A glucuronidase and its mutants and use in the production of bilirubin

Abstract

The application belongs to the technical field of bioengineering, and specifically discloses a glucuronidase and a mutant thereof and application of the glucuronidase and the mutant in production of bilirubin. The glucuronidase is derived from Escherichia coli, and an amino acid sequence of the glucuronidase is shown as SEQ ID NO. 1. An amino acid sequence of the mutant of the glucuronidase is shown as SEQ ID NO. 2, and the mutant is obtained by truncating amino acids 360-376 in the amino acid sequence of the glucuronidase. A coding gene of the glucuronidase or the mutant thereof is connected with an expression plasmid to construct an expression vector, the expression vector is introduced into a host strain to obtain a recombinant strain, and the recombinant strain is cultured to obtain whole-cell catalytic bacteria slurry. The recombinant strain or the whole-cell catalytic bacteria slurry can be used in preparation of bilirubin, has a high substrate conversion rate and less by-products, the preparation method is simple and convenient, production conditions are mild, environmental pollution is small, and the application has a good technical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a glucuronidase and a mutant thereof and application in the production of bilirubin. BACKGROUND

[0002] Bilirubin has multiple medicinal activities such as antipyretic, expectorant, sedative, anti-convulsive, antihypertensive, promoting hematin regeneration, inhibiting Japanese B encephalitis virus, etc. and is also an important raw material of artificial bezoar. At present, the main way to obtain bilirubin is to extract from the bile of pigs, cattle, sheep and other animals. Due to the shortage of raw material resources and the backwardness of production technology, the supply of bilirubin is severely insufficient, and the price is very high. Bilirubin is the product of hematin catabolism in living organisms, and exists in two forms of free bilirubin and conjugated bilirubin in bile, wherein the conjugated bilirubin accounts for more than 90% of the total bilirubin, but only the free bilirubin can be used for the production of drugs;

[0003] In industry, the free bilirubin is mainly produced by removing the glucuronic acid group of the conjugated bilirubin by chemical method, and in the production process, strong acid and strong base are used for hydrolysis, saponification and other reactions, and high temperature heating is accompanied, so the reaction conditions are harsh, and bilirubin is easily oxidized (according to statistics, about 20% of bilirubin is oxidized in the process), thereby greatly reducing the product yield;

[0004] The microbial fermentation method is a green and environmentally friendly production process, however, the glucuronidase activity in the microbial strain used for fermentation is low, and the expression amount is insufficient, and usually more than 48 hours of fermentation is needed, and the final yield of the obtained bilirubin product is between 0.01-0.014%, which is far lower than the production yield of the chemical method, in addition, a large amount of expensive antioxidant needs to be added in the fermentation process, thereby increasing the industrial production cost. Therefore, in view of the problems existing in the production of bilirubin in China, it is of great significance to study the technology of high-efficiency conversion of bile for producing bilirubin. SUMMARY

[0005] In order to solve the above problems, the primary purpose of the present application is to rationally design and optimize the protein expression of the glucuronidase from Escherichia coli, and to construct a recombinant strain with excellent catalytic performance, and to establish a whole-cell conversion method for efficiently producing bilirubin using the recombinant strain.

[0006] The specific technical scheme of the present application comprises:

[0007] The first purpose of the present application is to provide a glucuronidase, which is derived from Escherichia coli, the amino acid sequence of the glucuronidase is shown as SEQ ID NO. 1, and the coding gene of the glucuronidase is the nucleotide sequence shown as SEQ ID NO. 4.

[0008] The second object of the present application is to provide a glucuronidase mutant, wherein the amino acids at positions 360-376 in SEQ ID NO. 1 are truncated to obtain the glucuronidase mutant, the amino acid sequence of the glucuronidase mutant is shown in SEQ ID NO. 2, and the coding gene of the glucuronidase mutant is the nucleotide sequence shown in SEQ ID NO. 3.

[0009] The third object of the present application is to provide an application of glucuronidase or glucuronidase mutant in the production of bilirubin, wherein the glucuronidase is derived from Escherichia coli, and the amino acid sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the glucuronidase mutant is shown in SEQ ID NO. 2, which is a mutant obtained by truncating the amino acids at positions 360-376 in the amino acid sequence of the glucuronidase.

