CN116855551A

CN116855551A - Method for producing L-hydroxyproline by biological conversion

Info

Publication number: CN116855551A
Application number: CN202311062029.8A
Authority: CN
Inventors: 田朝勃; 李巧丽; 张梦娇; 庞义更; 巴红娟
Original assignee: Shijiazhuang Jirong Pharmaceutical Co ltd
Current assignee: Shijiazhuang Jirong Pharmaceutical Co ltd
Priority date: 2023-08-22
Filing date: 2023-08-22
Publication date: 2023-10-10

Abstract

The invention relates to the technical field of biological engineering, and discloses a method for producing L-hydroxyproline by biological conversion, which comprises the steps of preparing a culture medium by glucose, ammonium sulfate, yeast powder, monopotassium phosphate and other trace elements, feeding genetically engineered bacteria into a tank by stress, feeding ammonia water and 70% glucose into the line by bacterial culture, supplementing the culture medium and resistance, directly adding 0.8% lactose into an inducer, wherein the time is 11h, adopting CCTC as a surfactant, selecting immobilized cells, utilizing a pilot tank to perform conversion, adopting the feeding of substrate L-proline and alpha-ketoglutaric acid to regulate the pH of the system, removing MES and Tris, greatly reducing the ion concentration of the system, reducing the osmotic pressure of the system, accelerating the reaction speed, improving the total substrate concentration by 48h to be 37-40 g/L, and enabling the conversion rate to be more than 95%.

Description

Method for producing L-hydroxyproline by biological conversion

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for producing L-hydroxyproline by biological conversion.

Background

The production of L-hydroxyproline in China adopts a chemical hydrolysis extraction process, takes animal collagen as a raw material, has the yield of only 5 percent through the processes of strong acid hydrolysis, nitrous acid oxidation, ion exchange and the like, and has the problems of large raw material consumption, large three-waste discharge, high energy consumption, high production cost and the like. The process adopts animal source raw materials, nitrous acid oxidation process is used in the production process, and trace residual substances or impurities can generate potential harm to the intermediate or raw material medicine after subsequent derivatization. Foreign pharmaceutical companies have made demands on the biological origin of L-hydroxyproline, which it is desirable to limit the use of L-hydroxyproline of animal origin. All domestic enterprises adopting chemical hydrolysis processes cannot realize standard emission of three wastes, belong to severe pollution processes and face the embarrassment of stopping at any time. There is an urgent need to develop a clean and low-energy-consumption process, and the biocatalytic conversion method can replace the traditional chemical extraction process to obtain the high-purity L-hydroxyproline product, so that the large-scale production can be realized.

Disclosure of Invention

(one) solving the technical problems

Aiming at the defects of the prior art, the invention provides a method for producing L-hydroxyproline by biological conversion, which improves the conversion rate of L-hydroxyproline.

(II) technical scheme

A method for producing L-hydroxyproline by biological conversion, wherein the L-proline hydroxylase gene for producing L-hydroxyproline is derived from dactylosporium sp.rh1; the engineering bacteria BL21-CodonPlus (DE 3) strain capable of expressing the target genes is cultured, prepared by enzyme source fermentation and embedded and immobilized; the L-hydroxyproline is formed by catalytic conversion of immobilized cells to a conversion solution.

Preferably, the engineering bacteria construction steps are as follows:

(1) According to the gene phy-1 sequence information of the L-proline hydroxylase from Dactrosoporanium sp.RH1 disclosed in NCBI database;

(2) Adjusting according to the codon frequency of the escherichia coli, reducing the GC content of the whole DNA sequence, and obtaining an optimized gene (phy-2) of the L-proline hydroxylase;

(3) Respectively connecting an L-proline hydroxylase gene phy-1 of Dactylosporium sp.RH1 and an optimized gene phy-2 with an expression vector pET-M-3C to construct an expression vector of the L-proline hydroxylase; and then the expression vector is transformed into engineering bacteria BL21-CodonPlus (DE 3) capable of expressing the target gene to obtain the strain.

Preferably, the strain culture comprises:

(1) Culture medium: glucose as carbon source, yeast extract, peptone and ammonium sulfate as nitrogen source, K is selected ₂ HPO ₄ 、KH ₂ PO ₄ 、Mg2SO4、FeSO ₄ Four inorganic salts;

(2) Loading the stress strain into a tank: bacterial liquid dilution multiple: 10 ^-5 ～10 ^-8 The method comprises the steps of carrying out a first treatment on the surface of the Treatment temperature: 45-60 ℃; the treatment time is as follows: 4-7 min;

(3) Culture temperature: 28-30 ℃; the culture period is 10-16 hours;

during the culturing process, the mycelia form, pH value and light transmittance are sampled and checked.

Preferably, the optimal formula of the culture medium is as follows: glucose 2%, yeast extract 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.2%, magnesium sulfate 0.02%, ferrous sulfate 0.01%, and pH 6.8-7.2.

Preferably, the enzyme source fermentation comprises:

(1) Culturing the bacterial cells: air flow rate: 1:0.5-1.5; the rotating speed is 200-600 rpm; dissolved oxygen ratio: 10-40%; ammonia water, a culture medium, 70% glucose, resistance and the like are fed in;

(2) The induction process comprises the following steps: inducer: lactose; concentration of inducer: 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%; inducer addition time: 0h, 2h, 4h, 6h, 8h and 10h; induction time: 5h, 7h, 9h, 11h, 13h, 15h;

(3) Conversion solution: 200mM L-proline, 200mM alpha-ketoglutarate, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5MES buffer, and 1% CCTC; the CCTC concentration: 20. 24, 28, 32, 37mg/L.

