阅读说明：本技术 一种人工酶法高效生产硫酸软骨素a的方法 (Method for efficiently producing chondroitin sulfate A by artificial enzyme method ) 是由 康振 陈坚 堵国成 李江华 金学荣 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种人工酶法高效生产硫酸软骨素A的方法,属于生物工程技术领域。本方法经过微生物异源表达了一个双功能蛋白软骨素4-O-磺基转移酶C4ST和芳基磺基转移酶AST IV,用于催化软骨素合成硫酸软骨素A,结合酶的定点突变和反应体系组分的优化,加速硫酸软骨素的合成效率。(The invention discloses a method for efficiently producing chondroitin sulfate A by an artificial enzyme method, belonging to the technical field of biological engineering. The method expresses a bifunctional protein chondroitin 4-O-sulfotransferase C4ST and aryl sulfotransferase AST IV by microbes in a heterologous manner, is used for catalyzing chondroitin to synthesize chondroitin sulfate A, and accelerates the synthesis efficiency of the chondroitin sulfate by combining site-directed mutagenesis of enzyme and optimization of reaction system components.)

1. A method for efficiently producing chondroitin sulfate A by an artificial enzyme method is characterized in that chondroitin 4-O-sulfotransferase C4ST and aryl sulfotransferase ASTIV are connected by different linker sequences to construct a bifunctional protein with two enzyme activities, and the bifunctional protein is used for catalyzing chondroitin to synthesize chondroitin sulfate A in the presence of a stabilizer; the sequence of the joint is any one of the following sequences: 1, 2, 3 and 4; the sequence of the gene coding for chondroitin 4-O-sulfotransferase is shown in SEQ ID NO. 5.

2. The method for efficiently producing chondroitin sulfate A by using the artificial enzyme method as claimed in claim 1, wherein a gene carrying a code chondroitin 4-O-sulfotransferase C4ST, a linker sequence and aryl sulfotransferase AST IV is connected to a pPIC9K vector, the recombinant vector is transformed into Pichia pastoris GS115, the bifunctional protein is obtained by expression of the recombinant Pichia pastoris GS115, and the bifunctional protein is used as a catalyst to catalyze chondroitin to synthesize chondroitin sulfate.

3. The method for efficiently producing chondroitin sulfate A by using the artificial enzyme method as claimed in claim 2, wherein the finally optimized C4ST sequence encoding chondroitin 4-O-sulfotransferase and gene sequence of aryl sulfotransferase AST IV are connected to pPIC9K vector by fusion PCR and Gibson assembly to obtain pPIC9K-C4ST-AST IV plasmid, the nucleotide sequence encoding linker sequence is inserted by cyclization PCR or Gibson assembly, the recombinant vector is transformed into Pichia pastoris GS115, the recombinant Pichia pastoris GS115 is expressed to obtain the bifunctional protein, and the bifunctional protein is used as a catalyst to catalyze chondroitin sulfate to synthesize chondroitin sulfate.

4. The method for efficiently producing chondroitin sulfate A by using the artificial enzyme method according to claim 1, wherein the stabilizer is at least one of glycerol, trehalose and sorbitol.

5. The method for efficiently producing chondroitin sulfate A by artificial enzyme method according to any one of claims 1-4, wherein 1-5mg/mL of bifunctional protein, 1-20mg/mL of chondroitin, 0.2-1mM of PAP, 25-75mM of PNPS, and 40-200 mg/mL of glycerol are subjected to catalytic reaction to obtain chondroitin sulfate.

6. The method for efficiently producing chondroitin sulfate A by using the artificial enzyme method according to any one of claims 1-4, wherein 1-5mg/mL of bifunctional protein, 1-20mg/mL of chondroitin, 0.2-1mM of PAP, 25-75mM of PNPS, 40-200 mg/mL of glycerol and 2.0-15 mg/mL of trehalose are subjected to a catalytic reaction to obtain chondroitin sulfate.

7. The method for efficiently producing chondroitin sulfate A by using the artificial enzyme method according to any one of claims 1-4, wherein 1-5mg/mL of bifunctional protein, 1-20mg/mL of chondroitin, 0.2-1mM of PAP, 25-75mM of PNPS, 40-200 mg/mL of glycerol, 2.0-15 mg/mL of trehalose and 2.0-15 mg/mL of sorbitol are subjected to a catalytic reaction to obtain chondroitin sulfate.

8. A recombinant pichia pastoris for expressing chondroitin 4-O-sulfotransferase C4ST and aryl sulfotransferase ASTIV bifunctional enzymes is characterized in that genes of chondroitin 4-O-sulfotransferase C4ST, a linker sequence and aryl sulfotransferase AST IV are connected to a pPIC9K vector, the recombinant vector is transformed into pichia pastoris GS115, and the bifunctional protein is obtained by expression of the recombinant pichia pastoris GS 115; the sequence of the joint is any one of the following sequences: 1, 2, 3 and 4; the gene sequence of the coding chondroitin 4-O-sulfotransferase is shown as SEQ ID NO. 5.

