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CN117487781A - Beta-glucosidase mutant and application thereof in production of rare ginsenoside - Google Patents

Beta-glucosidase mutant and application thereof in production of rare ginsenoside Download PDF

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CN117487781A
CN117487781A CN202311455898.7A CN202311455898A CN117487781A CN 117487781 A CN117487781 A CN 117487781A CN 202311455898 A CN202311455898 A CN 202311455898A CN 117487781 A CN117487781 A CN 117487781A
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glucosidase
beta
ginsenoside
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pxbgl
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李丹
李晓磊
许文琪
段翠翠
马福敏
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Changchun University
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Abstract

A beta-glucosidase mutant and application thereof in producing rare ginsenoside belong to the technical field of genetic engineering and enzyme engineering. Aiming at the problems that the existing glycosidase capable of hydrolyzing ginsenoside Rb3 to form ginsenoside Rd has few types, low enzyme activity, long reaction time, large enzyme adding amount, poor specificity and the like, the invention adopts a site-directed mutagenesis method to mutate the 293 th asparagine of beta-glucosidase derived from xylan clostridium petroleum (Petroclostridium xylanilyticum) into lysine, and compared with wild type beta-glucosidase, the obtained mutant has 29 percent higher specific activity on ginsenoside Rb3, thereby being beneficial to the industrial production and application of converting ginsenoside Rb3 into ginsenoside Rd.

Description

Beta-glucosidase mutant and application thereof in production of rare ginsenoside
Technical Field
The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a beta-glucosidase mutant and application thereof in production of rare ginsenoside.
Background
Ginseng has been used as an ancient Chinese herbal medicine for many years as a food and a medicine to supplement nutrition and treat human diseases. The ginseng contains various active ingredients including ginsenoside, polysaccharide, peptide, phytosterol, polyacetylene alcohol and fatty acid. Wherein ginsenoside is the main active ingredient of Ginseng radix, and the total content of ginsenoside in the fine root is 142.49 + -1.14 mg/g. Up to now, 100 or more ginsenosides have been identified from ginseng, which are classified into protopanaxadiol saponin- (PPT-), protopanaxatriol saponin- (PPT-), oleanolic acid- (OA-), eur Kang Tiluo mol- (OT-), C17 side chain variant- (C17 SCV-) and other types. Ginsenoside Rb1, rb2, rb3, rc, rg1 and Re are abundant in wild ginseng root and pseudo-ginseng leaf, and are called main ginsenoside. The ginsenoside C-3, C-6 and C-20 have glycosyl side chains, so that the oral intake ginsenoside has lower film permeability in human gastrointestinal tract, the absorption efficiency is reduced, and the biological activity and bioavailability are greatly reduced. In vitro, the deglycosylation of the main ginsenoside can generate various rare ginsenosides with high biological activity, and the human body can take in the rare ginsenosides to improve the bioavailability of the ginsenosides, so that the rare ginsenosides have excellent application value in the fields of foods, medicines and the like.
The method for converting main ginsenoside into rare ginsenoside by enzyme method has the characteristics of mild reaction condition, less byproducts and the like. However, the conventional glycosidases capable of hydrolyzing ginsenoside Rb3 to ginsenoside Rd have the problems of low enzyme activity, long reaction time, large enzyme adding amount, poor specificity and the like. Beta-glucosidase (EC 3.2.1.21) can act on the glycosidic bond of the non-reducing end of the beta-1, 4 glucan sugar chain, releasing the glycosyl. Kyung-Chul Shin et al (Complete biotransformation ofprotopanaxadiol-type ginsenosides to-O-beta-glucopyranosyl-20 (S) -protopanaxadiol using a novel and thermostable beta-glucosidase. JAmatic Food chem.2018,66, 2822-2829.) found that beta-glucosidase derived from Caldicellulosiruptor bescii can recognize the xylose group at C-20 and the glucose group at C-3 on ginsenoside Rb3, which resulted in the inability to form specific ginsenoside Rd during transformation. Wenhua Yang et al (Combinatorial enzymatic catalysis forbioproduction ofginsenoside compoundK.JAgric Food chem.2023,71, 3385-3397) found that 2 beta-glucosidase enzymes (BG 19 and BG 23) produced in Aspergillus tubingensis JE0609 could hydrolyze a PPD-type ginsenoside mixture containing Rb3, converting almost all over 6 hours to yield ginsenoside Rd. Hui Zhang et al (Enzymatic biotransformation ofRb3 from the leaves ofPanax notoginsengto ginsenoside Rdby arecombinant beta-xylosidase from Thermoascus)
aurentiacus, world J Microbiol biotechnol.2022,39,21.) found a source of Thermoascus
Beta-xylosidase of aurentiacus catalyzes ginsenoside Rb3 to remove xylosyl under the optimal condition, and in the case of adding the enzyme again at 24h, the molar conversion rate of Rb3 reaches 56.93 percent in the reaction for 60 h. Therefore, the existing beta-glucosidase capable of converting ginsenoside Rb3 into ginsenoside Rd has the problems of long reaction time, large enzyme adding amount and poor specificity. Therefore, by means of genetic engineering and enzyme engineering, the modification of the existing beta-glucosidase and the improvement of the catalytic activity of the beta-glucosidase are of great significance.