Preferably, the inducer concentration: 0.8%; inducer addition time: lactose is directly added after inoculation; lactose induction time: 11h; CCTC concentration: 25mg/L.

Preferably, the embedding immobilization mode:

(1) Enzyme immobilization assay: sodium alginate immobilized enzyme, macroporous resin D201GF immobilized enzyme, macroporous resin D301R immobilized enzyme;

(2) Enzyme-containing whole cell immobilization assay: sodium alginate embeds cells.

Preferably, the enzyme activity yield of the immobilized enzyme: sodium alginate immobilized enzyme: 87.1%, macroporous resin D201GF immobilized enzyme 83.6%, macroporous resin D301R immobilized enzyme: 77.4% sodium alginate embedded cells: 87.9%; the use frequency of the immobilized bacterial cells is as follows: the conversion rate of the immobilized hydroxylase is reduced to below 71% after the immobilized hydroxylase is repeatedly used for five times, and the conversion rate of the immobilized cell is more than 92% after the immobilized hydroxylase is repeatedly used for five times.

Preferably, the selectively immobilized cells are used.

Preferably, the conversion reaction is:

(1) The conversion process comprises the following steps: utilizing a pilot plant tank for conversion, adopting the flowing addition of substrates L-proline and alpha-ketoglutaric acid to adjust the pH value of the system, and removing MES and Tris;

(2) Pretreatment of a conversion solution: filtering after conversion, collecting clear liquid, regulating pH to 3.0-3.2 with concentrated sulfuric acid, heating to above 95deg.C, centrifuging to remove impurity protein, collecting supernatant, concentrating under reduced pressure for 5-6 times, decolorizing with activated carbon, adding equal volume ethanol, removing protein, concentrating under reduced pressure, adding ethanol for crystallization, drying, and making into final product;

(3) Detecting the content by a liquid chromatography external standard method: 98.0% or more of the raw materials are qualified products.

(III) beneficial technical effects

The culture medium is prepared by taking single glucose as a carbon source, taking ammonium sulfate as a nitrogen source, adding a small amount of yeast powder, monopotassium phosphate and other microelements, and increasing the enzyme content from 1% to 2%. The genetically engineered bacteria are amplified, cultured and put in a pot under stress, and the dilution factor of bacterial liquid is 10 ^-5 ～10 ^-8 The treatment temperature is 45-60 ℃ and the treatment time is 4-7 min, so that the pilot experiment of the genetically engineered bacteria is successfully realized. Ammonia water and 70% glucose are fed in line for culturing the thalli, a culture medium and resistance are supplemented, and the bacterial amount is increased from 2% to 5%, and the maximum bacterial amount can reach 6%. The inducer is lactose, the concentration is 0.8%, the lactose is directly added after inoculation, the induction time is 11h, the lactose is both a carbon source and an inducer, and the induction is started after the consumption of glucose, so that the glucose flow processing technology is realized, the conversion rate is highest, the cost is controlled, and the technology is simple. The surfactant adopts CCTC, and compared with SDS, the surfactant has no foaming effect and more stable wall breaking effect. The conversion rate of the immobilized enzyme and the immobilized cells after five times of use is respectively below 71% and above 92%, and the immobilized cell preparation method has the advantages of less loss, simple separation, multiple times of repeated use, mild reaction conditions and simple operation. The pilot plant pot is used for conversion, the pH value of the system is regulated by adopting the flowing addition of the substrate L-proline and alpha-ketoglutarate, MES and Tris are removed, the ion concentration of the system is greatly reduced, the osmotic pressure of the system is reduced, the reaction speed is accelerated, the total substrate concentration is increased to 300MM for 48 hours, the L-hydroxyproline concentration reaches 37-40 g/L, and the conversion rate reaches more than 95%.

Drawings

FIG. 1 is a diagram of cis-L-hydroxyproline and L-proline targets.

FIG. 2 is a diagram of trans-L-hydroxyproline, cis-L-hydroxyproline, and L-proline targets.

FIG. 3 shows the reaction solution of the enzyme-catalyzed conversion.

FIG. 4 shows the effect of the concentration of inducer lactose on the expression level.

FIG. 5 shows the effect of induction time on the expression level.

Fig. 6 shows the surfactant type and concentration change.

FIG. 7 shows the conversion of L-proline by immobilized hydroxylase in different batches.

FIG. 8 is the residual enzyme activity of different batches of embedded cells for catalyzing L-proline.

FIG. 9 shows the conversion of L-proline to L-hydroxyproline.

FIG. 10 is a flow chart of biological enzyme preparation and immobilization.

FIG. 11 is a flow chart of an enzymatic conversion process.

