CN112029740A - 7 beta hydroxysteroid dehydrogenase mutant and application thereof - Google Patents

7 beta hydroxysteroid dehydrogenase mutant and application thereof Download PDF

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CN112029740A
CN112029740A CN202010967922.5A CN202010967922A CN112029740A CN 112029740 A CN112029740 A CN 112029740A CN 202010967922 A CN202010967922 A CN 202010967922A CN 112029740 A CN112029740 A CN 112029740A
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宋鹏
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Jiangxi Bontac Green Biocatalysis Ecoindustrial Park Co ltd
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Abstract

The invention relates to a 7 beta hydroxysteroid dehydrogenase mutant and application thereof, belonging to the technical field of enzyme engineering, and an amino acid sequence shown in SEQ ID NO. 2 is obtained by mutant library construction and a high-throughput screening method. And determining key amino acids influencing the activity of the 7 beta-HSDH by error-prone PCR, and carrying out site-directed saturation mutagenesis to improve the tolerance concentration of a 7 beta-HSDH substrate. The tolerance of the mutated 7 beta-HSDH substrate is obviously enhanced compared with the original 7 beta-HSDH substrate, and can tolerate the concentration of 200 mmol/L7-keto-lithocholic acid (7-KLCA) substrate, and the recombinant escherichia coli with the enhanced secretion capability of the mutant 7 beta-HSDH constructed by the invention can improve the enzyme activity of the 7 beta-HSDH by 2.47 times compared with the original strain. The modified genetically engineered bacterium has obviously improved enzyme production capability, is more suitable for industrial application, can reduce production cost and improve production efficiency.

Description

7 beta hydroxysteroid dehydrogenase mutant and application thereof
Technical Field
The invention relates to a 7 beta hydroxysteroid dehydrogenase (7 beta-HSDH) enzyme mutant modified by one or more error-prone PCR/site-directed saturation mutagenesis and application thereof, belonging to the technical field of enzyme engineering.
Background
Ursodeoxycholic acid (UDCA) is an effective drug for treating various cholelithiasis, various acute and chronic liver diseases. The traditional preparation method of UDCA is to extract from bear bile, but the method has the disadvantages of difficult extraction process, low product yield, too much means and violation of animal protection law.
At present, the method for industrially producing UDCA mainly comprises a chemical synthesis method and an enzyme method besides the bear gall extraction method, wherein the chemical synthesis method is gradually eliminated due to the defects of higher cost, more process steps, serious three wastes, harsh conditions and the like; the enzyme method has the advantages of low cost, simple process, greenness, no pollution, mild reaction conditions and the like, so that the enzyme method is increasingly paid attention.
Processes for the enzymatic preparation of UDCA are shown in FIG. 2 (references: last reduction in the synthesis of ursolic acid (UDCA): a critical review):
in combination with the enzymatic routes described above, it is necessary to use 7 β -HSDH as a biocatalyst in the UDCA preparation. However, 7 β -HSDH is very limited as a key enzyme in the UDCA preparation process, and researchers at home and abroad have screened out a great number of microorganisms producing 7 β -HSDH and cloned its coding gene, such as active Ruminococcus (Ruminococcus gnavus), Ruminococcus strawberrii (ATCC 35915), corynebacterium aeroginosum (collinella aerofaciens), Clostridium sardinieri (Clostridium sp.) and the like. However, these 7 β -HSDH enzymes are very unstable, and have good enzyme activity when detecting enzyme activity, but cannot bear slightly higher substrate concentration in actual catalysis at all, the substrate concentration used for synthesizing UDCA catalyzed by 7 β -HSDH from various sources is shown in table 1, the highest substrate concentration is less than 40g/L, and only the reaction amount in milliliter level can achieve higher conversion rate by consuming a large amount of enzyme, generally speaking, the enzyme conversion process at least reaches 50g/L or higher substrate concentration, and the enzyme catalysis system reaches kilogram level, which can be regarded as meaning of industrial production. The cost for extracting and purifying the enzyme protein is high; the stability of key enzyme is poor and the restriction factors such as insufficient substrate bearing capacity exist, the current improvement mode mainly adopts enzyme immobilization to improve the stability and the repeated utilization rate of the enzyme, and in addition, through the improvement of a reactor, a flow system is used for reducing partial enzyme activity of the enzyme lost due to immobilized mechanical stress. These improvements improve reaction efficiency and overall conversion to some extent, but still do not fundamentally solve the problem of poor stability of key enzymes, which greatly limits the development of UDCA production processes. Therefore, the method has very important significance in digging and screening the 7 beta-HSDH which has good catalytic performance and can tolerate high-concentration substrates.
TABLE 17 Current State of catalytic Synthesis of UDCA with beta-HSDH
Figure RE-GDA0002724945260000021
Figure RE-GDA0002724945260000031
With the development of genetic engineering, protein engineering, enzyme engineering and bioinformatics technology, many researchers use rational design, semi-rational design, directed evolution and other means to carry out mutation transformation on wild type 7 beta-HSDH. For example, in Chinese patent CN 105274070A, Liu Shi bin and the like mutate 7 beta-HSDH derived from Ruminobium liverwort, the activity of mutant RU-8C2 and RU-4F9 is improved to 9.5U/ml and 16.6U/ml from 5.1U/ml of wild type, and the corresponding amino acid residue changes are T210N and L3M/T219N respectively. For example, in Chinese patent CN 106636285A, wild type 7 beta-HSDH from Turneriella parva was mutated by Friedel et al, the activity of mutant V38R + V39R was increased from 254.8U/ml to 412.8U/ml, the reaction temperature was increased from 25 ℃ to 30 ℃, the amount of enzyme solution and NADP were added+The dosage is obviously reduced.
Although researchers at home and abroad use genetic engineering and protein engineering techniques to mutate and transform 7 beta-hydroxysteroid dehydrogenase so as to improve the activity of the dehydrogenase by times, researches on enhancing the substrate tolerance are still rarely reported.
Disclosure of Invention
In order to solve the problems, the invention starts from a clostridium (Clostridium) 7 beta-hydroxysteroid dehydrogenase sequence SEQ ID NO. 1, and obtains a mutant SEQ ID NO. 2 with obviously improved substrate tolerance by screening error-prone PCR.
2 is used as an initial sequence, beneficial mutants are further screened by virtual amino acid site-directed saturation mutagenesis, and a foundation is laid for practical application. The beneficial mutants were verified by measuring enzyme activity by shake flask fermentation.
The invention aims to provide one or more 7 beta-HSDH mutants which have an amino acid sequence shown in SEQ ID NO. 2.
The 7 beta-HSDH mutant is SEQ ID NO. 2 which replaces valine at position 207 of 7 beta-HSDH with methionine or other beneficial amino acids, wherein the amino acid sequence of the 7 beta-HSDH is shown as SEQ ID NO. 1.
The 7 beta hydroxysteroid dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: and 2.
SEQ ID NO: and 2, the application of the mutant amino acid sequence in the preparation of ursodeoxycholic acid.
The invention has the advantages of
(1) The invention determines the key amino acid influencing the activity of the 7 beta-HSDH through error-prone PCR and carries out site-specific saturation mutation, thereby improving the tolerance concentration of the 7 beta-HSDH substrate. The tolerance of the mutated 7 beta-HSDH substrate is obviously enhanced compared with the original 7 beta-HSDH, and the mutated 7 beta-HSDH substrate can tolerate 200 mmol/L7-keto-lithocholic acid (7-KLCA) substrate.
(2) The recombinant escherichia coli constructed by the invention and having enhanced secretion capability of the mutant 7 beta-HSDH can improve the enzyme activity of the 7 beta-HSDH by 2.47 times compared with that of an original strain. The enzyme production capacity of the modified genetically engineered bacteria is obviously improved, the enzyme activity of 7 beta-HSDH produced by shake flask fermentation reaches 80U/mL, the genetically engineered bacteria is more suitable for industrial application, the production cost can be reduced, and the production efficiency is improved.
Drawings
FIG. 1 shows the mutant structure of 7. beta. -HSDH.
FIG. 2 is a process scheme for the enzymatic preparation of UDCA.
Detailed Description
The following examples and descriptions thereof are provided to illustrate the present invention, but are not to be construed as limiting the invention. The methods used in the examples described below in this application are conventional methods, unless otherwise specified, and are carried out as described in the molecular cloning Experimental guidelines, J. SammBruk, D.W. Lassel, Huang Peyer, Wangjia seal, Zhu Hou, et al, 3 rd edition, Beijing: scientific Press, 2002. Meanwhile, the amino acids in the present invention are designated by their abbreviations or symbols unless otherwise specified.
Example 1
The mutant library construction and high throughput screening method comprises the following steps:
construction of mutant library:
in order to improve the activity of wild 7 beta-HSDH enzyme, a random mutant library is constructed by taking a recombinant expression vector PET28a (+) -RT-7 beta-HSDH as a DNA template through an error-prone PCR method, and Mg in an error-prone PCR reaction system is adjusted2+And Mn2+The concentration and dCTP and dTTP oligonucleotide concentration make the base mismatching rate of the mutant library five per thousand, i.e. ensure that 1 to 3 amino acids of one mutant are mutated, and the concrete process for constructing the mutant library is as follows. Error-prone PCR reaction system and conditions:
error-prone PCR reaction system:
Figure BDA0002683023040000051
the error-prone PCR reaction conditions were: pre-denaturation at 95 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 1min, and annealing at 72 ℃ for 1.5min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.
And (3) cutting the error-prone PCR product, recovering and purifying the cut rubber, connecting the product with a prokaryotic expression vector pET28a (+), and transforming recombinant genetic engineering bacteria to obtain a mutant library with large library capacity.
Screening a mutant library:
the high-throughput screening method of the 7 beta-HSDH mutant library adopts an NADPH (nicotinamide adenine dinucleotide phosphate) measuring method, the maximum absorption peak is formed at 340nm, 200 mmol/L7-KLCA is used as a substrate to screen mutagens, an enzyme labeling instrument is used for measuring the light absorption value of the mutant at 340nm, the smaller the numerical value is, the higher the enzyme activity is, the stronger the high-concentration substrate tolerance is, and the mutant is a beneficial mutant.
About 20000 clones were screened from the above mutant library to obtain 10 mutants with significant changes in number. These 10 mutants were then screened in shake flasks. The specific process is as follows: the 10 mutants are inoculated in a 500ml shake flask containing 100ml LB culture medium for fermentation and induction, the activity of catalytic 200 mmol/L7-KLCA is measured by a colorimetric method to obtain 1 optimal mutant which is named as SEQ ID NO. 2, and sequencing results show that the amino acid at the 207 th site is changed and is mutated into methionine from valine.
Example 2
Mutational crystal structure simulation:
a three-dimensional structure diagram of a 7 beta-HSDH mutant (SEQ ID NO:2) of Clostridium Clostridia (Clostridium) was constructed using a reported 7 beta-HSDH enzyme (PDB code: ID:5FYD) derived from C.aerofaciens as a template (published in Structural and biochemical information intro 7 beta-hydrolytical enzyme stereospecificity.2016) (the amino acid similarity between the two is 76%), and using a SWISS-MODEL online server (http:// www.swissmodel.expasy.org /). Substrates 7-KLCA and NADPH were docked into the mutator structure using AutoDock software (FIG. 1). 7-KLCA and NADPH three-dimensional stereotactic mechanism by Pubchem database download (https:// Pubchem. ncbi. nlm. nih. gov /).
Pseudo saturation mutation of 207 amino acid:
the amino acid 207 of SEQ ID NO. 2 was virtually saturated with a mutation and the binding affinity of the enzyme protein-ligand complex was analyzed by means of Discovery Studio.
The method comprises the following steps:
the Protein structure is introduced into Discovery Studio → Protein is dehydrated → preparation Protein (renamed Ligard) → entering into Simulation (Change Forcefield, Apply Forcefield in turn), and the Protein is endowed with CHARMM force field → Macromolecules → Design Protein → calcium Mutation Energy (binding).
The results of the free energy of binding of the mutated enzyme protein to the ligand are shown in the following table:
Figure BDA0002683023040000061
Figure BDA0002683023040000071
when M207 is changed to an amino acid other than D and E, the affinity between the receptor and the ligand is improved. From the prediction of the mutation mark, reasonable amino acid mutation can be carried out in the next step, and a mutant with improved enzyme activity and high substrate concentration tolerance is screened from the reasonable amino acid mutation.
Example 3
Determination of 7 beta-HSDH enzyme activity:
2.7mL of 50mM phosphate buffer solution (pH 8.0) at 25 ℃, 0.2mL of 7-ketolithocholic acid (7-KLCA) (the final concentration is 200mmol/L) (dissolved in buffer), 0.05mL of 7 beta-HSDH crude enzyme liquid sample diluted by a certain multiple of 50mM phosphate buffer solution (pH 8.0) are mixed in a cuvette and placed in an ultraviolet spectrophotometer, and the absorption value is reduced to zero.
Adding 0.05mL NAD (P) H (50mg/mL) into a cuvette, uniformly mixing, timing for 2min, reading an absorption change value with a wavelength of 340nm within 2min, and calculating delta OD/min;
blank control: the procedure is as above, but the enzyme in the reaction system is replaced by an equal amount of Tris/HCl buffer, and the result is determined to be a negative control.
Definition of enzyme activity unit:
enzyme activity (U/mL) ═ Δ OD/min Vt/(6.22 × 1.0 × Vs)
Vt reaction Total volume 3.05mL
Df: dilution factor
6.22: extinction coefficient of NADPH at 340nm wavelength
1.0: measuring optical path
Vs: volume of 7 beta-HSDH enzyme solution (0.05mL)
Influence of 7 beta-HSDH 207 site mutation on 7 beta-HSDH enzyme activity expression
The 7 beta-HSDH mutant gene is synthesized by using the whole gene, and is connected with a pET-28a (+) vector to construct a recombinant expression plasmid, the recombinant expression plasmid is transformed into a competent E.coil JM109, a kanamycin LB plate is coated, and a positive colony is selected. After shaking overnight at 37 ℃, plasmids were extracted and then transferred into E.coli BL21(DE3) to obtain recombinant strains with site-directed mutations.
The activity of the wild 7 beta-HSDH enzyme is measured to be 5.2U/mL by adopting the method; the enzyme activity of the mutant was 12.8U/mL.
Example 4
The 7 beta-HSDH mutant and glucose dehydrogenase from Bacillus subtilis are used in combination to transform 7-KLCA.
Dissolving 7-KLCA in phosphate buffer solution with pH 8.5 to a final concentration of 200 mmol/L; adding 7 beta-HSDH (total enzyme amount is 1 mg/mL); adding 360mmol/L glucose; adding glucose dehydrogenase (total enzyme amount is 3 mg/mL); adding NADP+3 mmol/L; the reaction is carried out for 4 to 6 hours at 35 ℃, and the conversion rate is calculated by an HPLC method. The results show that: the conversion of mutant 7 β -HSDH was 58%.
Sequence listing
<110> Jiangxi Bangtai Green biosynthesis ecological Industrial park development Co., Ltd
<120> 7 beta hydroxysteroid dehydrogenase mutant and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 264
<212> PRT
<213> SEQ ID NO:1 (Artificial sequence)
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Gly Lys Ser Ile Ser Glu Thr Tyr Gly Val Asp His Met Val Ile Arg
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Ala Asp Phe Ala Gln Ser Asp Cys Thr Asp Lys Ile Phe Glu Ala Thr
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Asn Val Asn Val Met Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Gly
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Ile Phe Ala Lys Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Leu
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Thr Ala Ile Ser Ser Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
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Ser Tyr Ile Lys Lys Leu Thr Glu Ala Val Ala Ala Glu Cys Glu Ser
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met
195 200 205
Lys Thr Ala Met Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Asp Asn
210 215 220
Leu Gly Lys Ser Leu Ser Val Ile Ala Gly Glu His Asn Lys Ala Asn
225 230 235 240
Val His Asn Trp Gln Ala Asn Lys Thr Asp Asp Glu Tyr Ile Arg Tyr
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Met Gly Ser Phe Tyr Ser Asn Asn
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<210> 2
<211> 264
<212> PRT
<213> SEQ ID NO:2 (Artificial sequence)
<400> 2
Met Asn Leu Arg Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Ala Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Ser Val Val Leu Val Gly Arg Arg Glu Glu Lys Leu Gln Glu Leu
35 40 45
Gly Lys Ser Ile Ser Glu Thr Tyr Gly Val Asp His Met Val Ile Arg
50 55 60
Ala Asp Phe Ala Gln Ser Asp Cys Thr Asp Lys Ile Phe Glu Ala Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
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Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Ile
100 105 110
Asn Val Asn Val Met Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Gly
115 120 125
Ile Phe Ala Lys Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Leu
130 135 140
Thr Ala Ile Ser Ser Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Lys Lys Leu Thr Glu Ala Val Ala Ala Glu Cys Glu Ser
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
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Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Met Met
195 200 205
Lys Thr Ala Met Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Asp Asn
210 215 220
Leu Gly Lys Ser Leu Ser Val Ile Ala Gly Glu His Asn Lys Ala Asn
225 230 235 240
Val His Asn Trp Gln Ala Asn Lys Thr Asp Asp Glu Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Ser Asn Asn
260

Claims (3)

1. A mutant 7 β hydroxysteroid dehydrogenase enzyme characterized by: the 7 beta hydroxysteroid dehydrogenase mutant is prepared by reacting a mutant with a mutant amino acid sequence shown in SEQ ID NO: the substitution of valine at position 207 of 7 β -HSDH shown in figure 1 with another amino acid.
2. The mutant 7 β hydroxysteroid dehydrogenase according to claim 1, wherein: the 7 beta hydroxysteroid dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: and 2.
3. Having the sequence shown in SEQ ID NO: and 2, the application of the amino acid sequence in the preparation of ursodeoxycholic acid.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113416717A (en) * 2021-07-14 2021-09-21 江西邦泰绿色生物合成生态产业园发展有限公司 7 beta hydroxysteroid dehydrogenase mutant suitable for industrial production
CN114231508A (en) * 2021-12-28 2022-03-25 宋建芳 7 beta-hydroxysteroid dehydrogenase mutant and application thereof
CN114438046A (en) * 2021-11-30 2022-05-06 山东省药学科学院 Preparation method of high-purity ursodeoxycholic acid

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CN108546691A (en) * 2018-05-09 2018-09-18 华东理工大学 7 beta-hydroxy sterol dehydrogenase mutants and its application in preparing ursodesoxycholic acid
CN109182284A (en) * 2018-09-28 2019-01-11 湖南福来格生物技术有限公司 A kind of 7beta-Hydroxysteroid dehydrogenase mutant, coded sequence, recombinant expression carrier, genetic engineering bacterium and application
CN110776572A (en) * 2019-11-14 2020-02-11 无锡佰翱得生物科学有限公司 Fusion protein of 7 β -HSDH enzyme and DPS

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CN108546691A (en) * 2018-05-09 2018-09-18 华东理工大学 7 beta-hydroxy sterol dehydrogenase mutants and its application in preparing ursodesoxycholic acid
CN109182284A (en) * 2018-09-28 2019-01-11 湖南福来格生物技术有限公司 A kind of 7beta-Hydroxysteroid dehydrogenase mutant, coded sequence, recombinant expression carrier, genetic engineering bacterium and application
CN110776572A (en) * 2019-11-14 2020-02-11 无锡佰翱得生物科学有限公司 Fusion protein of 7 β -HSDH enzyme and DPS

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113416717A (en) * 2021-07-14 2021-09-21 江西邦泰绿色生物合成生态产业园发展有限公司 7 beta hydroxysteroid dehydrogenase mutant suitable for industrial production
CN113416717B (en) * 2021-07-14 2023-12-29 江西邦泰绿色生物合成生态产业园发展有限公司 7 beta hydroxysteroid dehydrogenase mutant suitable for industrial production
CN114438046A (en) * 2021-11-30 2022-05-06 山东省药学科学院 Preparation method of high-purity ursodeoxycholic acid
CN114438046B (en) * 2021-11-30 2023-08-22 山东省药学科学院 Preparation method of high-purity ursodeoxycholic acid
CN114231508A (en) * 2021-12-28 2022-03-25 宋建芳 7 beta-hydroxysteroid dehydrogenase mutant and application thereof
CN114231508B (en) * 2021-12-28 2022-11-11 宋建芳 7 beta-hydroxysteroid dehydrogenase mutant and application thereof

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