Disclosure of Invention
The invention adopts genetic engineering technology in the early stage, and heterologously expresses beta-glucosidase Pxbgl from xylan clostridium petroleum (Petroclostridium xylanilyticum). It was found that 0.3U/mLPxbgl could convert 1mM of a mixture of ginsenoside Rb1, rb2, rb3 and Rc to ginsenoside Rd in 3 hours. Wherein, the enzyme can also rapidly remove the xylosyl on the ginsenoside Rb3 within 10min, and the yield of the generated ginsenoside Rd is 5883.65 mu M/h, which is 340.1 times of the beta-xylosidase from Thermoascus aurantiacus. In order to further improve the catalytic capability of the beta-glucosidase Pxbgl on the reaction, the invention obtains the beta-glucosidase mutant with improved specific activity through gene site-directed mutagenesis, and the mutant can more efficiently realize the conversion of rare ginsenoside Rd, thereby having important industrial application prospect and production value.
In order to solve the technical problems and realize the corresponding technical effects, the invention provides the following technical scheme:
the first object of the invention is to provide a beta-glucosidase mutant, which takes wild-type beta-glucosidase with an amino acid sequence shown as SEQ ID NO.2 as a parent, and mutates the 293 th asparagine of the parent into lysine.
It is a second object of the present invention to provide a coding sequence for the above-mentioned β -glucosidase mutant.
In one embodiment of the invention, the coding sequence is shown in SEQ ID NO. 3.
A third object of the present invention is to provide a recombinant vector comprising the above coding sequence.
The fourth object of the present invention is to provide a recombinant bacterium comprising the above recombinant vector.
A fifth object of the present invention is to provide a method for preparing the above-mentioned β -glucosidase mutant, comprising the steps of:
s1, designing a mutation primer for site-directed mutagenesis according to a determined mutation site, and carrying out site-directed mutagenesis by taking a vector carrying a wild beta-glucosidase gene as a template to construct a plasmid vector containing a coding mutant gene;
s2, transforming a plasmid vector containing the coding mutant gene into a host cell;
s3, selecting positive clones for culture, and centrifugally collecting cells after induction culture, wherein cell wall-broken supernatant is crude enzyme liquid of the beta-glucosidase mutant.
In one embodiment of the present invention, the nucleotide sequence of the wild-type β -glucosidase gene is shown in SEQ ID No. 1.
The sixth object of the invention is to provide the application of the beta-glucosidase mutant in the production of rare ginsenoside Rd.
The seventh object of the invention is to provide the application of the coding sequence in the production of rare ginsenoside Rd.
An eighth object of the present invention is to provide an application of the recombinant vector in the production of rare ginsenoside Rd.
The ninth object of the invention is to provide an application of the recombinant bacterium in the production of rare ginsenoside Rd.
The invention has the beneficial effects that:
the invention carries out site-directed mutagenesis on beta-glucosidase Pxbgl from xylan clostridium petroleum (Petroclostridium xylanilyticum) to obtain beta-glucosidase mutant N293K, the mutant can realize high-efficiency expression in escherichia coli BL21 (DE 3), compared with wild type beta-glucosidase Pxbgl, the specific activity of the mutant N293K is improved by 29 percent, and compared with wild type beta-glucosidase Pxbgl, the mutant N293K obtained by the invention is more suitable for industrial production of converting ginsenoside Rb3 into ginsenoside Rd, and has wide application prospect.