Detailed Description

Example 1

1. Strain construction

According to the gene phy-1 sequence information of the L-proline hydroxylase from Dactrosoporanium sp.RH1 disclosed in NCBI database, the GC content of the whole DNA sequence is reduced by adjusting according to the codon frequency of escherichia coli, and the optimized gene (phy-2) of the L-proline hydroxylase is obtained. Then, respectively connecting the gene phy-1 of the L-proline hydroxylase of Dactyosporium sp.RH1 and the optimized gene phy-2 with an expression vector pET-M-3C to construct an expression vector of the L-proline hydroxylase; and then the expression vector is transformed into engineering bacteria BL21-CodonPlus (DE 3) capable of expressing the target gene to obtain the strain.

2. Strain culture and enzyme source preparation

2.1 preparation of culture Medium for shake flask strains

The shake flask strain culture medium adopts LB culture medium, peptone 10g, yeast extract 5g, sodium chloride 7g and water, stirring and dissolving, dissolving to 1000mL, and subpackaging to 9cm double-dish, 50mL and 500mL shake flask. The filling amount is 15mL, 20mL and 100mL, and the autoclave is used for sterilization. Sterilizing at 120-121 deg.c for 20min. And (5) standby.

2.2 inoculation

And taking out the glycerol pipe from the refrigerator at the temperature of minus 80 ℃, marking the glycerol pipe on a double dish, culturing in an incubator, picking single bacterial colonies, inoculating into 20mL shake flask for culturing, sucking 5mL to 500mL shake flask from 50mL for culturing, and inoculating into a fermentation tank for culturing after growing.

2.3 culture temperature: 28-30 ℃; the culture period is 10-16 hours.

2.4 fermentative preparation of hydroxylase

Fermentation tank culture operation: glucose 1.4kg, yeast extract 0.7kg, sodium chloride 1.05kg and water were added to a 30L fermenter, stirred uniformly, dissolved to 16L, and covered. Controlling the temperature to be 120-122 ℃ and sterilizing for 20-30 min. Introducing air into the tank, opening cold water to cool, and starting inoculation after the tank is cooled. And (3) sterilizing the surrounding environment before inoculation, rapidly pouring the resistance, the shake flask seed liquid and lactose into a tank under the protection of flame, putting an inoculation cap on the tank, and immediately introducing air for culture.

Tank temperature: 25-29 DEG C

Flow rate: the air flow after inoculation is controlled to be 10L/min m according to the period stage by stage ³ The culture period is 45-48 h above the fermentation broth.

In the culture process, taking a sterile sample and a biochemical sample to check the mycelium form, the pH value and the light transmittance, and before transplanting for 2 hours, judging whether bacteria are infected by the bacteria by microscopic examination, and sampling and measuring the pH and the light transmittance before transplanting.

In the fermentation process, ammonia water is fed in to maintain the pH value at 6.8-7.0, 70% glucose is fed in, and foam is eliminated by using a defoaming agent to obtain a cultured enzyme source. And (3) centrifugally filtering and collecting solid thalli for preparing immobilized enzyme, and directly entering filtered waste liquid into a sewage treatment device.

2.5 enzyme Activity assay

2.5.1 determination of hydroxylase enzyme Activity

Taking 10mL of fermentation liquor, centrifuging at 12000r/min for 5min, discarding the supernatant, collecting thalli, suspending the thalli in 1mL of Tris-HCl with pH of 7.5 and 20mmol/L, crushing the thalli by using an ultrasonic crusher with crushing power of 200W for 10min (ultrasonic wave for 5s and interval of 10 s), centrifuging at 12000r/min for 5min, and collecting the supernatant, thus obtaining crude enzyme liquid.

E.coli BL21/pET-M-3C expressed recombinant protein with 6-His tag and Ni ²⁺ Purifying by using an affinity chromatography column. Balancing Ni with 20mmol/L phosphate buffer solution at pH7.8 ²⁺ Column, sample volume is 3mL, wash with 10mmol/L imidazole solution with pH7.8, 6-9 mL, removeEluting the mixed protein with 200mmol/L imidazole solution with pH of 7.8 to obtain purified enzyme solution, and collecting effluent liquid, which is generally about 3mL, and refrigerating for standby. Freeze-drying the collected liquid to obtain hydroxylase freeze-dried powder.

After the freeze-dried powder is added into buffer solution for dissolution, SDS-PAGE electrophoresis can be used for detecting the purity of hydroxylase. The content determination method comprises measuring colorimetric value (OD 595) at 595nm with ultraviolet spectrophotometer using Bovine Serum Albumin (BSA) as standard protein, and preparing hydroxylase content standard curve according to colorimetric result.

The conversion activity of hydroxylase was measured at the same protein concentration, and 80mM MES buffer, 4mM proline, 8mM 2-ketoglutarate, 2mM ferrous sulfate, 4mM ascorbic acid and 35℃were added to the reaction solution for 10min. The conversion solution is subjected to derivatization reaction and is detected and analyzed by HPLC.

2.5.2 immobilized cell bacteria the enzyme activity was detected in the same manner.

The results showed that a single clear band was present at 31kDa in SDS-PAGE electrophoresis and the purified protein content was about 0.147mg/mL.1g wet bacterial enzyme activity is 22.0U/g,1mg pure protease activity is

1.63U/mg, and it was calculated that about 13.5mg of pure enzyme was obtained from 1g of recombinant E.coli wet cells. The conversion rate of 40mM L-proline can reach 98%.

