CN113403245A

CN113403245A - Recombinant escherichia coli immobilized cell and application thereof

Info

Publication number: CN113403245A
Application number: CN202110680783.2A
Authority: CN
Inventors: 朱云峰; 苗华明; 夏俊刚; 孙剑峰; 袁风娇; 张立志; 李鹤松
Original assignee: Disha Jinan Pharmaceutical Research Co ltd; Dijia Pharmaceutical Group Co ltd
Current assignee: Disha Jinan Pharmaceutical Research Co ltd; Dijia Pharmaceutical Group Co ltd
Priority date: 2021-06-19
Filing date: 2021-06-19
Publication date: 2021-09-17

Abstract

The invention relates to a recombinant escherichia coli immobilized cell and application thereof in preparation of micromolecular hyaluronic acid or salt thereof. The technical scheme of the invention is that a recombinant escherichia coli immobilized cell containing hyaluronidase is prepared by constructing a recombinant plasmid containing a natural hyaluronidase coding gene, introducing escherichia coli competent cells, screening to obtain recombinant escherichia coli, and immobilizing the recombinant escherichia coli to obtain the immobilized cell. The invention provides a recombinant escherichia coli immobilized cell and application thereof in preparation of micromolecular hyaluronic acid or salt thereof.

Description

Recombinant escherichia coli immobilized cell and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant escherichia coli immobilized cell and application thereof in preparation of micromolecular hyaluronic acid or salt thereof.

Background

Hyaluronic acid is an unbranched glycosaminoglycan consisting of a disaccharide unit consisting of N-acetylglucosamine and D-glucuronic acid, exists in animal intercellular substances and certain bacterial capsules, and has wide application in the fields of cosmetics, foods and medicines. According to the molecular weight, the hyaluronic acid or the salt thereof can be divided into macromolecules (1800-3000 kDa), medium molecules (1000-1800 kDa), small molecules (10-1000 kDa) and micro molecules (below 10 kDa), and the hyaluronic acids with different molecular weights have different water locking, penetrating and biological activity functions. The hyaluronic acid with medium and small molecular weight is beneficial to the transportation of protein and polypeptide substances, is applied to the healing of chronic wounds, can permeate into the basal layer of skin, effectively inhibits the release of inflammatory factors, relieves the sensitive state of the skin caused by various factors, eliminates free radicals, repairs the damage of skin cells caused by the stimulation of ultraviolet rays or chemical substances, realizes deep moisturizing and repairs damaged cells, and can kill various kinds of cancer cells by damaging the combination of receptors and the hyaluronic acid.

With the application and popularization of low molecular weight hyaluronic acid, the degradation and preparation technology becomes a hot point of research. Common degradation methods include physical degradation, such as heating, ultrasonication, radiation, etc.; chemical degradation, such as oxidative hydrolysis, acid hydrolysis, alkaline hydrolysis, etc., but these two degradation methods have the disadvantages of wide molecular weight distribution range of oligosaccharides, complicated purification procedure, easy damage of oligosaccharide structure, high requirement on equipment, etc., reduce the preparation efficiency of oligosaccharides with specific molecular weight, and are difficult to mass-produce in industrial scale. The reaction condition for preparing the low-molecular hyaluronic acid by the biological enzyme method is mild, and the preparation efficiency is high.

The patent CN 103255187B discloses a method for preparing low molecular hyaluronic acid, which is generated by bacillus with the preservation number of CGMCC No.5744A hyaluronidase produced by fermentation is used for degrading hyaluronic acid or its salt with molecular weight of more than 1000 kDa, and the enzyme amount is 10 per 1 kg hyaluronic acid or its salt⁶～10⁷IU, the preparation steps comprise bacillus seed culture, bacillus fermentation and enzyme production, enzyme liquid extraction, enzymolysis, inactivation, filtration, precipitation, dehydration and drying.

The patent CN 109897876B discloses a preparation method of small molecular hyaluronic acid or salt thereof, which uses hyaluronidase produced by fermenting lactobacillus plantarum with the strain preservation number of CGMCC No.16836 to carry out enzymolysis on the hyaluronic acid or salt with the molecular weight of 500 KDa-700 KDa, and the enzyme amount is 10 per 1 kg of the hyaluronic acid or salt⁵～10⁸IU, the preparation steps comprise lactobacillus plantarum seed culture, enzyme production by fermentation, enzyme liquid extraction, enzymolysis, inactivation, activated carbon adsorption impurity removal, microporous filter membrane filtration and low-temperature spray drying to obtain the micromolecular hyaluronic acid or the salt thereof.

Patent CN 110331178B discloses a method for preparing small molecular hyaluronic acid by enzyme cleavage, which uses a collection number of CCTCC NO: the hyaluronidase is prepared by fermenting M2018452 Arthrobacter globiformis HL6, has enzymolysis molecular weight of more than 600 KDa hyaluronic acid or its salt, and the enzyme amount is 10 per 1 kg hyaluronic acid or its salt⁵～5*10⁸U, the preparation steps comprise arthrobacterium globiformis seed culture, enzyme production by fermentation, enzyme liquid extraction, enzymolysis, inactivation, filtration, precipitation, dehydration and drying.

The patent CN 104263666B discloses a recombinant pichia pastoris for producing small-molecule hyaluronic acid and a construction method thereof, hyaluronic acid synthase has A from streptococcus zooepidemicus and UDP-glucose dehydrogenase tuaD from bacillus subtilis are adopted to realize the production of the hyaluronic acid in a recombinant pichia pastoris host, meanwhile, the hyaluronidase from leeches is integrated on a pichia pastoris genome and respectively placed under constitutive promoters with different strengths for secretory expression, and the small-molecule hyaluronic acid with different molecular weights is prepared by controlling the secretion amount of the hyaluronidase. And (3) fermenting for 48-96 hours at 20-30 ℃ by using glycerol, methanol, sorbitol or glucose as a carbon source.

The method comprises the steps of producing hyaluronidase by using wild strains for fermentation in CN 103255187B, CN 109897876B and CN 110331178B, adding enzyme liquid into high-molecular hyaluronic acid or salt solution, incubating and degrading at constant temperature, inactivating enzyme at high temperature, and extracting oligomeric hyaluronic acid or salt.

The enzyme adding amount is 10 per 1 kg of hyaluronic acid or its salt prepared by the above process technology⁵-5*10⁸U, and the enzyme solution can not be reused, thus the preparation cost is increased invisibly; moreover, high-temperature enzyme deactivation is needed after degradation is completed, and the process easily causes browning or structural damage of the hyaluronic acid oligosaccharide solution, so that the product quality is unstable; meanwhile, after the enzymolysis is finished, an ethanol precipitation process is used for extraction, the extraction steps are relatively complicated, and the problem of large ethanol consumption exists.

The recombinant pichia pastoris constructed in the patent CN 104263666B is fermented to produce the hyaluronic acid with low molecular weight by a one-step method, the fermentation period is longer, the longest time can reach 96 h, the energy consumption cost in the fermentation process is higher, the control requirement on the fermentation process is higher, and the method is not easy to realize in an industrialized scale.

The method provides a more efficient hyaluronidase, and simplifies the extraction process of micro-molecular sodium hyaluronate, which is one of the key factors for reducing the production cost of micro-molecular sodium hyaluronate and realizing industrial production.

Disclosure of Invention

The purpose of the invention is as follows: provides a recombinant escherichia coli immobilized cell and a preparation method thereof, simplifies the preparation process of micromolecular hyaluronic acid or salt thereof, and reduces the production and preparation cost thereof.

The technical scheme is as follows:

in order to achieve the above object, the present invention provides a recombinant E.coli immobilized cell containing hyaluronidase.

The technical scheme of the invention is as follows: a recombinant escherichia coli immobilized cell containing hyaluronidase is prepared by constructing a recombinant plasmid containing a natural hyaluronidase coding gene, introducing escherichia coli competent cells, screening to obtain recombinant escherichia coli, and immobilizing the recombinant escherichia coli to obtain the immobilized cell.

In order to improve the enzyme activity unit of hyaluronidase, the invention also utilizes an error-prone PCR technology-based in vitro evolution technology, takes recombinant plasmid pET28(a +) -PF as a template, uses the error-prone PCR technology to obtain a linear gene fragment containing base mutation, carries out double enzyme digestion (EcoRI and Not I), purification, ligation and transformation on PCR products and pET28a (+) expression plasmids respectively to obtain competent cells of escherichia coli BL21(DE3), then carries out PCR verification and induced expression, and obtains mutant strains with obviously improved enzyme activity units through mass screening.

The preparation method of the recombinant escherichia coli immobilized cell containing hyaluronidase comprises the following steps:

first step of synthesisPenicillium funiculosumThe natural hyaluronidase encoding gene (GenBank: AB 355480) is connected to plasmid pET28(a +) through EcoR I and Not I enzyme cutting sites to obtain recombinant plasmid pET28(a +) -PF.

In the second step, the recombinant plasmid pET28(a +) -PF was transformed into competent E.coli cells by heat shock transformation (90 sec at 42 ℃ and 5 min in ice bath)E.coli DH5 alpha, obtaining recombinant Escherichia coli containing hyaluronidase by kanamycin resistance screeningE.coli DH5α-pET28(a+)-PF。

Third step pickingE.coliA single colony of DH5 alpha-pET 28(a +) -PF is inoculated into Luria-Bertani culture medium (LB culture medium), activated and cultured for about 15 h at 37 ℃ and 220 r/m, and a plasmid miniprep kit is used for extracting the recombinant plasmid pET28(a +) -PF.

Fourthly, the recombinant plasmid pET28(a +) -PF is transformed into Escherichia coli by heat shock transformation (42 ℃ for 90 seconds and 5 minutes in ice bath)E.coli BL21(DE3) competent cells, through kanamycin resistance screening, obtain recombinant Escherichia coli containing hyaluronidaseE.coli BL21-pET28(a+)-PF。

The fifth stepE.coliBL21-pET28(a +) -PF fermentation culture, specifically as follows:

preparing a culture medium: preparing Luria-Bertani culture medium (LB culture medium), wherein the final concentration of kanamycin is 50-100 mg/L.

(II) seed culture: and (3) selecting a recombinant escherichia coli single colony, inoculating the recombinant escherichia coli single colony to the culture medium in the step (I), and performing shake culture at 37 ℃ at 220 r/m for 8-15 hours.

(III) fermentation culture: inoculating the culture medium in the step (I) with an inoculum size of 2-6%, performing shake culture at 37 ℃ and 220 r/m until the fermentation broth has an absorbance value (OD) at 600 nm₆₀₀) 0.6 to 1.0.

(IV) inducing expression: adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.05-0.1 mmol/L, and carrying out induced expression for 8-12 h at the temperature of 18-25 ℃.

(V) post-treatment: firstly, centrifuging fermentation liquor at 8000-12000 r/m for 10-20 min, and removing supernatant liquor; then, shaking and suspending the lower-layer thalli by using a 0.85% sodium chloride solution, centrifuging for 10-20 min at 8000-12000 r/m once, and removing the supernatant; then, repeatedly washing and centrifuging once by using 0.85% sodium chloride solution; and finally, suspending the thalli by using 0.85% sodium chloride solution of 1/20-1/10 volume of the original fermentation liquor to obtain wet thalli cells.

Sixthly, preparing the immobilized cells, which comprises the following steps:

preparing an immobilization carrier: respectively weighing 0.5-6 g of sodium alginate and 1-10 g of polyvinyl alcohol, dissolving with 100-150 mL of hot water, sterilizing at 115 ℃ for 20 min, and cooling to room temperature;

(II) preparing immobilized cells: and (3) mixing the wet bacterial cells obtained in the fifth step (V) with the immobilized carrier in the previous step according to the ratio of 1: 1-1: fully mixing the raw materials according to the volume ratio of 10, dropwise adding the mixed solution into 0.1-4% of calcium chloride and 1-5% of boric acid solution at the temperature of 0-4 ℃ for solidification, then placing the mixture at the temperature of 0-4 ℃ for cooling and forming for 12-24 hours, and controlling the particle size of immobilized particles to be 2-10 mm.

(III) filtering and washing: filtering the immobilized cells with a 200-300 mesh screen, and washing with sterile water for 2-3 times.

The application of the recombinant escherichia coli immobilized cell of the hyaluronidase in the preparation of micromolecule (5000-10000 Da) hyaluronic acid or salt thereof is characterized by comprising the following steps:

in the first step, preparing a high molecular weight (1000 KDa-2000 KDa) hyaluronic acid or salt solution thereof with the concentration of 2-5% (m/V).

And secondly, adding 10-30% (m/V) of the immobilized cells obtained in the sixth step, reacting for 4-10 hours at 30-40 ℃ under the condition of 100-150 r/m, and filtering the reaction solution by using a 200-300-mesh screen after the reaction is completed to respectively obtain micro-molecular hyaluronic acid or a salt solution thereof and immobilized cell particles.

And thirdly, washing the immobilized cells by using a sterile 0.85% sodium chloride solution, placing the cells in a refrigerator at 4 ℃ for storage, concentrating the low-molecular-weight hyaluronic acid or salt solution thereof by using an ultrafiltration membrane with the molecular weight cutoff of 3 KDa, then drying the concentrated solution under reduced pressure, crushing the solution after the water content is less than 5%, and sieving the dried solution by using a 100-mesh sieve to obtain micromolecular sodium hyaluronate.

Has the advantages that:

the invention provides a recombinant escherichia coli immobilized cell and application thereof in preparation of micromolecular hyaluronic acid or salt thereof. The technical scheme is simple and easy to implement, the immobilized particles have excellent performance, can be repeatedly used for 30 times, still can keep about 90% of catalytic activity, the preparation cost of the immobilized particles is reduced by more than 30%, and the immobilized particles have high industrial application value.

[ description of the drawings ]

FIG. 1 units of viability of recombinant E.coli wild-type enzyme strains and mutant enzyme strains.

FIG. 2 shows the relative enzyme activity of the recombinant E.coli-immobilized cells as a function of the number of uses.

[ detailed description ] embodiments

The invention is further described below with reference to specific embodiments, but the scope of protection of the invention is not limited thereto:

example 1 was carried out: construction of recombinant Escherichia coli containing wild-type hyaluronidase

(1) FromObtained from NCBI Gene BankPenicillium funiculosumA natural hyaluronidase encoding gene (GenBank: AB 355480);

(2) carrying out codon optimization according to the codon preference of escherichia coli to obtain an optimized gene sequence, carrying out whole-gene synthesis, introducing an EcoR I enzyme cutting site at the 5 'end, introducing a Not I enzyme cutting site at the 3' end, integrating the EcoR I enzyme cutting site into a plasmid pET28(a +) to obtain a recombinant plasmid, and transforming the recombinant plasmid into escherichia coli DH5 alpha host bacteria to obtain the escherichia coli DH5 alpha host bacteriaE.coli DH5α-pET28(a+)-PF。

(3) Will be provided withE.coli DH5 alpha-pET 28(a +) -PF is inoculated into LB liquid medium, activated and cultured for 15 h at 37 ℃ and 220 r/m, and the recombinant plasmid pET28(a +) -PF is extracted by using a plasmid miniprep kit.

(4) Escherichia coli BL21(DE3) competent cells were transformed with the recombinant plasmid pET28(a +) -PF by heat shock method, plated on LB solid medium containing 50 mg/L kanamycin sulfate, and cultured at 37 ℃ for 15 hours. Selecting a plurality of single colonies to an LB culture medium (containing 50 mg/L kanamycin sulfate), culturing at 37 ℃ overnight, carrying out PCR amplification by using bacterial liquid as a template, verifying an amplification product by using 1% agarose gel electrophoresis, and obtaining a strain with correct PCR verification, namely recombinant escherichia coli containing wild-type hyaluronidase, which is named as hyalosidaseE.coli-BL21-pET28(a+)-PF。

Example 2 was carried out: construction and screening of escherichia coli containing mutant type high-expression hyaluronidase

According to the description of example 1, using recombinant plasmid pET28(a +) -PF containing the gene encoding the parent wild-type hyaluronidase as a template, and Primer 5.0 to design and synthesize primers at both ends (Table 1), PCR products containing linear gene fragments were obtained by using error-prone PCR technique (materials and concentrations are shown in Table 2, reaction conditions are shown in Table 3), and these PCR products and pET28a (+) expression plasmid were double digested (EcoRI, Not I), purified, ligated and transformed into competent cells of E.coli BL21(DE3), respectively, plated on LB agar plates containing 50 mg/L kanamycin sulfate, and cultured overnight at 37 ℃.

TABLE 1 random mutation primer sequences

。

TABLE 250 μ L error-prone PCR Material System

。

TABLE 3 error prone PCR reaction conditions

。

Selecting a single colony growing on the culture dish, inoculating the single colony in an LB liquid culture medium containing 50 mu g/mL kanamycin sulfate, carrying out shaking culture at 37 ℃ for 15 h, centrifugally collecting thalli, carrying out plasmid extraction, PCR identification and double enzyme digestion identification (detection by using agarose gel electrophoresis), naming a correct recombinant plasmid as pET28a (+) -A-Z, and carrying out induced expression on escherichia coli containing the correct recombinant plasmid, wherein the specific operation is as follows: the above-mentioned bacterial solution was transferred to 100 mL of LB liquid medium containing 50. mu.g/mL kanamycin sulfate, and cultured with shaking at 37 ℃ to OD₆₀₀When the concentration is 0.8, adding an inducer isopropyl-beta-D-thiogalactoside (IPTG) to the final concentration of 0.05 mmol/L, performing induction expression at 18 ℃ for 12 h, taking out bacterial liquid, centrifuging at 10000 r/m for 15 min, collecting thalli, and resuspending the thalli by using 0.85% sodium chloride solution of the original fermentation liquid volume of 1/50-1/10 to obtain whole cell enzyme liquid. The whole cell enzyme solution is used for measuring the enzyme activity of hyaluronidase to obtain a strain with obviously improved enzyme activity.

Example 3 of implementation: enzyme activity assay for hyaluronidase

Definition of enzyme activity: the amount of enzyme required to degrade and release 1. mu.g of reducing sugar is one unit of enzyme activity per hour from a sodium hyaluronate solution at a concentration of 2 mg/mL at 38 ℃ and pH 5.5.

Standard blank: adding 200 mu L of pure water into a test tube with a plug, adding 800 mu L of sodium hyaluronate substrate solution (2 mg/mL), shaking and uniformly mixing, accurately reacting in a 38 ℃ water bath for 20 min, then quickly putting into a boiling water bath for accurately reacting for 2 min to stop the enzyme reaction, quickly cooling to room temperature, adding 2 mL of DNS developer, accurately reacting in the boiling water bath for 5 min, quickly cooling to room temperature, adding 7 mL of pure water, shaking and uniformly mixing;

enzyme blank: adding 200 mu L of preheated whole-cell enzyme solution obtained in the example 2 into a test tube with a plug, adding 2 mL of DNS color developing agent, adding 800 mu L of sodium hyaluronate substrate solution (2 mg/mL), shaking and mixing uniformly, carrying out accurate reaction in 38 ℃ water bath for 20 min, then quickly putting into boiling water bath for accurate reaction for 5 min, quickly cooling to room temperature, adding 7 mL of pure water, shaking and mixing uniformly, taking a standard blank sample as a control, and measuring the light absorption value (A) at 540 nm (A is measured)_B)；

Sample preparation: adding 200 mu L of preheated whole-cell enzyme solution obtained in the example 2 into a test tube with a plug, adding 800 mu L of sodium hyaluronate substrate solution (2 mg/mL), shaking and uniformly mixing, accurately reacting in a 38 ℃ water bath for 20 min, then quickly putting into a boiling water bath for accurately reacting for 2 min to stop the enzyme reaction, quickly cooling to room temperature, adding 2 mL of DNS color developing agent, accurately reacting in the boiling water bath for 5 min, quickly cooling to room temperature, adding 7 mL of pure water, shaking and uniformly mixing, measuring the light absorption value (A) at 540 nm by taking a standard blank as a control_E)；

The enzyme activity calculation formula is as follows:

X_Dactivity of hyaluronidase in the sample, U/mL

A_EAbsorbance of the enzyme reaction solution

A_BAbsorbance of enzyme blank

C₀Intercept of the standard curve

Slope of the K- -standard curve

t- -reaction time, 20 min

V- -volume of enzyme solution added, 200. mu.L

1000- -transforming factor, 1 mg = 1000. mu.g

N- -sample dilution factor

Selecting positive mutant with better enzyme activity than female parent, repeating the operation of example 2, and screening for multiple timesObtaining a mutant strain with obviously improved enzyme activity, and naming the mutant strainE.coli BL21-pET28a (+) -B93, the results of the enzyme activity unit are shown in Table 4, the corresponding amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.

TABLE 4 mutant strains with significantly improved enzyme activity

。

Example 4 of implementation: fermentation culture of high-yield enzyme strain

Will be provided withE.coli BL21-pET28a (+) -B93 single colony is inoculated to LB liquid culture medium containing kanamycin with the final concentration of 50 mg/L and cultured for 15 h at 37 ℃ and 220 r/m; inoculating to new LB liquid medium containing kanamycin to 50 mg/L at 2%, culturing at 37 deg.C and 220 r/m to OD₆₀₀Up to 0.8; adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.05 mmol/L, and carrying out induced expression for 8 h at 16 ℃; centrifuging fermentation liquor at 10000 r/m for 10 min, discarding supernatant, shaking and suspending thallus precipitate with 0.85% sodium chloride solution, centrifuging, discarding supernatant, washing precipitate repeatedly, centrifuging once, and suspending thallus with 0.85% sodium chloride solution to obtain wet thallus cells.

Example 5 was carried out: immobilization of high-enzyme-producing recombinant Escherichia coli cells

Preparing an immobilized carrier: respectively weighing 3 g of sodium alginate and 2 g of polyvinyl alcohol, dissolving with 100 mL of hot water, sterilizing at 115 ℃ for 20 min, and cooling to room temperature;

preparing immobilized cells: 20 g of the wet bacterial cells obtained in example 4 were taken and mixed with the immobilized carrier, the mixture was added dropwise to 500 mL of a solution containing 4% calcium chloride and 5% boric acid at 4 ℃ to solidify, and then the mixture was cooled and molded at 4-8 ℃ for 24 hours with the particle size of the immobilized particles being controlled to be 5 mm.

Filtering and washing: filtering the immobilized cells by a 200-mesh screen, and washing with sterile water for 2 times to obtain the immobilized recombinant escherichia coli cells.

Examples 6 to 10: preparation of micromolecular sodium hyaluronate by using recombinant escherichia coli immobilized cells

Weighing 2-5 g of high molecular weight sodium hyaluronate with molecular weight of 1000-2000 KDa, adding 100 mL of purified water, starting stirring, adding 10-30 g of the immobilized Escherichia coli cells prepared in example 5, controlling the stirring speed to be 100-150 r/m, incubating for 5-6 h at 38 +/-0.5 ℃, filtering the reaction solution by using a 200-mesh screen to obtain immobilized cells and filtrate, washing the immobilized cells by using a sterile 0.85% sodium chloride solution, storing in a refrigerator at 4 deg.C, concentrating the filtrate with ultrafiltration membrane with molecular weight cutoff of 3 KDa, concentrating by 5-10 times, drying the concentrated solution under reduced pressure until the water content of the material is less than 5%, pulverizing the material, sieving with 100 mesh sieve to obtain micro-molecular sodium hyaluronate, and measuring the molecular weight of the micro-molecular sodium hyaluronate with 4000-10000 Da by using Ubbelohde viscometer method. The results of the relevant experiments are shown in Table 5.

TABLE 5 examples 6-10 feed and product conditions

Description of the drawings: 1. the high molecular weight sodium hyaluronate in the table is sodium hyaluronate with a molecular weight of 1000-2000 KDa.

2. The immobilized recombinant E.coli cells described in the table were the immobilized recombinant E.coli cells obtained in example 5.

3. The "product" in the table refers to micro-molecular weight sodium hyaluronate obtained after degradation of high molecular weight sodium hyaluronate.

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gatggcctgt atagcaccag caataatcag atttatctgg gcgtggataa aaccaccgtg 240

ctggatagca gcagcaccgg ccgtaatagc gtgcgcgttt atagccagaa tacctttagc 300

agcggcattc tgattaccga ttttgcgcat ctgccggtga gcgtgtgcgg catttggccg 360

gcgtattgga ccattaataa tcaggcgaat ccgtatggcg aaattgatat ttatgaagcg 420

tatgacgatg tggcgggcgc gtatgtgagc ctgcatacca gtaataccca caccctgagc 480

aatcgcaatt ttaccggcac cgatacccgc accgattgca ccttaagcag cggcggtggt 540

tgcggcgttc agagcaccag tagtcaattt ggcgcgggct ttaatgcggc gggcggcggt 600

gtttgggttt tgagcttaga aaatagcctg cagctgtggg tgtttccgcg caatcagatt 660

ccggcggata ttaccaatgg cagcccgaat ccgagcagct ggggcacccc attatttgaa 720

tttgatagca ataatggctg cgacgtgagc agcaatttta ttgatcagac cgtgattttt 780

aacctggact tctgcggcca gaatggcgcg ggcggtcaag aatggagcga ttggaccgat 840

tgcgcgacca ccaccggcca aagcacttgt aatgcgtatg tggcggcgaa tagcagcgcg 900

tatagcgaaa cctattttag cattaatagc atcaagctgt atcagtga 948

Claims

1. A recombinant escherichia coli immobilized cell containing hyaluronidase is characterized in that a recombinant plasmid containing a natural hyaluronidase coding gene is constructed firstly, then escherichia coli competent cells are introduced, recombinant escherichia coli is obtained through screening, and the recombinant escherichia coli is immobilized to obtain the immobilized cell.

2. The recombinant escherichia coli immobilized cell of hyaluronidase according to claim 1, wherein the error-prone PCR-based in vitro evolution technology is characterized in that a linear gene fragment containing base mutation is obtained by using an error-prone PCR technology with a recombinant plasmid pET28(a +) -PF as a template, PCR products and a pET28a (+) expression plasmid are subjected to double digestion (EcoRI, Not I), purification, ligation and transformation of escherichia coli BL21(DE3) competent cells, and then PCR verification and inducible expression are performed, and a mutant strain with significantly improved enzyme activity unit is obtained by mass screening.

3. The method for preparing the recombinant E.coli immobilized cell of hyaluronidase of claim 1, comprising the steps of:

first step of synthesisPenicilliumfuniculosumA natural hyaluronidase encoding gene (GenBank: AB 355480) which is connected to a plasmid pET28(a +) through EcoR I and Not I enzyme cutting sites to obtain a recombinant plasmid pET28(a +) -PF;

in the second step, the recombinant plasmid pET28(a +) -PF was transformed into competent E.coli cells by heat shock transformation (90 sec at 42 ℃ and 5 min in ice bath)E.coliDH5 alpha, obtaining recombinant Escherichia coli containing hyaluronidase by kanamycin resistance screeningE.coliDH5α-pET28(a+)-PF；

Third step pickingE.coliA single colony of DH5 alpha-pET 28(a +) -PF is inoculated into Luria-Bertani culture medium (LB culture medium), activated and cultured for about 15 h at 37 ℃ and 220 r/m, and a plasmid miniprep kit is used for extracting recombinant plasmid pET28(a +) -PF;

fourthly, the recombinant plasmid pET28(a +) -PF is transformed into Escherichia coli by heat shock transformation (42 ℃ for 90 seconds and 5 minutes in ice bath)E.coliBL21(DE3) competent cells, through kanamycin resistance screening, obtain recombinant Escherichia coli containing hyaluronidaseE.coliBL21-pET28(a+)-PF；

preparing a culture medium: preparing a Luria-Bertani culture medium (LB culture medium), wherein the final concentration of kanamycin is 50-100 mg/L;

seed culture: selecting a recombinant escherichia coli single colony, inoculating the recombinant escherichia coli single colony to the culture medium in the step (I), and performing shake culture at 37 ℃ at 220 r/m for 8-15 hours;

fermentation culture: inoculating the culture medium in the step (I) with an inoculum size of 2-6%, performing shake culture at 37 ℃ and 220 r/m until the fermentation broth has an absorbance value (OD) at 600 nm₆₀₀) 0.6 to 1.0;

inducing expression: adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.05-0.1 mmol/L, and carrying out induced expression for 8-12 h at the temperature of 18-25 ℃;

and (3) post-treatment: firstly, centrifuging fermentation liquor at 8000-12000 r/m for 10-20 min, and removing supernatant liquor; then, shaking and suspending the lower-layer thalli by using a 0.85% sodium chloride solution, centrifuging for 10-20 min at 8000-12000 r/m once, and removing the supernatant; then, repeatedly washing and centrifuging once by using 0.85% sodium chloride solution; finally, suspending the thalli by using 0.85% sodium chloride solution of 1/20-1/10 of the volume of the original fermentation liquor to obtain wet thalli cells;

sixthly, preparing the immobilized cells, which comprises the following steps:

preparing an immobilized carrier: respectively weighing 0.5-6 g of sodium alginate and 1-10 g of polyvinyl alcohol, dissolving with 100-150 mL of hot water, sterilizing at 115 ℃ for 20 min, and cooling to room temperature;

preparing immobilized cells: and (3) mixing the wet bacterial cells obtained in the fifth step (V) with the immobilized carrier in the previous step according to the ratio of 1: 1-1: fully mixing the raw materials according to a volume ratio of 10, dropwise adding the mixed solution into 0.1-4% of calcium chloride and 1-5% of boric acid solution at 0-4 ℃ for solidification, then placing the mixture at 0-4 ℃ for cooling and forming for 12-24 hours, and controlling the particle size of immobilized particles to be 2-10 mm;

filtering and washing: filtering the immobilized cells with a 200-300 mesh screen, and washing with sterile water for 2-3 times.

4. The use of the recombinant E.coli immobilized cell of hyaluronidase of claim 1 for preparing micromolecular (5000-10000 Da) hyaluronic acid or its salt, which comprises the following steps:

preparing a high molecular weight (1000-2000 KDa) hyaluronic acid or a salt solution thereof with the concentration of 2-5% (m/V);

secondly, adding 10-30% (m/V) of the immobilized cells obtained in the sixth step, reacting for 4-10 hours at 30-40 ℃ under the condition of 100-150 r/m, and filtering the reaction solution by using a 200-300-mesh screen after the reaction is completed to respectively obtain micromolecular hyaluronic acid or a salt solution thereof and immobilized cell particles;