Drawings
FIG. 1 is a reaction structural formula of ginsenoside Rb3 under the action of beta-glucosidase mutant Pxbgl-N293K to generate ginsenoside Rd;
FIG. 2 is a protein electrophoretogram of wild-type β -glucosidase Pxbgl and its mutant N293K; in FIG. 2, lane M is a protein molecular weight standard, lane 1 is a wild-type beta-glucosidase Pxbgl crude enzyme, lane 2 is a wild-type beta-glucosidase Pxbgl pure enzyme, lane 3 is a mutant N293K crude enzyme, and lane 4 is a mutant N293K pure enzyme.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description and the accompanying drawings. The experimental procedures used in the examples below were conventional, and the materials, reagents, and apparatus used, unless otherwise indicated, were conventional in the art and are commercially available to those skilled in the art.
Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the methods and applications described herein can be modified or adapted and combined to implement and utilize the technology of this invention without departing from the spirit and scope of this invention.
The xylan Clostridium petroleum used in the present invention (Petroclostridium xylanilyticum) is described in the literature: zheng, z.z., liu, l.y., deng, y., zhang, h, & Cheng, l (2018) Petroclostridium xylanilyticum gen.nov., sp.nov., a xylan-degrading bacterium isolated from an oilfield, and reclassification of clostridial cluster III members into four novel genera in a new Hungateiclostridiaceae fam.nov.international journal of systematic and evolutionary microbiology,68 (10), 3197-3211.
Example 1: amplification of wild-type beta-glucosidase Pxbgl gene and construction of recombinant plasmid containing coding sequence thereof
Extracting genome DNA of xylan clostridium petroleum (Petroclostridium xylanilyticum), and designing primer for amplifying beta-glucosidase gene according to the gene sequence corresponding to beta-glucosidase amino acid sequence (NCBI Reference Sequence: WP_ 198543882.1) in the strain. The gene corresponding to beta-glucosidase Pxbgl is amplified by Polymerase Chain Reaction (PCR) by taking the genome DNA of xylan clostridium petroleum as a template and Pxbgl-F and Pxbgl-R as primers (the nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO. 2). The PCR amplification conditions were as follows: pre-denaturation at 96℃for 3min; 15s at 95 ℃,15 s at 58 ℃,20 s at 72 ℃ and 23 cycles; finally, the extension is carried out for 1min at 72 ℃.
Pxbgl-F(SEQ ID NO.5)：
5’-AATTTTGTTTAACTTTAAGAAGGAGATATACATATGGAAAATCAAAATAGAAAGAAGGAGATGCTGTACCTGGATGCAACTCAGCCTGTG-3’；
Pxbgl-R(SEQ ID NO.6)：
5’-AGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCTAACCTCTACTTGAGTAGAAAAAGTTTTACATTTACTTATATCTGTTACTTCCC-3’。
The amplified target gene is inserted into a vector pET-21b (+) by a homologous recombination method. The reaction system is as follows: mu.L of purified PCP product, 5 mu LNdeI-XhoI digestion vector, 10 mu L of 2X MultiF Seamless Assembly Mix (WU.S. Pat. No. 2) were ligated with the above ligation solution at a constant temperature of 52℃for 30min, and the ligation product was transformed into E.coli MC1061 to obtain recombinant bacteria.
And extracting plasmids of the recombinant strain for sequencing and identification. By comparison, the beta-glucosidase gene containing xylan clostridium petroleum was determined and the recombinant plasmid was named pET Pxbgl.
Example 2: construction of beta-glucosidase Pxbgl mutant N293K
In order to further improve the enzyme activity of beta-glucosidase Pxbgl, the invention utilizes Autodock Tools 1.5.7 to carry out molecular docking on wild beta-glucosidase Pxbgl and ginsenoside Rb3, shows that the amino acid residue asparagine at 293 position on Pxbgl forms hydrogen bond with ginsenoside Rb3, and predicts that the amino acid at the position plays an important role in hydrolyzing ginsenoside Rb 3. The amino acid sequence of wild-type beta-glucosidase Pxbgl is searched by utilizing BLAST tools in NCBI to obtain a series of amino acid sequences with different similarity, and enzymes capable of converting ginsenoside are selected, wherein the enzymes comprise beta-glucosidase from Caldicellulosiruptor bescii, beta-glycase from Dictyoglomus turgidum and the like. The sequence alignment was performed using Clustal W software, and the result showed that the amino acid corresponding to position 293 of Pxbgl was lysine in the amino acid sequence of β -glucosidase derived from Caldicellulosiruptor bescii. Therefore, asparagine at position 293 was selected as a subject and mutated to a different amino acid. Then the mutant is subjected to molecular docking again, and the result shows that the catalytic amino acid of the active center of the mutant N293K (the 293 th mutation is lysine) forms a hydrogen bond with the xylosyl on the ginsenoside Rb3, and the mutant N293K is predicted to be more beneficial to hydrolyzing the ginsenoside Rb3, so the invention constructs the mutant N293K next.