2.6 embedding (immobilization) of enzyme-containing Whole cells

The fermentation broth was filtered with a ceramic membrane to collect 1.1kg of cells, and the supernatant was centrifuged to obtain an enzyme source. The enzyme source was added to 0.9% physiological saline to prepare 1.4L of a bacterial suspension having a bacterial content of 80%.

Adding the mixture into 7L of melted embedding liquid, and stirring for 20-30 min. The embedding liquid contains 5% of sodium alginate and 2% of polyvinyl alcohol, and the embedding liquid which is uniformly mixed is slowly dripped into an immobilization kettle for molding.

The immobilization kettle contains CaCl with the equal volume of 0.1M ₂ And standing the solution for 3-4 hours, and fixing the enzyme source. And (5) carrying out suction filtration by using a suction filter and washing with deionized water to obtain immobilized cells.

3. Conversion reaction step

Adding the immobilized cells into a conversion reaction kettle according to the mass and volume ratio of 1kg:360L of the conversion solution, and performing enzyme catalytic conversion.

Preparing a conversion solution: 1400L of the conversion solution contains 47.46kg of L-proline; 33.81kg of a-ketoglutarate; 2.338kg of ferrous sulfate heptahydrate; vitamin C0.742 kg.

And (3) process control: maintaining pH at 6.5-6.7 and temperature at 28-30 deg.c, stirring at 140rpm to react, detecting L-proline converting rate, feeding converting liquid, feeding 1400L for 40-48 hr, and adding immobilized cell to 30kg. The concentration of the L-hydroxyproline reaches 37-40 g/L, and after the reaction is finished, the centrifugal solid-liquid separation is carried out to obtain a conversion reaction solution containing the hydroxyproline. After the reaction is finished, the mixture is pumped into a conversion liquid storage tank.

4. Separation and purification step

Stirring and adding sulfuric acid in a conversion liquid storage tank to adjust the pH value to 3.0-3.1; heating to above 95 ℃ and preserving heat for 1-2 h; cooling, centrifuging and measuring the pH value to 3.5-3.6; the supernatant was collected.

5. Charcoal stripping procedure

And removing pigment, impurity protein and other trace impurities by adopting an activated carbon adsorption technology.

Pumping the clear liquid into a primary decolorizing kettle filled with active carbon, wherein the carbon content is 2.5-3%, stirring, heating to 60-65 ℃, decolorizing for 2h, filtering, removing carbon, and collecting filtrate into a filtrate storage tank.

6. Purifying, concentrating, crystallizing and centrifuging

Concentrating the decolorized solution in the previous step by 5-6 times through a concentrating flash evaporator under reduced pressure, cooling the vapor generated in the process through a concentrating condenser, and collecting the vapor into a receiving water tank. Pumping into a concentrating kettle, adding ethanol with equal volume to generate flocculent protein, centrifuging, and collecting the clear liquid.

Pumping the concentrated solution in the previous step into a secondary concentrating kettle, concentrating under reduced pressure, cooling the vapor generated in the process by a secondary concentrating condenser, and collecting the vapor in a receiving tank.

And (3) placing the secondary concentrated solution reaching the end point in the last step into a crystallization kettle, adding ethanol, and introducing cold brine into a jacket for cooling and crystallizing. And (3) placing the crystallization stock solution into a crystallization centrifuge for solid-liquid separation to obtain a crude L-hydroxyproline product, and collecting mother liquor into a crystallization mother liquor storage tank.

7. Refining and drying process

And (3) heating the crude L-hydroxyproline to 70 ℃ in a secondary decoloring kettle to dissolve, adding active carbon for decoloring, and filtering to remove carbon. The filtrate is decompressed and concentrated by a secondary concentrating kettle, and steam generated in the process is cooled by a secondary concentrating condenser and then is collected in a receiving tank.

And (3) after the concentrated solution reaches the concentration end point, putting the concentrated solution into a recrystallization kettle for cooling and crystallizing, and centrifuging the concentrated solution by a recrystallization centrifuge to obtain an L-hydroxyproline wet product.

And (3) carrying out biconical vacuum drying on the wet L-hydroxyproline product to obtain the L-hydroxyproline product.

Example 2

(1) Product standard formulation and index

L-hydroxyproline product index

Project	Index (I)
		Appearance state	White crystalline solid powder
Content (%)	≥98.0
		Specific optical rotation	-74.0°～-77.0°
Proline content	≤1.0％

The product is detected to meet the enterprise standard requirement. The medicine is used by users, and meets the requirements of medicine production.

(2) Confirmation of L-hydroxyproline optical isomer

As shown in HPLC spectrogram, spectrogram 1 is a standard product of cis-4-hydroxy-L-proline and L-proline, spectrogram 2 is a standard product of trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline and L-proline, and spectrogram 3 is a sample reaction solution.

As can be seen from the L-proline conversion reaction solution, only trans-L-hydroxyproline was produced, and the catalytic conversion selectivity of the enzyme was specific.

Example 3 optimization of biosynthesis of L-hydroxyproline Medium and enzyme Source Induction Process conditions

(1) Optimization of basal medium

In the small test stage, the LB culture medium for the enzyme source induction culture medium is complicated and valuable in nutrient components of yeast powder and peptone, so that the industrialization dosage is large, the quality of raw materials is difficult to stabilize even the quality of products of the same company, and unstable production can be caused. Glucose with lower price and single component is used as a carbon source, ammonium sulfate is used as a nitrogen source, a small amount of yeast powder, monopotassium phosphate and other microelements are added, and various nutritional components and proportions of the culture medium are determined through an orthogonal test:

A. optimizing nitrogen sources

Yeast extract, peptone and ammonium sulfate; as a nitrogen source, an Lg (33) orthogonal table was used for the test.

