereafter. Since the wet cells cannot be reused, the optimal cell amount can be selected to be 14% or slightly lower than 12% in order to reduce the production cost.

Example 2

Process for synthesizing uracil by using pseudomonas aeruginosa on 100L scale

1. Preparation of cells

1.1 activation of cells and enzyme production culture

Activating a culture medium: 5g/L of yeast extract, 10g/L of sodium chloride, 10g/L of peptone and pH7.0;

the culture conditions are as follows: 35 ℃, 200rpm and 24 hours;

enzyme production culture medium: 75g/L of yeast extract, 15g/L of sodium chloride, 60mL/L of corn steep liquor, 0.8g/L of calcium chloride, 0.6g/L of magnesium sulfate heptahydrate, 0.8g/L of manganese sulfate monohydrate and 0.1g/L of zinc chloride, wherein the pH value is 7.0;

the culture conditions are as follows: the DO is more than or equal to 50 percent at 38 ℃ for 20 hours.

1.2 Collection of cells

The enzyme-producing culture solution is subjected to microfiltration concentration treatment, then wet thalli are collected by centrifugation, and the wet thalli are stored at the temperature of minus 20 ℃ for standby.

2. Enzymatic reaction

Preparing a reaction system: 40Kg of cytosine; the wet bacterial amount is 12 Kg; 100L of purified water; pH: 7.0. stirring at 200rpm after the preparation of the reaction system is finished, carrying out water bath reaction at 15-75 ℃ for 60h, then cooling, crystallizing, and carrying out suction filtration to obtain a crude uracil product.

3. Product refinement

Dissolving wet uracil crude product in hot water, suction filtering, cooling the collected filtrate for crystallization, and suction filtering to obtain white solidDrying to obtain 38.1Kg pure uracil, HPLC: 99.97 percent of the total weight of the steel,¹HNMR and¹³CNMR was consistent with the standard samples.

Claims

1. The method for synthesizing uracil by using pseudomonas aeruginosa is characterized by comprising the following steps: taking cytosine as a raw material, and adopting pseudomonas aeruginosa to convert and synthesize uracil; the Pseudomonas aeruginosa has the latin name of Pseudomonas aeruginosa and is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO. 16110; the uracil synthesizing process includes thallus preparation, enzyme reaction and product refining; the enzymatic reaction includes: reacting 50-600g/L cytosine and 10-200g/L wet thallus in a purified water system at 15-75 ℃ to obtain uracil.

2. A method of synthesizing uracil according to claim 1, wherein: the preparation of the thallus comprises the following steps: the first stage, thallus activation and spawn production culture; and a second stage: and (4) collecting thalli.

3. A method of synthesizing uracil according to claim 2, wherein: the thallus activation and spawn production culture operations are as follows: strain: pseudomonas aeruginosa; activating a culture medium: 10g/L beef extract, 10g/L peptone, 5g/L sodium chloride and pH7.0; the culture conditions are as follows: 35 ℃, 200rpm and 24 hours; enzyme production culture medium: 75g/L beef extract, 15g/L sodium chloride, 60mL/L soybean meal hydrolysate, 0.6g/L magnesium sulfate heptahydrate, 0.4g/L manganese sulfate monohydrate, 0.6g/L calcium chloride, 0.05g/L zinc chloride and pH7.0; the culture conditions are as follows: DO is more than or equal to 50% at 38 ℃ for 24 h.

4. A method of synthesizing uracil according to claim 2, wherein: the thallus collection comprises the following steps: centrifuging the enzyme-producing culture solution, washing wet thallus with 20mmol/L potassium dihydrogen phosphate buffer solution with pH of 7.0, and freezing and storing at-20 deg.C.

5. A method of synthesizing uracil according to claim 1, wherein: the enzymatic reaction includes: stirring the reaction system at 200rpm, reacting in water bath for 24-72h, cooling, crystallizing, and filtering to obtain uracil.

6. A method of synthesizing uracil according to claim 1, wherein: the refining stage is as follows: dissolving uracil in hot water, filtering, cooling the collected filtrate to crystallize, filtering and drying to obtain pure uracil product.

7. The method of synthesizing uracil according to claim 6, wherein: the filtration adopts suction filtration or plate-and-frame filter pressing.