Primers for introducing the N293K codon mutation were designed and synthesized according to the sequence of the beta-glucosidase Pxbgl gene shown in SEQ ID No. 1. The PCR is utilized, the recombinant plasmid pET Pxbgl is used as a template for mutation, and the N293K mutant site-directed mutagenesis primer is used as follows:
forward primer (SEQ ID No. 7):
5’-TGCTATTAAGATGCTTTATGATTACCATCGTTTAAAG-3’
reverse primer (SEQ ID NO. 8):
5’-AAAGCATCTTAATAGCAAAATAATCCGATACTACCAGC-3’
the PCR reaction system is as follows: 5 XPrimeSTAR Buffer 10. Mu.L, 2.5mM dNTPs 4. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu.L, template DNA 0.5. Mu.L, high fidelity PrimeSTARTM HSDNA polymerase (2.5U/. Mu.L) 1. Mu.L, and sterilized water 33. Mu.L were added.
The PCR amplification conditions were as follows: pre-denaturation at 98 ℃ for 30s; then denaturation at 98℃for 10s, annealing at 57℃for 15s, elongation at 72℃for 7min 48s, and elongation at 72℃for 10min, 20 cycles were performed.
After digestion of the PCR product with DpnI for 5min, the linearized digestion product was subjected to circularized recombination with homologous recombination enzymes. The reaction system is as follows: 4. Mu.L (100 ng) of the digested product was linearized, 2X MultiF Seamless Assembly Mix. Mu.L and 6. Mu.L of sterilized water.
The assembled product was transferred into competent cells of E.coli MC1061, plated onto LB solid medium containing ampicillin, and cultured overnight at 37 ℃. The single colony is picked up and cultured in LB liquid medium at 37 ℃ for 6 hours, and then plasmid is extracted and sequenced for verification. The mutant recombinant plasmid was designated pET Pxbgl-N293K.
The nucleotide sequence of the mutant N293K is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO.4.
Example 3: expression and purification of wild-type beta glucosidase Pxbgl and mutant N293K
E.coli BL21 (DE 3) was transformed with the recombinant plasmid pET Pxbgl of example 1 and the mutant plasmid pET Pxbgl-N293K of example 2, respectively, and the recombinant E.coli was cultured overnight at 37℃on LB solid medium containing 0.1mg/mL ampicillin. Single colonies were then inoculated into LB liquid medium containing 0.1mg/mL ampicillin, and cultured with shaking at 37℃and 250rpm for 7 hours. Subsequently, isopropyl- β -d-thiogalactoside (IPTG) was added at a final concentration of 0.16mM, and the mixture was subjected to shaking culture at 16℃and 150rpm for 16 hours to induce the expression of β -glucosidase. The recombinant E.coli cell culture solution was centrifuged at 7000 Xg at 4℃for 20min, the pellet was suspended in a buffer solution containing 50mM Tris-HCl (pH 7.4), 500mM sodium chloride, 20mM imidazole, and then 50mL of the suspension was placed in ice water, and the suspension was broken with a 750W ultrasonic breaker (Sonics & Materials, newtown, CT, USA) for 45min. Centrifuging at 4deg.C and 9000 Xg for 20min, and collecting supernatant to obtain crude enzyme solutions of wild type beta-glucosidase Pxbgl and its mutant N293K.
The crude enzyme was filtered through a 0.45 μm filter membrane and passed through Ni Sepharose TM Purification was performed with a 6 Fast Flow affinity column and nucleic acid protein detector, eluting with a buffer solution containing 50mM Tris-HCl (pH 7.4), 500mM sodium chloride, 500mM imidazole, to obtain pure enzymes of wild-type β -glucosidase Pxbgl and mutant N293K.
Analysis of the crude and pure enzymes of wild-type β -glucosidase Pxbgl and mutant N293K by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the pure enzymes of wild-type β -glucosidase Pxbgl and mutant N293K exhibited a single band (see FIG. 2).
Example 4: comparative analysis of wild-type beta-glucosidase Pxbgl and mutant N293K
(1) Determination of enzyme Activity
The enzyme activity was determined by detecting the decrease in ginsenoside. In 50mM Na containing 0.4mM ginsenoside Rb3 2 HPO 4 -NaH 2 PO 4 (pH 6.0) buffer solution, adding pure enzyme solution of wild type beta-glucosidase Pxbgl or mutant N293K, reacting at 60 ℃ for 10 minutes, and stopping the reaction by using an equal volume of water saturated N-butanol solution. 1U is defined as the amount of enzyme required to reduce 1. Mu. Mol of ginsenoside per minute. The reaction structural formula of ginsenoside Rb3 for generating ginsenoside Rd under the action of beta-glucosidase mutant Pxbgl-N293K is shown in figure 1.