TABLE 1

B. Optimizing inorganic salts

Experiment selects K ₂ HPO ₄ 、KH ₂ PO ₄ 、Mg ₂ SO ₄ 、FeSO ₄ Four inorganic salts were tested in an Lg (34) orthometric design.

TABLE 2

Sequence number	K ₂ HPO ₄ (A％)	KH ₂ PO ₄ (B％)	Mg ₂ SO ₄ (C％)	FeSO ₄ (D％)
					1	0.2	0.5	0.5	0.01
2	0.5	1	1	0.03
					3	0.8	1.2	2	0.05

Through orthogonal experiments, the optimal formula of the culture medium is determined as follows: glucose 2%; yeast extract powder 0.5%; ammonium sulfate 0.5%; potassium dihydrogen phosphate 0.1%; sodium chloride 0.2% magnesium sulfate 0.02%; ferrous sulfate 0.01%; the pH is 6.8-7.2.

The amount of the thalli is increased by optimizing the culture medium, and the enzyme content is increased by one time from 1% to 2%.

(2) Stress optimization strain feeding tank

The problem that the fermentation failure is caused by the plasmid loss is a common problem of engineering bacteria amplification because of the expansion and the tank loading of the genetically engineered bacteria, and in order to solve the problem, experiments are carried out for a long time, and the temperature stress experiment is finally utilized by a plurality of professionals for solving the problem. Orthogonal test method Lg (34) was used.

TABLE 3 orthogonal test table for temperature stress

Project	1	2	3	4
					1 dilution of bacterial liquid	10 ^-5	10 ^-6	10 ^-7	10 ^-8
2 treatment temperature (. Degree. C.)	45	50	55	60
					3 treatment time (minutes)	4	5	6	7

The concentration, temperature and treatment time of the thalli suitable for stress are found through a plurality of experiments, so that the pilot experiment of the genetically engineered bacterium is successfully realized.

(3) Thallus culture and induction process condition optimization

The air flow rate is optimized to be 1:0.5-1:1.5, the stirring rotating speed is adjusted to be 200-600 r/min, the dissolved oxygen is comprehensively regulated and controlled to be 10-40%, the concentration of oxygen is controlled in stages, the pH is controlled by online feeding of ammonia water, the culture medium is supplemented, 70% glucose is fed, the resistance is supplemented and the like, and the bacterial density is improved. The bacterial amount is increased from 2% to 5%, and the maximum bacterial amount can be up to 6%.

A. Optimization of inducers

isopropyl-B-D-galactoside (IPTG) is used as an inducer, but the IPTG has high cost and pollutes the environment, and the process control condition is harsh and is not suitable for industrial production. Based on long-term pilot plant work, a fermentation process for inducing recombinant proteins by lactose instead of IPTG was developed. Unlike IPTG, where lactose is both a carbon source and an inducer, lactose induction begins after glucose consumption, a very important regulatory mechanism that enables the glucose processing process to be practiced by trying to essentially replace IPTG completely with lactose and achieve a fairly good result.

B. Influence of the concentration of inducer lactose on the expression level

Inoculating seed solution into fermentation culture medium according to 1% ratio, when shaking bacteria at 28 deg.C and 140rpm about 2hA600 at 0.6-0.8, respectively adding inducer lactose into culture flask to make final concentration be 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2%, induction culturing for 9h, centrifugally collecting bacterial body, adding conversion solution (containing 200mM L-proline, 200mM alpha-ketoglutaric acid, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5MES buffer solution and 1% SDS) into bacterial body according to the ratio of 1g to 100mL (bacterial body quantity: conversion solution), making conversion to make liquid chromatography analysis and analysis, and making experiment result, when inducer concentration is between 0.6-1.2%, its conversion rate is not greatly influenced, cost principle is considered, inducer concentration is defined as 0.8%.

C. Influence of the addition time of inducer lactose on the expression level

In shake flask culture, induction is generally considered to be suitable at low cell concentrations, because cells are in the logarithmic growth phase at low cell concentrations, and actively grow, facilitating protein expression.

Inoculating the seed solution into a fermentation culture medium according to the proportion of 1%, carrying out liquid chromatography analysis on the seed solution from 0h, 2h, 4h, 6h, 8h and 10h after inoculation, adding 8g/L of inducer lactose, carrying out induction culture for 9h, centrifugally collecting thalli, adding a conversion solution (containing 200mM L-proline, 200mM alpha-ketoglutaric acid, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5MES buffer solution and 1% SDS) into the thalli according to the mass volume ratio of 1g to 100mL (the quantity of the thalli: the conversion solution), carrying out three parallel experiments in each group, and carrying out the transformation analysis on the lactose, wherein experimental results show that lactose addition has little influence on the conversion rate after 0-10 h inoculation, has no requirement on an OD value of 0.6-0.8, and reduces the process difficulty, so lactose is directly added after inoculation.