9. The chondroitin 4-O-sulfotransferase mutant is characterized in that the gene sequence for coding the mutant is shown as SEQ ID NO. 5.

10. The gene for coding the chondroitin 4-O-sulfotransferase mutant is characterized by having a sequence shown as SEQ ID NO. 5.

Technical Field

The invention relates to a method for efficiently producing chondroitin sulfate A by an artificial enzyme method, belonging to the technical field of biological engineering.

Background

Chondroitin Sulfate A (CSA) is a proteoglycan with important biological functions, widely distributed in cartilage tissue. The skeleton of chondroitin sulfate A is linear polysaccharide formed by alternately connecting D-glucuronic acid and N-acetylgalactosamine. Chondroitin sulfate is formed by sulfonating modification of chondroitin by different sulfotransferases, and can be divided into the following four types according to the position of the sulfonating modification: chondroitin sulfate A (4-O-sulfonated), chondroitin sulfate C (6-O-sulfonated), chondroitin sulfate D (2, 6-di-O-sulfonated), and chondroitin sulfate E (4, 6-di-O-sulfonated). The chondroitin sulfate has excellent biocompatibility and can be widely applied to the fields of medical health, health products, foods and cosmetics. Different sulfonated forms of chondroitin sulfate have different biological activities and application fields. CSA and CSCs are often used to treat arthritis, and chondroitin sulfate E promotes neurite outgrowth of primary neurons.

Chondroitin sulfate, which is currently commercially produced, is mainly obtained by extraction from animal tissues. The production method has various problems, such as potential animal virus cross infection, environmental pollution of waste liquid generated by treating animal tissues, uneven structure height of chondroitin sulfate products, and the like. In order to obtain chondroitin sulfate with uniform structure and biological safety, the problem can be effectively avoided by using the biological enzyme method to catalyze and synthesize the chondroitin sulfate. Among them, the preparation of chondroitin sulfate a by the bio-enzymatic method requires chondroitin sulfate transferase C4ST and a sulfate group donor PAPS. Wherein the sulfate donor PAPS is formed by PNPS and PAP catalyzed by aryl sulfotransferase AST IV.

Previous literature reports require about 48h for complete conversion of 100mg chondroitin (Zhou, et al (2018), A microbial-enzymatic synthesis for producing chondroitin sulfate, Biotechnology and Bioengineering,115(6),1561 1570), catalytic reactions are too long in duration, and small in scale to be truly practical. Meanwhile, many studies are still being conducted on the expression of C4ST activity using mammalian cells, and this method is expensive and cannot be used for actual large-scale production.

Disclosure of Invention

[ problem ] to

The chondroitin sulfate prepared by the traditional tissue extraction method has various defects, and the conventional enzyme method for catalyzing and synthesizing the chondroitin sulfate has low efficiency and is complex.

[ solution ]

The invention provides a method for efficiently producing chondroitin sulfate A by an artificial enzyme method, which is characterized in that chondroitin 4-O-sulfotransferase C4ST and aryl sulfotransferase AST IV are connected by utilizing different linker sequences to construct a bifunctional protein with two enzyme activities, the two proteins are fused into the bifunctional protein through the linker sequences, the spatial distance between two enzyme catalysis structural domains is shortened on the molecular level, the transfer efficiency of an intermediate substrate is improved, and the catalysis efficiency of enzyme cascade reaction can be effectively improved. Then, by adding a stabilizer to the reaction system, the enzyme molecule can maintain a certain high activity for a long time. The synthesis of chondroitin sulfate A can be efficiently realized by utilizing the optimization of the bifunctional protein and a stabilizer in a catalytic system. The nucleotide sequence for coding the linker sequence is any one of the following sequences: 1, 2, 3 and 4. The gene for coding chondroitin 4-O-sulfotransferase is optimized, and the optimized gene sequence is shown as SEQ ID NO. 5.

In one embodiment of the invention, a gene carrying a code chondroitin 4-O-sulfotransferase C4ST, a linker sequence and aryl sulfotransferase AST IV is connected to a pPIC9K vector, a recombinant vector is transformed into Pichia pastoris GS115, the recombinant Pichia pastoris GS115 expresses the bifunctional protein, and the bifunctional protein is used as a catalyst to catalyze chondroitin to synthesize chondroitin sulfate.