Compared with the wild type beta-glucosidase Pxbgl, the mutant N293K has 14% improved enzyme activity on ginsenoside Rb3, and the result is shown in Table 1.
TABLE 1 enzyme activities of wild-type beta-glucosidase Pxbgl and mutant N293K
(2) Protein content determination
Protein content was determined by coomassie brilliant blue method and calculated, and the results are shown in table 2.
TABLE 2 protein content of wild-type beta-glucosidase Pxbgl and mutant N293K
(3) Comparison of enzyme specific Activity
The enzyme activities of the pure enzyme solutions of the wild-type β -glucosidase Pxbgl or mutant N293K were determined according to the methods described above and used to calculate the specific activities of the wild-type and mutant N293K.
The specific activity was calculated according to the following formula:
specific activity (U/mg) =enzyme activity (U/mL)/protein content (mg/mL)
Compared with the wild beta-glucosidase Pxbgl, the specific activity of the mutant N293K to the ginsenoside Rb3 is improved by 29%.
TABLE 3 specific Activity of wild-type beta-glucosidase Pxbgl with its mutant N293K
(4) Comparison of conversion of ginsenoside Rb3
To investigate the conversion of ginsenoside Rb3 by wild-type β -glucosidase Pxbgl and mutant N293K, 1mM ginsenoside Rb3 standard was reacted with 0.3U/mL wild-type β -glucosidase Pxbgl and mutant N293K at 60℃and pH 6.0 for 10min, respectively, and TLC and HPLC analysis were performed. The results showed that the molar conversion of wild-type β -glucosidase Pxbgl to ginsenoside Rb3 was 98.17% and that the molar conversion of mutant N293K to ginsenoside Rb3 was 100%. It can be seen that under the same enzyme activity and reaction conditions, the conversion rate of the mutant N293K to ginsenoside Rb3 is higher compared with that of the wild-type beta-glucosidase Pxbgl, and the conversion of ginsenoside Rb3 into Rd is facilitated.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A beta-glucosidase mutant is characterized in that the mutant takes wild beta-glucosidase with an amino acid sequence shown as SEQ ID NO.2 as a parent, and the 293 th asparagine of the parent is mutated into lysine.
2. A coding sequence for a β -glucosidase mutant according to claim 1.
3. The coding sequence of claim 2, wherein the coding sequence is set forth in SEQ ID No. 3.
4. A recombinant vector comprising the coding sequence of any one of claims 2 or 3.
5. A recombinant bacterium comprising the recombinant vector of claim 4.
6. A method for preparing the β -glucosidase mutant of claim 1, comprising the steps of:
s1, designing a mutation primer for site-directed mutagenesis according to a determined mutation site, and carrying out site-directed mutagenesis by taking a vector carrying a wild beta-glucosidase gene as a template to construct a plasmid vector containing a coding mutant gene;
s2, transforming a plasmid vector containing the coding mutant gene into a host cell;
s3, selecting positive clones for culture, and centrifugally collecting cells after induction culture, wherein cell wall-broken supernatant is crude enzyme liquid of the beta-glucosidase mutant.
7. Use of the beta-glucosidase mutant according to claim 1 for the production of rare ginsenoside Rd.
8. Use of a coding sequence according to any one of claims 2 or 3 for the production of rare ginsenoside Rd.
9. The use of the recombinant vector of claim 4 for producing rare ginsenoside Rd.
10. The recombinant bacterium of claim 5, which is used for producing rare ginsenoside Rd.
CN202311455898.7A 2023-11-03 2023-11-03 Beta-glucosidase mutant and application thereof in production of rare ginsenoside Pending CN117487781A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118006590A (en) * 2024-02-23 2024-05-10 南京师范大学常州合成生物学产业研究院有限公司 Microbacterium dextranolyticum beta-glucosidase mutant, method and application
CN118291430A (en) * 2024-05-16 2024-07-05 广东金骏康生物技术有限公司 Glucoamylase mutant and preparation method and application thereof
CN118325874A (en) * 2024-05-16 2024-07-12 广东金骏康生物技术有限公司 Glucoaptation hydrolase combined mutant and preparation method and application thereof

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