D. Influence of lactose Induction time on expression level

Inoculating the seed solution into a fermentation culture medium according to the proportion of 1%, adding an inducer lactose to the final concentration of 8g/L for induction culture, carrying out induction for 5h, 7h, 9h, 11h, 13h and 15h, centrifugally collecting thalli, adding a conversion solution (containing 200mM L-proline, 200mM alpha-ketoglutarate, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5MES buffer solution and 1% SDS) into the thalli according to the mass volume ratio of 1g to 100mL (the quantity of the thalli: the conversion solution), carrying out OD value measurement on each group of three parallel experiments, and carrying out liquid chromatography analysis on the conversion.

The experimental result analysis shows that the OD value is always increased from 5 to 9 hours, the conversion rate is always increased, the OD value is 11 to 13 hours, the conversion rate starts to be reduced, and the conversion rate is slightly reduced and the change is not obvious. Therefore, the induction time of lactose was determined to be once at OD after 11h and 30 minutes after 11h, and the induction was stopped when the OD value was decreased.

Example 4 surfactant species and concentration optimization

The small test of the surfactant adopts SDS all the time, but when the small test is converted by a pilot scale test tank, air is directly introduced due to the increase of the charging coefficient, stirring is started, the foam is very large to cause serious liquid escape, the wall breaking effect of the SDS is weakened by adding the defoaming agent, and the CCTC without the foaming effect is added for wall breaking to carry out gradient test.

Inoculating the seed solution into a fermentation culture medium according to the proportion of 1%, adding an inducer lactose to the final concentration of 8g/L for induction culture, carrying out induction for 20 hours, centrifuging to collect thalli, adding a conversion solution (containing 200mM L-proline, 200mM alpha-ketoglutaric acid, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5MES buffer solution and CCTC) into the thalli according to the mass volume ratio of 1g to 100mL (the thalli amount: the conversion solution), and respectively selecting three parallel experiments of final concentrations of 20, 24 and 28, 32 and 37mg/L for conversion, wherein the concentration conversion rate of the experiment results of 20 and 24mg/L is not influenced, the conversion rate of 37mg/L is reduced, and the concentration experiment of 25mg/L is adopted in consideration of production cost and production stability.

Example 5 immobilization assay of enzyme

(1) Preparing a crude enzyme solution: centrifuging the fermentation liquor at 12000r/min for 5min, discarding the supernatant, collecting thalli, and washing twice with physiological saline to obtain resting cells. 1g of the resting cells thus obtained were resuspended in 5mL of water, homogenized under high pressure, and disrupted to give a crude enzyme solution. And freeze-drying the crude enzyme solution by a freeze dryer to obtain freeze-dried crude enzyme powder.

(2) Enzyme immobilization method one: mixing 1.5% (w/v) sodium alginate with the crude enzyme solution, and thawing in a water bath at 37deg.C; after fully mixing, adding 1% (v/v) glutaraldehyde, shaking for 30min in a shaking table, and standing in a refrigerator at 4 ℃ for 3h to obtain a mixed solution. Then instilling the mixed solution into 2% (w/v) calcium chloride solution to form gel pellets; filtration and washing twice with 0.9% (w/v) sodium chloride solution gave immobilized hydroxylase.

(3) The immobilization method of the enzyme is as follows: crude hydroxylase enzyme solution was obtained by the same method as described above for cell treatment. The macroporous resin D201GF is washed 3 times with deionized water, soaked for 4 hours with 4% NaOH, washed with deionized water to be neutral, soaked for 4 hours with 4% HCl, converted into Cl type, washed with deionized water to be neutral, and finally soaked with deionized water with volume more than 2 times for standby. Taking treated macroporous ion exchange resin, re-suspending the macroporous ion exchange resin by using a phosphate buffer solution with the pH of 7.0, adding glutaraldehyde solution to the final concentration of 0.1-0.3%, slightly vibrating for 1-5h, and then washing off excessive glutaraldehyde by using deionized water; suspending the cross-linked macroporous ion exchange resin in hydroxylase crude enzyme liquid according to the mass ratio of 1:5-1:10, fixing for 5-15h at the temperature of 0-16 ℃, and washing with deionized water to remove free enzyme, thus obtaining the immobilized hydroxylase.

(4) Enzyme immobilization method three: crude hydroxylase enzyme solution was obtained by the same method as described above for cell treatment. Repeatedly cleaning macroporous resin D301R with 50-60deg.C hot water, soaking in 3-5% hydrochloric acid for 24-48 hr, washing with water to neutrality, soaking in 3-5% NaOH for 24-48 hr, washing with water to neutrality, balancing with phosphate buffer solution of pH 7.5, and washing with deionized water. And dissolving chitosan in an acetic acid solution with the mass fraction of 10-15% to obtain a chitosan saturated solution. Dripping the chitosan into a three-mouth bottle filled with D301R macroporous resin, and absorbing the chitosan on the inner and outer surfaces of the resin for 3-5h under negative pressure. The carrier is separated by decompression filtration, washed to be neutral by distilled water, then 5 to 8 percent of glutaraldehyde is added for treatment, washed to be neutral by water, and dried in vacuum. Dissolving a certain amount of hydroxylase in a phosphate buffer solution with pH of 4.5, adding a certain amount of prepared macroporous resin chitosan membrane carrier, standing at 0-20 ℃ for 5-10h, and drying to obtain the immobilized hydroxylase.