In one embodiment of the present invention, the chondroitin 4-O-sulfotransferase C4ST gene is optimized as follows:

(1) firstly, removing 1-62 amino acids from the N end of a sequence of a GenBank accession number NP-067414.2, then carrying out codon optimization and artificial synthesis, assembling and connecting a synthesized original sequence to a pPIC9K plasmid to obtain a pPIC 9K-delta 62C4ST plasmid, linearizing the plasmid by using a fast-cutting enzyme SalI, and transferring the linearized plasmid into pichia pastoris GS115 to obtain a recombinant strain S001;

(2) fusing a SUMO tag protein sequence at the N end of a delta 62C4ST sequence by a fusion PCR technology, then connecting the fused sequence SUMOC4 to a pPIC9K vector by Gibson assembly to obtain a pPIC9K-SUMOC4ST plasmid, linearizing the plasmid by a fast-cutting enzyme SalI, and transferring the linearized plasmid into Pichia pastoris GS115 to obtain a recombinant strain S002;

(3) mutating L134 of C4ST gene in pPIC9K-SUMOC4ST plasmid into L134E by site-directed mutagenesis technology, linearizing the plasmid with fast-cutting enzyme SalI, and transferring into Pichia pastoris GS115 to obtain recombinant strain S003;

(4) mutating F182 of C4ST gene in pPIC9K-SUMOC4ST plasmid into F182S by site-directed mutagenesis technology, linearizing the plasmid with fast-cutting enzyme SalI, and transferring into Pichia pastoris GS115 to obtain recombinant strain S004;

(5) superposing the two mutations in (3) and (4) in a plasmid by a site-directed mutagenesis technology to obtain an L134E/F182S double-mutation plasmid, linearizing the plasmid by using a fast-cutting enzyme SalI, and transferring the linearized plasmid into pichia pastoris GS115 to obtain a recombinant strain S005.

In one embodiment of the invention, the finally optimized C4ST sequence encoding chondroitin 4-O-sulfotransferase and gene sequence of aryl sulfotransferase AST IV are connected to a pPIC9K vector through fusion PCR and Gibson assembly to obtain pPIC9K-C4ST-AST IV plasmid, a nucleotide sequence encoding a linker sequence is inserted through cyclization PCR or Gibson assembly, the recombinant vector is transformed into Pichia pastoris GS115, the bifunctional protein is expressed by the recombinant Pichia pastoris GS115, and the bifunctional protein is used as a catalyst to catalyze chondroitin to synthesize chondroitin sulfate.

In one embodiment of the invention, 1-5mg/mL of bifunctional protein, 1-20mg/mL of chondroitin, 0.2-1mMPAP, 25-75mM of PNPS and 40-200 mg/mL of glycerol are subjected to a catalytic reaction to obtain chondroitin sulfate.

In one embodiment of the invention, 1-5mg/mL of bifunctional protein, 1-20mg/mL of chondroitin, 0.2-1mMPAP, 25-75mM of PNPS, 40-200 mg/mL of glycerol and 2.0-15 mg/mL of trehalose are subjected to a catalytic reaction to obtain chondroitin sulfate.

In one embodiment of the invention, 1-5mg/mL of bifunctional protein, 1-20mg/mL of chondroitin, 0.2-1mMPAP, 25-75mM of PNPS, 40-200 mg/mL of glycerol, 2.0-15 mg/mL of trehalose and 2.0-15 mg/mL of sorbitol are subjected to catalytic reaction to obtain chondroitin sulfate.

The invention also provides recombinant pichia pastoris for expressing the chondroitin 4-O-sulfotransferase C4ST and aryl sulfotransferase AST IV bifunctional enzymes, which is obtained by connecting the chondroitin 4-O-sulfotransferase C4ST, a linker sequence and the aryl sulfotransferase AST IV gene to a pPIC9K vector, transforming the recombinant vector into pichia pastoris GS115 and expressing the pichia pastoris GS 115. The nucleotide sequence for coding the linker sequence is any one of the following sequences: 1, 2, 3 and 4. The gene encoding chondroitin 4-O-sulfotransferase is optimized, and the optimized gene sequence is shown as SEQ ID NO. 5. The gene sequence of the coded aryl sulfotransferase AST IV is shown as SEQID NO. 6.

[ advantageous effects ]

1. Compared with the chondroitin sulfate obtained by the traditional tissue extraction method, the chondroitin sulfate synthesized by the biological enzyme method has the advantages of uniform product structure, no potential pathogenic factor and guaranteed quality safety of the product.

2. The invention utilizes the bifunctional protein constructed by the linker sequence and adds some specific stabilizers to synthesize chondroitin sulfate, and the distance between two enzyme catalysis structural domains can be effectively shortened by the fusion of the linker sequence to form an ion channel, thereby accelerating the transmission of the intermediate product of the enzyme cascade reaction; meanwhile, the stabilizer added in the reaction system can keep higher activity of the enzyme for a long time; compared with other in vitro enzymatic synthesis methods for synthesizing chondroitin sulfate, the method has the advantages of obviously improving the synthesis efficiency and reducing the cost.

3. The invention can effectively synthesize the chondroitin sulfate with the scale of about 20g, and lays a foundation for the industrial production of the chondroitin sulfate by the biological enzyme method.

4. The invention carries out site-directed mutagenesis on chondroitin 4-O-sulfotransferase C4ST, the single-point mutagenesis of L134 and F182 respectively improves the enzyme activity by 60 percent and 50 percent, and after further combined mutagenesis, the enzyme activity is improved to about 10 times.

Drawings

FIG. 1 shows relative enzyme activities optimized for expression of chondroitin 4-O-sulfotransferase C4 ST.

FIG. 2 shows the relative enzyme activity and chondroitin sulfate A conversion rate of the bifunctional protein.

FIG. 3 shows the conversion of chondroitin sulfate A with different stabilizers.

FIG. 4 is UPLC-MS identification of chondroitin and chondroitin sulfate A disaccharide. (a) Chondroitin liquid phase mass spectrogram; (b) chondroitin sulfate A liquid phase mass spectrogram.

Detailed Description

Materials:

1. pichia pastoris GS115 was purchased from the NTCC type culture Collection.

PrimeSTAR DNA polymerase, phosphorylase, DNA Marker, Solution I and other enzyme reagents from TaKaRa (Dalian).

Clonexpress one-step directed cloning kit was purchased from Vazyme Biotech (Nanjing).

4. Gel recovery kit, fast-cutting enzyme SalI, etc. were purchased from Thermofeisher Scientific Co.

5. The plasmid extraction kit was purchased from bioengineering (Shanghai) Co., Ltd.

6. Various analytical reagents were purchased from the national pharmaceutical group.

7. Pichia pastoris GS115 competent preparation method and transformation procedure refer to Thermo Fisher Invitrogen's sPichia EasyComp Kit.

8. Culture medium:

YPD medium (g/L): peptone 20, yeast powder 10 and glucose 20.

BMGY medium (g/L) peptone 20, yeast powder 10, 100mM phosphoric acid solution, pH 6.0, YNB 13.4, biotin 4 × 10^-4And 10 of glycerol.

BMMY culture medium (g/L) comprises peptone 20, yeast powder 10, 100mM phosphoric acid solution, pH 6.0, YNB 13.4, and biotin 4 × 10^-4Methanol 10 (v/v).

The liquid quality detection method of chondroitin sulfate A comprises the following steps: and adding 5 mu L of chondroitin sulfate lyase ABCI into 500 mu L of the sample after the catalytic reaction is finished, and incubating at 37 ℃ for 12 h. Heating the cracked solution in boiling water for 10min to inactivate and denature protein, centrifuging, and collecting the supernatant for UPLC-MS detection. UPLC-MS detection was performed using an Acquisty UPLC BEH Amide column (1.7 μm, 2.1X 100mm, Waters, MA, USA). Eluent A was acetonitrile, eluent B was ultrapure water, and the pH was adjusted to 10.4 with ammonia water. The elution gradient used was set as follows: 0-2 min, 5% B; 2-3 minutes, 5-30% B; 30-60% B for 3-6 min; 6-8 min, 60% B. The column temperature was maintained at 40 ℃ and the flow rate was 0.2 mL/min. The mass range of m/z 300-800 is scanned and monitored in the negative ion mode.

Conversion rate of chondroitin sulfate a: the mass ratio of the chondroitin sulfate A to the chondroitin mass initially added in the reaction is shown.

The enzyme activity definition and enzyme activity detection method of C4ST comprises the following steps: c4ST enzyme activity is defined as: the amount of enzyme required to synthesize 1. mu. MPNP per hour at 37 ℃. Adding 50mM PNPS,0.5mM PAP,2mg AST IV, 10% (v/v) glycerol, 20mg chondroitin and 500 mu l C4ST enzyme solution into the reaction system, diluting the reaction system to 1.5mL by using a 20mM phosphate buffer system (pH7.0), incubating at 37 ℃ for 2h, and detecting the experimental group and the control group A₄₀₀Difference in absorbance values. In the control group, the C4ST enzyme solution was heated in boiling water for 5 minutes for inactivation, and the other operations were the same as those in the experimental group.

The AST IV enzyme activity definition and enzyme activity detection method comprises the following steps: AST IV enzyme activity is defined as: the amount of enzyme required to synthesize 1. mu.M PNP per minute at 37 ℃. 50mM PNPS and 0.5mM PAP solution are placed in a 37 ℃ metal bath for incubation, 900 μ l of AST IV enzyme solution is added, the mixture is placed in a 37 ℃ metal bath for incubation for 2 minutes, and the increase value of the A400 absorbance is detected by a spectrophotometer. In the control group, the AST IV enzyme solution is heated in boiling water for 5 minutes for inactivation, and the rest operations are the same as those in the experimental group.