[0010] The fourth object of the present application is to provide an expression vector containing the coding gene of the glucuronidase or the glucuronidase mutant.

[0011] As a further optimization scheme of the present application, the expression vector is obtained by connecting the coding gene of the glucuronidase or the glucuronidase mutant to the pET22b plasmid, the expression vector expresses the coding gene of the glucuronidase mutant using a promoter mode, the promoter is one of T7 promoter, Tac promoter or Trc promoter, and the expression plasmid of the present application can also be the Escherichia coli-Corynebacterium glutamicum shuttle expression plasmid pXMJ19, or the pDXW series vector, or the pBL1 series vector, or other vectors of the Escherichia coli expression plasmid pET series, or the pBAD series vector, or the Bacillus subtilis expression plasmid pHT01, or the pMA5 vector, etc.

[0012] The fifth object of the present application is to provide a recombinant strain of glucuronidase, which is obtained by introducing the expression vector as described in any of the above into a host strain, and the host strain is Escherichia coli BL21 (DE3), and the host strain can also be selected from other Corynebacterium, Escherichia or Bacillus, for example, Corynebacterium glutamicum or Bacillus subtilis.

[0013] The sixth object of the present application is to provide a whole-cell catalytic bacterial slurry containing the recombinant strain of glucuronidase as described above.

[0014] As a further optimization scheme of the present application, the preparation method of the whole-cell catalytic slurry is as follows: the glucuronidase recombinant strain is inoculated into a seed culture medium for culture to obtain a seed liquid, then the seed liquid is inoculated into a fermentation culture medium for fermentation, isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to induce expression of the target protein, and the bacterial cell is collected to obtain the whole-cell catalytic slurry.

[0015] A seventh object of the present application is to provide a method for preparing bilirubin, which uses animal bile as a substrate and adds a glucuronidase recombinant strain or a whole-cell catalytic slurry for catalytic reaction to obtain bilirubin.

[0016] In summary, the present application has the following beneficial effects:

[0017] The glucuronidase provided by the present application is derived from Escherichia coli, and the amino acid sequence is shown as SEQ ID NO. 1. The amino acid sequence of the glucuronidase mutant is shown as SEQ ID NO. 2, which is a mutant obtained by truncating the 360th-376th amino acid in the amino acid sequence of the glucuronidase. The expression vector is introduced into a host strain to obtain a recombinant strain, and then the whole-cell catalytic slurry is obtained by culturing the recombinant strain. When the recombinant strain or the whole-cell catalytic slurry is used for the preparation of bilirubin, it has a high substrate conversion rate and less by-products, and the preparation method is simple and convenient, the production conditions are mild, the environmental pollution is small, and it has a good technical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 SDS-PAGE analysis of protein GUS wild type and GUSMu mutant recombinant expression;

[0019] Figure 2 Whole-cell transformation of bile to produce bilirubin schematic diagram;

[0020] Figure 3a Whole-cell transformation of extract HPLC analysis spectrum;

[0021] Figure 3b GUS wild type and GUSMu mutant catalyze the conversion rate of conjugated bilirubin in bile. DETAILED DESCRIPTION

[0022] The following further describes the present application with reference to the accompanying drawings. It is necessary to point out that the following detailed description is only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0023] I. Reagents and materials

[0024] Escherichia coli DH5a, Escherichia coli BL21(DE3) and Corynebacterium glutamicum are commercially available, Escherichia coli DH5a is used for cloning of all genes in the present application, and BL21(DE3) and Corynebacterium glutamicum are used for gene protein expression and whole cell transformation production of bilirubin in the present application. Escherichia coli DH5a, BL21(DE3) competent cells and Corynebacterium glutamicum competent cells are prepared according to the conventional method. The test methods in the following examples without specific conditions are carried out according to the conventional conditions, for example, the conditions described in "Molecular Cloning: Laboratory Manual" or the conditions recommended by the manufacturer of the corresponding biological reagents.

[0025] II. Method

[0026] 1. Construction of glucuronidase wild type and mutant expression vectors

[0027] The coding gene (SEQ ID No. 4) of glucuronidase (SEQ ID No. 1) from Escherichia coli was entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for gene synthesis, and the gene was connected between the restriction enzyme cutting sites Ndel and Xhol of plasmid pET22b to obtain an expression plasmid regulated by T7 promoter, and named as pET22b-GUS. The GUS was truncated, i.e. the 360th-376th amino acids were removed, and the amino acid sequence of the glucuronidase mutant is shown in sequence SEQ ID No. 2.

[0028] According to the codon usage frequency of Escherichia coli, the nucleotide sequence is shown in SEQ ID No. 3, and the gene synthesis was entrusted to Shengong Bioengineering (Shanghai) Co., Ltd., and the gene was connected between the restriction enzyme cutting sites Ndel and Xhol of plasmid pET22b to obtain an expression plasmid regulated by T7 promoter, and named as pET22b-GUSMu.

[0029] The selected expression plasmid in the present application is pET22b as an induced expression plasmid, and the plasmid itself contains a strong promoter T7 related sequence (including the operator sequence lacO), when an inducer such as isopropyl-β-D-thiogalactopyranoside (IPTG) or lactose is added, it will promote the repressor protein to leave the operator sequence and thus initiate gene expression.

[0030] 2. Construction of glucuronidase wild type and mutant expression recombinant strains and expression analysis

[0031] The recombinant plasmids pET22b-GUS and pET22b-GUSMu obtained above are extracted, and are transformed into E. coli BL21 (DE3) by a chemical transformation method to obtain a wild-type expression recombinant strain of glucuronidase (denoted as GUS wild type) and a mutant expression recombinant strain of glucuronidase (denoted as GUSMu mutant). The specific method is as follows: 100 μL of E. coli BL21 (DE3) competent cells are taken, about 1 μL of the expression plasmid is added, and the mixture is uniformly mixed and placed on ice for 30 min. Then, the competent cells are heat-shocked in a 42°C water bath for 90 s, and then are placed on ice for 2 min. 1 mL of liquid LB medium is added, and the mixture is incubated at 37°C for 45 min. The mixture is spread on an LB solid medium plate containing 100 μg / mL of ampicillin, and is cultured at 37°C for 9-12 h. After single colonies are grown, verification is performed.

[0032] The recombinant E. coli containing the expression plasmid obtained above is subjected to protein induction expression, and the expression of glucuronidase is analyzed. The specific method is as follows: single colonies are inoculated in 5 mL of LB liquid medium, and are cultured at 37°C and 200 r / min overnight. Subsequently, the overnight culture is transferred to 50 mL of LB liquid medium at an inoculation amount of 1%, and is cultured at 37°C and 200 r / min until the bacterial concentration OD 600 is about 0.6. IPTG is added at a final concentration of 0.01-1.0 mM, and the mixture is cultured at 200 r / min for 2-12 h to induce the expression of the target protein. After induction is completed, 5 mL of the bacterial liquid is subjected to ultrasonic disruption, and then SDS-PAGE gel electrophoresis is used to detect the expression of glucuronidase in the recombinant bacteria. As shown in FIG. 1, the wild-type and mutant glucuronidases are completely soluble under the above conditions, and the expression amount is high. Figure 1

[0033] 3. Preparation of whole-cell catalytic slurry by fermentation culture

[0034] The preparation method of the whole-cell catalytic slurry is as follows: the expression recombinant strain is inoculated in 100 mL of LB liquid medium in a 500 mL flask, and is cultured at 37°C for 15-18 h. The culture is transferred to 600 mL of LB liquid medium in a 2000 mL flask at an inoculation amount of 5%, and is cultured at 37°C and 200 r / min. When the bacterial concentration OD 600 is about 0.3-1.5, IPTG is added at a final concentration of 0.01-1 mM to induce the expression of the target protein, and the induction time is about 2-12 h. After the induction process is completed, the fermentation culture is centrifuged, and the bacterial cells are collected to obtain the whole-cell catalytic slurry.

[0035] 4. Establishment of a whole-cell transformation process for producing bilirubin​

[0036] The whole cell catalytic bacteria slurry obtained above is used as a whole cell catalyst to establish a process for the biological preparation of bilirubin. A schematic diagram of the whole cell conversion of bile to produce bilirubin is shown in Figure 2 The specific method is as follows: 1L of a whole cell catalytic system is established in a triangular flask, 10-20g of wet whole cell catalytic bacteria slurry is added to 1L of pig bile, and the catalytic reaction is carried out at pH = 6.0-8.0, 30-55°C, 50-100r / min on a shaking table for 4-16h. After the reaction is completed, the components of the catalytic reaction are quantitatively analyzed by diazo analysis and liquid chromatography.

[0037] The specific method of liquid chromatography is as follows: the whole cell conversion reaction liquid is extracted with chloroform, and the extract is determined by high performance liquid chromatography. The chromatographic conditions are as follows: a C18 chromatographic column is used as the analysis column, methanol is used as the mobile phase, the flow rate is 1mL / min, and the detection wavelength is 450nm.

[0038] The diazo analysis method specifically includes the following steps:

[0039] (1) Preparation of diazotization reagent: Solution A: 1.0g of p-aminobenzenesulfonic acid is weighed, a suitable amount of deionized water is added, heated in a water bath until completely dissolved, 15mL of concentrated hydrochloric acid is added, and deionized water is added to 1000mL. Solution B: 0.5g of NaNO2 is weighed and deionized water is added to 100mL. 10mL of solution A and 0.3mL of solution B are mixed to obtain diazotization reagent (this reagent is prepared before use).

[0040] (2) 200μL of the supernatant of the whole cell conversion liquid after centrifugation is taken, 700μL of ethanol and 100μL of diazotization reagent are added in turn, and it is placed in the dark at room temperature for 1h. The absorbance at 520nm is measured by an enzyme marker instrument, and the concentration of bilirubin is calculated by the internal standard method.

[0041] The results are shown in Figure 3a and Figure 3b The extract composition is relatively simple after the whole cell conversion liquid is extracted with chloroform. The expression strain obtained based on the glucuronidase mutant shows high bilirubin production capacity. After 12h of whole cell conversion reaction, the molar efficiency of the conversion of conjugated bilirubin in bile to free bilirubin can reach 95%, which is 22% higher than that of the wild type. In addition, the bilirubin production method provided by the present application does not require the addition of strong acid, strong base and antioxidant, which can greatly save the production cost and simplify the process flow.

[0042] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation to the patent scope of the present application. It should be noted that, for those skilled in the art, several improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. Use of a glucuronidase mutant in the production of bilirubin, characterized in that, The glucuronidase mutant is obtained by truncating amino acids 360-376 in the amino acid sequence of the glucuronidase derived from Escherichia coli, the amino acid sequence of the glucuronidase mutant is shown as SEQ ID NO. 2, and the coding gene of the glucuronidase mutant is a nucleotide sequence shown as SEQ ID NO.

3.

2. A method of preparing bilirubin, characterized by, The bilirubin is obtained by catalytic reaction of animal bile as a substrate, and a glucuronidase recombinant strain or whole-cell catalytic slurry containing the glucuronidase mutant coding gene with the nucleotide sequence shown as SEQ ID NO.

3.

3. The method of claim 2, wherein the method is characterized by, The glucuronidase recombinant strain is obtained by linking the glucuronidase mutant coding gene to the pET22b plasmid to obtain an expression vector, and then introducing the expression vector into a host strain, and the host strain is Escherichia coli BL21 (DE3).

4. The method of claim 3, wherein the method is characterized by, The preparation method of the whole-cell catalytic slurry is inoculating the glucuronidase recombinant strain into a seed culture medium to culture, obtaining a seed liquid, then inoculating the seed liquid into a fermentation culture medium to ferment, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) to induce expression of the target protein, collecting bacterial cells to obtain the whole-cell catalytic slurry.

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