(5) Immobilization method enzyme activity assay:

1g of immobilized enzyme prepared by different methods is added into a reaction system of L-proline with the final concentration of 5g/L, and the reaction is carried out for 20min under 35 ℃ magnetic stirring. The reaction solution was used for derivatization, the peak area of the product was measured by HPLC, the concentration of the product was calculated from the peak area, and the enzyme activity was calculated as shown in Table 4.

TABLE 4 enzyme Activity of free enzyme and different immobilized enzymes

Numbering device	Enzymes	Yield of enzyme activity (%)	Specific enzyme activity (U/g)
				1	Free enzyme	100	134
2	Macroporous resin D301R immobilized enzyme	77.4	157
				3	Macroporous resin D201GF immobilized enzyme	83.6	183
4	Sodium alginate immobilized enzyme	87.1	201

(6) Frequency of use of immobilized enzyme

20g of immobilized hydroxylase prepared by different methods are added to 100mL of reaction buffer, L-proline with a final concentration of 200mM is added and reacted under magnetic stirring at 35 ℃. After 24 hours of reaction, the reaction was stopped and the conversion was measured by HPLC. After the reaction, the mixture was filtered with gauze, and the residue was washed three times with 10mL of 80% (v/v) ethanol, filtered, and the residue was recovered to obtain immobilized enzyme, which was used for the next batch reaction (as shown in Table 5).

TABLE 5 conversion of L-proline catalyzed by immobilized enzymes of different batches

Batch of	1	2	3
				Macroporous resin D301R immobilized enzyme	>95％	>81％	>68％
Macroporous resin D201GF immobilized enzyme	>97％	>83％	>70％
				Sodium alginate immobilized enzyme	>98％	>85％	>72％

From the results, it was found that the number of times of use of the immobilized enzyme was still to be increased.

EXAMPLE 6 enzyme-containing Whole cell immobilization assay

(1) Embedding of whole cells: centrifuging the fermentation liquor at 12000r/min for 5min, discarding the supernatant, collecting thalli, and washing twice with physiological saline to obtain resting cells. The resting cells 1g were resuspended in 5mL of water to give a bacterial suspension.

The bacterial suspension content was 400mg/g as measured by the Bradford method. Mixing 5% (w/v) sodium alginate with 2% (w/v) polyvinyl alcohol for melting; after fully and evenly mixing, adding the bacterial suspension, putting into a shaking table to shake for 30min, and standing for 3h in a refrigerator at the temperature of 4 ℃ to obtain a mixed solution. Then instilling the mixed solution into 2% (w/v) calcium chloride solution to form gel pellets; filtering, washing twice with 0.9% (w/v) sodium chloride solution to obtain embedded immobilized cells.

(2) Determination of the Activity of the embedded cells:

1g of the prepared enzyme-embedded enzyme was added to a reaction system of L-proline having a final concentration of 200mM, and reacted at 30℃under magnetic stirring for 20 minutes. The reaction solution was used for derivatization, the peak area of the product was measured by HPLC, the concentration of the product was calculated from the peak area, and the enzyme activity was calculated as shown in Table 6.

TABLE 6 enzyme Activity of free and immobilized enzymes and embedded cells

Numbering device	Enzymes	Yield of enzyme activity (%)	Specific enzyme activity (U/g)
				1	Free enzyme	100	434
2	Immobilized enzyme	87.4	757
				3	Embedding cells	87.9	798

The experiments in the table show that the activity of the free enzyme is the highest, and the activity of the immobilized enzyme and the activity of the embedded cells are not very different.

(3) Frequency of use study of immobilized cell

To 100mL of reaction buffer, 20g of immobilized hydroxylase and 20g of embedded cells were added, and L-proline was added to a final concentration of 200mM, and reacted under magnetic stirring at 30 ℃. The reaction was stopped after the conversion was measured to 99% by HPLC during the reaction or after 24 hours of the reaction. After the reaction is finished, filtering the mixture by using gauze, washing filter residues by using 10mL of 80% (v/v) ethanol for three times, filtering, recovering the filter residues to obtain immobilized enzyme, and measuring residual activity for the next batch of reaction.

As shown in FIGS. 7 and 8, it was found that the conversion rate was reduced to 71% or less after the immobilized hydroxylase was repeatedly used five times. The transformation rate of the immobilized cells is still more than 92% after the immobilized cells are repeatedly used for five times. The preparation method has the advantages of less enzyme activity loss of the embedded cells, simple separation, multiple repeated utilization times, mild reaction conditions and simple and convenient operation. The immobilized somatic cells are selected for use.

(4) Transformation study of immobilized cells on L-proline substrate concentration in the reaction system.

1g of the immobilized cells was added to 100mL of the transformation solution and reacted for 24 hours. The concentrations of L-proline in the conversion solutions were respectively (5, 10, 20, 40, 60 g/L), and after the reaction was completed, the conversion rate of L-proline was measured.

TABLE 7

Substrate concentration (g/L)	Reaction time (h)	Conversion (%)
			5	24	>99
10	24	>99
			20	24	>98
40	24	>95
			50	24	>90

Analysis of results: the L-proline conversion rate of the immobilized cells prepared by the method is slightly reduced when the immobilized cells convert 40g/L of L-proline, and the conversion rate is 95%. The conversion rate was reduced to 72.89% at 50g/L of L-proline. The optimal L-proline conversion concentration is selected to be 40g/L in consideration of the difficulty in extracting and separating the hydroxyproline from the proline.

Example 7 conversion reaction conditions process optimization

(1) In the small test stage, the thallus culture and induction are both carried out on a shaking table, the dissolved oxygen amount is optimized by the combined action of regulating the air flow and the stirring rotating speed on a pilot scale test tank, the pH is controlled on line, the conversion of the 300MM substrate is optimized for more than 95% in 48 hours, and the period is shortened by 24 hours.

(2) Implementation and optimization of conversion reaction fed-batch process

The existence of the substrate alpha-ketoglutarate makes the pH value of the reaction system very low, and the consumption of the alpha-ketoglutarate in the reaction process can make the pH value of the reaction system very high. The pH value of the original reaction system can be regulated on line by utilizing pilot scale tank conversion, wherein the original reaction system has 2.558 percent of MES and about 7-9 percent of Tris to stabilize the pH value of the system, the pH value of the system is regulated by adopting the flowing addition of the substrate L-proline and alpha-ketoglutaric acid, the MES and the Tris are removed, the ion concentration of the system is greatly reduced, the osmotic pressure of the system is reduced, the reaction speed is accelerated, the total substrate concentration is increased to 300MM, the L-hydroxyproline concentration reaches 37-40 g/L, and the conversion rate reaches more than 95 percent.

EXAMPLE 8L-hydroxyproline extraction

(1) Pretreatment of conversion solution

Filtering after conversion, collecting clear liquid, regulating pH to 3.0-3.2 with concentrated sulfuric acid, heating to above 95 ℃, centrifuging to remove impurity-removed protein, collecting supernatant, concentrating under reduced pressure for 5-6 times, decolorizing with active carbon, adding equal volume ethanol, removing protein twice, concentrating under reduced pressure, adding ethanol for crystallization, drying, and obtaining the final product.

(2) Detecting the content by a liquid chromatography external standard method: 98.0% or more of the raw materials are qualified products.

In a word, the enzyme yield of the system is improved from 1% to 5% by optimizing the induction process and the conversion system, the substrate concentration is improved from 200MM to 300MM, the conversion rate is over 95%, and the qualified product is obtained by separation and purification.

Claims

1. A method for producing L-hydroxyproline by biological conversion, which is characterized by comprising the following steps: the L-proline hydroxylase gene for producing L-hydroxyproline is derived from dactylosporium (Dactylosporium sp.RH1); the engineering bacteria BL21-CodonPlus (DE 3) strain capable of expressing the target genes is cultured, prepared by enzyme source fermentation and embedded and immobilized; the L-hydroxyproline is formed by catalytic conversion of immobilized cells to a conversion solution.

2. The L-proline hydroxylase gene for producing L-hydroxyproline according to claim 1, which is derived from a sporangium, characterized in that: the engineering bacteria construction steps are as follows:

3. The method for culturing and fermenting the engineering bacteria strain capable of expressing the target gene and preparing and embedding and immobilizing the target gene according to claim 1, which is characterized in that: the strain culture comprises the following steps:

(3) Culture temperature: 28-30 ℃; the culture period is 10-16 hours;

4. A culture medium according to claim 3, characterized in that: the optimal formula of the culture medium is as follows: glucose 2%, yeast extract 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.2%, magnesium sulfate 0.02%, ferrous sulfate 0.01% and pH 6.8-7.2.

5. The method for culturing and fermenting the engineering bacteria strain capable of expressing the target gene and preparing and embedding and immobilizing the target gene according to claim 1, which is characterized in that: the enzyme source fermentation comprises the following steps:

(3) Conversion solution: 200mM L-proline, 200mM alpha-ketoglutarate, 6mM ferrous sulfate, 6mM L-ascorbic acid, 80mM pH 6.5MES buffer, 1% CCTC; the CCTC concentration: 20. 24, 28, 32, 37mg/L.

6. The enzyme source fermentation according to claim 5, wherein: the inducer concentration: 0.8%; inducer addition time: lactose is directly added after inoculation; lactose induction time: 11h; CCTC concentration: 25mg/L.

7. The method for culturing and fermenting the engineering bacteria strain capable of expressing the target gene and preparing and embedding and immobilizing the target gene according to claim 1, which is characterized in that: the embedding immobilization mode comprises the following steps:

8. The entrapping immobilization method according to claim 7, wherein: yield of enzyme activity of the immobilized enzyme: sodium alginate immobilized enzyme: 87.1%, macroporous resin D201GF immobilized enzyme 83.6%, macroporous resin D301R immobilized enzyme: 77.4% sodium alginate embedded cells: 87.9%; the use frequency of the immobilized bacterial cells is as follows: the conversion rate of the immobilized hydroxylase is reduced to below 71% after the immobilized hydroxylase is repeatedly used for five times, and the conversion rate of the immobilized cell is more than 92% after the immobilized hydroxylase is repeatedly used for five times.

9. The immobilized enzyme and cell of claim 8, wherein: the selectively immobilized cells are used.

10. The catalytic conversion of L-hydroxyproline by immobilized cells to a conversion solution according to claim 1, wherein: the conversion reaction is as follows: