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CN118207186B - Beta-glucuronidase and recombinant expression vector, engineering bacteria, starter and method for mass production of glycyrrhetinic acid thereof - Google Patents

Beta-glucuronidase and recombinant expression vector, engineering bacteria, starter and method for mass production of glycyrrhetinic acid thereof Download PDF

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CN118207186B
CN118207186B CN202410627081.1A CN202410627081A CN118207186B CN 118207186 B CN118207186 B CN 118207186B CN 202410627081 A CN202410627081 A CN 202410627081A CN 118207186 B CN118207186 B CN 118207186B
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glucuronidase
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CN118207186A (en
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吕波
陈林浩
赖俊杰
李春
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Beijing Institute of Technology BIT
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Abstract

The invention relates to beta-glucuronidase and a recombinant expression vector, engineering bacteria, a starter and a method for mass production of glycyrrhetinic acid thereof, belonging to the technical fields of enzyme engineering and microorganisms. The beta-glucuronidase is selected from the group consisting of: mutants of beta-glucuronidase, acGUS1, acGUS, acGUS3, acGUS; acGUS is an enzyme obtained by deleting 1 st amino acid, 7 th alanine, 66 th arginine, 256 th alanine, 270 th threonine, 332 th valine, 363 th aspartic acid, 506 th methionine and valine of the amino acid sequence of GenBank accession AEK 69352.1. The invention is simple and efficient, and can prepare 18 alpha-GA and 18 beta-GA on an industrial scale.

Description

Beta-glucuronidase and recombinant expression vector, engineering bacteria, starter and method for mass production of glycyrrhetinic acid thereof
Technical Field
The invention belongs to the technical fields of enzyme engineering and microorganisms, and in particular relates to beta-glucuronidase, a recombinant expression vector thereof, engineering bacteria, a starter and a method for mass production of glycyrrhetinic acid.
Background
Licorice is used as a traditional plant with homology of medicine and food, and is widely applied to industries such as medicine, food, cosmetics and the like. According to the history of the history, liquorice is used for over four thousand years, which can be called as 'towards the middle aged, liquorice in medicine'. The licorice has the effects of clearing heat and detoxicating, tonifying spleen and replenishing qi, moistening lung and relieving cough, harmonizing various medicines and the like, can be used for treating symptoms such as sore throat, deficiency-cold of spleen and stomach, peptic ulcer and the like, and has wide application in the aspects of anti-inflammation, liver protection, antioxidation, antivirus, anti-tumor, antidiuretic and the like. Glycyrrhrizae radix or its effective components are added into compound preparation of many modern Chinese medicinal materials.
Glycyrrhizic acid (glycyrrhizic acid, GL) and glycyrrhetinic acid (GLYCYRRHETINIC ACID, GA) are the main and active components of Glycyrrhrizae radix, respectively. At present, the reported production of glycyrrhetinic acid mainly comprises a chemical method and a biological conversion method, CN101817867A reports that glycyrrhetate is used as a raw material, acetyl glycyrrhetinic acid is generated under the catalysis of sulfuric acid and high-concentration acetic acid, and then the acetyl glycyrrhetinic acid is further deacetylated to obtain the glycyrrhetinic acid, and a large amount of strong acid, strong alkali and organic solvent are required to be used for the reaction, so that the problems of high energy consumption, low yield, high environmental protection pressure and the like exist. The bioconversion method not only has the advantages of mild condition, high yield, environmental protection and the like, but also can provide medicinal precursors with higher purity for preparing different derivatives, and is an industrial production mode with great development prospect. There are a great deal of literature reports on the application of GA in anti-inflammatory, liver-protecting, antioxidant, antiviral, antitumor and antidiuretic aspects. Research shows that the liver distribution capacity of 18 alpha-GA is stronger than that of 18 beta-GA, the curative effect on hepatitis is better, the side effect is weaker than that of 18 beta-GA, and the safety is better.
In the report of the prior bioconversion method, mainly the method for producing 18 beta-GA by hydrolyzing glycyrrhizic acid by using beta-glucuronidase is adopted, and 18 alpha-glycyrrhetinic acid and 18 beta-glycyrrhetinic acid can be respectively obtained after the glycyrrhizic acid substrate is hydrolyzed by two steps of beta-glucuronidase (shown in figure 1). Early results of the study group where the inventors were: chinese patent No. 109628427B reports a recombinase AtGUS-mix obtained based on domain replacement, the final concentration of the recombinase reaches 16.3g/L at maximum when the recombinase is used for producing 18 beta-GA under different conditions, the reaction period is 96 hours, but the problem of reaction inhibition easily caused by accumulation of an intermediate 18 beta-GAMG is not solved.
In order to seek higher affinity, enzyme activity, catalytic efficiency, conversion rate and better reduce 18 beta-GAMG accumulation, further reduce the production cost of glycyrrhetinic acid, there is a need in the art to develop new beta-glucuronidase for industrial preparation of glycyrrhetinic acid.
Disclosure of Invention
The invention provides a beta-glucuronidase and a recombinant expression vector, engineering bacteria, a starter and a method for mass production of glycyrrhetinic acid thereof, which aim to solve the problems that the prior art pursues higher affinity, enzyme activity, catalytic efficiency and conversion rate and better reduces 18 beta-GAMG accumulation and is suitable for mass production.
The technical scheme of the invention is as follows:
A beta-glucuronidase selected from the group consisting of: beta-glucuronidase AcGUS, and/or beta-glucuronidase AcGUS1, and/or beta-glucuronidase AcGUS2, and/or beta-glucuronidase AcGUS3, and/or beta-glucuronidase AcGUS3 mutants;
The beta-glucuronidase AcGUS is an enzyme obtained by deleting 1 st amino acid, 7 th alanine, 66 th arginine, 256 th alanine, 270 th threonine, 332 th valine, 363 rd aspartic acid, 506 th methionine and valine of an amino acid sequence with GenBank accession number AEK 69352.1;
The beta-glucuronidase AcGUS, acGUS2 and AcGUS are enzymes obtained by truncating 1-39 amino acids at the nitrogen end of the beta-glucuronidase AcGUS;
The beta-glucuronidase AcGUS mutant is selected from the group consisting of: a mutant obtained by mutating the 461 st glycine of AcGUS to cysteine, a mutant obtained by mutating the 462 st glutamine of AcGUS to histidine, a mutant obtained by mutating the 575 nd isoleucine of AcGUS to lysine, or a mutant obtained by mutating the 461 st glycine of AcGUS to cysteine, the 462 st glutamine to histidine and the 575 nd isoleucine to lysine.
The amino acid sequence of the beta-glucuronidase AcGUS is shown as SEQ ID NO. 1;
preferably, the gene sequence of the beta-glucuronidase AcGUS is shown as SEQ ID NO. 2;
preferably, the beta-glucuronidase AcGUS is an enzyme obtained by truncating the nitrogen end of the beta-glucuronidase AcGUS by 10 amino acids;
Preferably, the beta-glucuronidase AcGUS is an enzyme obtained by truncating 20 amino acids at the nitrogen end of the beta-glucuronidase AcGUS;
preferably, the beta-glucuronidase AcGUS is an enzyme obtained by truncating the nitrogen end of the beta-glucuronidase AcGUS by 30 amino acids;
Preferably, the mutants obtained by mutating the 461 st glycine of AcGUS to cysteine, the 462 st glutamine to histidine and the 575 nd isoleucine to lysine are beta-glucuronidase AcGUS M1.
A recombinant expression vector, which is connected with the expression vector of the gene sequence of the beta-glucuronidase.
The expression vector is selected from pET28a and pGAPZ alpha A, pPIC9K, pPICZ alpha;
Preferably, the recombinant expression vector is selected from recombinant expression vector pGAPZαA-AcGUS, and/or recombinant expression vector pGAPZαA-AcGUS3, and/or recombinant expression vector pGAPZαA-AcGUS M1.
An engineered bacterium selected from pichia pastoris strain AcGUS, and/or pichia pastoris strain AcGUS, and/or pichia pastoris strain AcGUS M1, and/or pichia pastoris strain dG-GA1;
the pichia pastoris strain AcGUS can express the beta-glucuronidase AcGUS;
the pichia pastoris strain AcGUS can express the beta-glucuronidase AcGUS;
The pichia pastoris strain AcGUS M1 can express the beta-glucuronidase AcGUS M1;
The pichia pastoris strain dG-GA1 can simultaneously express the beta-glucuronidase AcGUS M1 and the beta-glucuronidase AtGUS derived from the aspergillus terreus (Aspergillus terreus) strain Li-20.
The pichia pastoris strain AcGUS contains the recombinant expression vector pGAPZalpha A-AcGUS;
preferably, the pichia pastoris strain AcGUS contains the recombinant expression vector pGAPZαA-AcGUS;
Preferably, the pichia pastoris strain AcGUS M1 contains the recombinant expression vector pgapzαa-AcGUS M1;
Preferably, the pichia pastoris strain dG-GA1 contains the recombinant expression vector pGAPZalpha A-AcGUS M1 modified by the recombinant expression vector pGAPZalpha A-AcGUS M1-NrsR and the recombinant expression vector pGAPZalpha A-AtGUS connected with a beta-glucuronidase AtGUS gene sequence derived from the aspergillus terreus (Aspergillus terreus) strain Li-20;
Preferably, the modification means: the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS M1 was replaced by NrsR resistance.
A starter comprising a starter active ingredient comprising said one beta-glucuronidase and/or said one recombinant expression vector and/or said one engineering bacterium.
The fermenting agent further comprises: auxiliary materials.
A method for mass production of glycyrrhetinic acid comprises fermenting a substrate with the beta-glucuronidase and/or the recombinant expression vector and/or the engineering bacteria.
The substrate is selected from: 18 alpha-GL or 18 beta-GL;
Preferably, the glycyrrhetinic acid is selected from the group consisting of: 18 a-GA or 18 β -GA;
Preferably, the conditions of the fermentation production include: 42.5 ℃, pH5.5, stirring rotation speed 300rpm, and charging concentration of 20g/L.
In one aspect, the invention provides a gene fragment encoding said β -glucuronidase AcGUS.
Based on the gene fragment, the beta-glucuronidase AcGUS mutant with remarkably improved catalytic efficiency is obtained.
A construction method of beta-glucuronidase AcGUS mutant carries out rational cutting operation on 1-39 amino acids at the nitrogen end of beta-glucuronidase AcGUS, and the preferable cutting number of the amino acids of a truncated AcGUS3 is 30.
Compared with AcGUS, the beta-glucuronidase AcGUS3 obtained by heterologous expression of the truncated AcGUS and pichia pastoris has the advantages that the enzyme activity is improved by 7 times in the aspect of preparing glycyrrhetinic acid, and meanwhile, the accumulation of the intermediate product mono glucuronic acid glycyrrhetinic acid is reduced by 3.74 times.
And the truncated body AcGUS is characterized in that the 461 th glycine of AcGUS is mutated into cysteine, the 462 th glutamine is mutated into histidine, and the 575 th isoleucine is mutated into lysine, so that a novel mutant AcGUS M1 is obtained.
The preferred AcGUS M1 mutant is characterized by an 11.02-fold and 6.10-fold increase in activity in hydrolyzing substrates, polyglucuronate glycyrrhetinate and glycyrrhizic acid, respectively.
A method for constructing a strain with improved activity of hydrolyzing 18 alpha-glycyrrhizic acid or 18 beta-glycyrrhizic acid comprises the step of introducing plasmids of pGAPZalpha A-AcGUS M1 mutants into a Pichia pastoris AtGUS host to obtain a Pichia pastoris strain dG-GA1.
The pichia pastoris strain dG-GA1 is characterized in that when glycyrrhizic acid is used as a substrate, accumulation of intermediate product polyglucuronate glycyrrhetinic acid can be effectively inhibited, and the system ratio in the fermentation process is always lower than 4%.
The final concentration of the prepared 18 alpha-glycyrrhetinic acid reaches 41.09g/L, and the conversion rate is 96.57%;
The final concentration of the prepared 18 beta-glycyrrhetinic acid reaches 48.73g/L, and the conversion rate is 97.26 percent.
The invention provides a coding gene and application of a beta-glucuronidase mutant, and provides a strain construction method for efficiently preparing glycyrrhetinic acid, wherein the constructed engineering bacteria are used for industrial scale production of the glycyrrhetinic acid. The β -glucuronidase AcGUS and its mutant provided by the present invention are different from the existing β -glucuronidase which is preferred to hydrolyze the substrate GL outside glycoside, the former is preferred to hydrolyze the substrate GAMG (i.e., GL inside glycoside), and they exhibit extremely excellent activity. The AcGUS mutant is applied to the construction method of the beta-glucuronidase combined engineering bacteria provided by the invention, and the verification shows that the obtained combined engineering bacteria have excellent industrial application potential when hydrolyzing 18 alpha-GL and 18 beta-GL in a 5L fermentation tank and a 1000L fermentation tank respectively, and have the advantages of high catalytic efficiency, strong specificity, short process period, environmental protection and the like. Thus, the invention provides a simple and efficient method for preparing 18 alpha-GA and 18 beta-GA on an industrial scale for the field.
Drawings
FIG. 1 is a schematic diagram of two-step conversion of 18α -glycyrrhizic acid and 18β -glycyrrhizic acid by β -glucuronidase, wherein A:18 a-GL preparation of 18 a-GA; b:18 beta-GL preparation 18 beta-GA.
FIG. 2 is a diagram showing agarose gel electrophoresis verification of AcGUS gene amplification in experimental example 2 of the present invention; among them, lanes 1 and 2 are AcGUS PCR product bands, and lane M is DNA MARKER.
FIG. 3 is a liquid phase assay of GAMG accumulation during 18 beta-glycyrrhizic acid conversion according to Experimental example 3 of the present invention.
FIG. 4 shows eukaryotic expression of 5 β -glucuronidases from Pichia pastoris AcGUS, acGUS1, acGUS, acGUS3 and AcGUS4 according to Experimental example 4: the relative enzymatic activities of AcGUS, acGUS1, acGUS, acGUS3 and AcGUS respectively hydrolyze GL to produce a bar graph of GA and GAMG, the abscissa marks the above 5 β -glucuronidases respectively.
FIG. 5 shows the wild type AcGUS (WT) and prokaryotic AcGUS3 mutants of Experimental example 4: acGUS 3A comparison chart of enzyme activities of G461C、AcGUS3Q462H、AcGUS3I575K、AcGUS3G461C/I575K、AcGUS3G461C/Q462H/I575K. Wherein, the test substrates of the upper graph and the lower graph are 18 beta-GL and 18 beta-GAMG respectively; the abscissa marks correspond to the wild type and each mutant, respectively, described above.
FIG. 6 is a comparative graph of the relative enzyme activities of the beta-glucuronidase expressed by different Pichia pastoris engineering bacteria in experimental example 7 of the present invention, with the abscissa marks listed below: atGUS is beta-glucuronidase AtGUS of known strain Aspergillus terreus (Aspergillus terreus) strain Li-20 described in Chinese patent application CN106047839A, auGUS is beta-glucuronidase AuGUS of known strain Aspergillus glaucus (Aspergillus ustus) strain Li-62 described in Chinese patent application CN106047839A, atGUS-mix is beta-glucuronidase AtGUS-mix described in patent CN109628427B, acGUS is beta-glucuronidase AcGUS described in the present invention, acGUS3M1 is beta-glucuronidase AcGUS M1 described in the present invention, dG-GA1 is beta-glucuronidase AcGUS M1 expressed simultaneously by Pichia pastoris strain dG-GA1 described in the present invention, and beta-glucuronidase AtGUS derived from Aspergillus terreus (Aspergillus terreus) strain Li-20.
FIG. 7 shows the production of GA by amplifying the dG-GA1 combination engineering bacterium in examples 8 and 9 of the present invention. Wherein, A is a graph showing the change of the concentration of the substrate and the product for preparing 18 alpha-GA by converting 18 alpha-GL in a 5L fermenter; panel B shows the substrate and product concentration profiles for 18. Beta. -GA production in 1000L fermentors 18. Beta. -GL.
Detailed Description
The present invention will be further illustrated by the following specific examples and experimental examples. It is to be understood that the examples are given by way of illustration and description only and are not to be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples and experimental examples are conventional methods, and the reagents, materials, etc. used are commercially available.
Sources and documentations of biological materials
1. The A.pyromellea CLH-22 strain mentioned in experimental example 1 is a known strain reported in Chinese patent application CN 115786133A.
2. The E.coli Top10 competence used in experimental examples 2 and 5 of the present invention is commercially available.
3. Pichia pastoris GS115 used in Experimental example 3 of the present invention is commercially available.
4. Competent e.coli BL21 (DE 3) and JM109 (DE 3) used in experimental example 4 of the present invention are commercially available.
5. The Pichia pastoris AtGUS used in experimental example 6 of the present invention is constructed in the laboratory of the present invention, and the engineering bacterium Pichia pastoris AtGUS can be obtained by cloning and transforming beta-glucuronidase AtGUS derived from the strain Li-20 of Aspergillus terreus (Aspergillus terreus) from the strain Li-20 of Aspergillus terreus (Aspergillus terreus) and into Pichia pastoris according to the description of the "reagent and consumable" item (4) of the present invention by a person skilled in the art, without technical obstacle.
Reagent and consumable
1. The following materials were used in the examples:
(1) Coli (e.coli): top10, BL21 (DE 3), JM109 (DE 3).
(2) Pichia pastoris (Pichia pastoris): GS115.
(3) AcGUS is derived from the A.pyrogallol (Aspergillus calidoustus) strain CLH-22, a known strain reported in Chinese patent application CN 115786133A.
(4) AtGUS is derived from Aspergillus terreus (Aspergillus terreus) strain Li-20, which is a known strain reported in Chinese patent application CN106047839A, genBank number is JF894133.1.
(5) AuGUS is derived from Aspergillus niger (Aspergillus ustus) strain Li-62, a known strain reported in China patent application CN106047839A, sequence ID No. JN247805.1.
(6) Pichia AtGUS and pichia AuGUS, stored in applicant's laboratory.
(7) Atgusmix another patent of the invention recorded in the inventor subject group is an engineering bacterium GA108/PGAPZ alpha A-Atgusmix for industrialized production of glycyrrhetinic acid, and the strain preservation number is CGMCC No.16731.
(8) The expression vectors of (1) and (2) are pGAPZαA, which are commercially available.
2. Culture medium
LB medium (per liter): 10g of tryptone, 5g of sodium chloride and 5g of yeast extract are weighed, deionized water is added to a volume of 1L, sterilization is carried out for 15min at 121 ℃, and 20g of agar powder is added to LB solid medium. YPD medium (per liter): 20g of tryptone, 20g of dextrose monohydrate and 10g of yeast extract, adding deionized water to a volume of 1L, sterilizing at 115 ℃ for 15min, and adding 20g of agar powder into the YPD solid culture medium.
(2) Solution preparation for construction of pichia pastoris engineering bacteria
1M sorbitol solution: 182.17g of sorbitol is weighed, 900mL of deionized water is added, the volume is fixed to 1L after the solution is fully stirred and dissolved, and the solution is sterilized at 115 ℃ for 20 min. Yeast lysate: 0.372g EDTA disodium salt, 2g NaOH was weighed separately, 5mL Triton X-100 was added, deionized water was added for dissolution and the volume was set to 1L. 200mL of deionized water is measured as sterile water, the sterile water is sealed in an conical flask by gauze, sterilized at 121 ℃ for 15min and stored in a refrigerator at 4 ℃.
(3) Validation solution and reaction buffer
4G/L GL-YPD validation solution was prepared by weighing 4g of glycyrrhizic acid, 20g of tryptone, 20g of glucose monohydrate, and 10g of yeast extract, respectively. 50mM HAc-NaAc reaction buffer (pH 5.5) of 5g/L GL: weighing 5g of monoammonium glycyrrhizinate and 4.1g of anhydrous sodium acetate, adding 950mL of purified water, adjusting pH to 5.5 with glacial acetic acid, adding deionized water for dissolution, fixing volume to 1L, sterilizing at 115 ℃ for 15min, and preserving at 4 ℃.5g/L GAMG in 50mM HAc-NaAc reaction buffer (pH 5.5): 5g of 18 beta-GAMG and 4.1g of anhydrous sodium acetate are weighed, 950mL of purified water is added, the pH is regulated to 5.5 by glacial acetic acid, deionized water is added for dissolution and volume fixation to 1L, sterilization is carried out for 15min at 115 ℃, and the mixture is preserved at 4 ℃. 50mM HAc-NaAc reaction buffer (pH 5.5) at 10g/L GL: 1g of monoammonium glycyrrhizinate and 0.41g of anhydrous sodium acetate are weighed, 95mL of purified water is added, the pH is regulated to 5.5 by glacial acetic acid, deionized water is added for dissolution and volume setting to 100mL, and after heating and dissolution at 60-80 ℃, stirring is carried out for 5min, and cooling to room temperature is carried out for preparation.
Based on the beta-glucuronidase AcGUS and the mutant thereof provided by the invention, when the combined engineering bacteria are constructed for GA production by a combination method, the combination is carried out according to different beta-glucuronidases, or the conventional adjustment and selection of the fermentation scale fall into the protection scope of the invention.
Group 1 example beta-glucuronidase of the invention
The present set of examples provides a beta-glucuronidase. The present set of embodiments all share the following common features: the one beta-glucuronidase is selected from: beta-glucuronidase AcGUS, and/or beta-glucuronidase AcGUS1, and/or beta-glucuronidase AcGUS2, and/or beta-glucuronidase AcGUS3, and/or beta-glucuronidase AcGUS3 mutants;
The beta-glucuronidase AcGUS is an enzyme obtained by deleting 1 st amino acid, 7 th alanine, 66 th arginine, 256 th alanine, 270 th threonine, 332 th valine, 363 rd aspartic acid, 506 th methionine and valine of an amino acid sequence with GenBank accession number AEK 69352.1;
The beta-glucuronidase AcGUS, acGUS2 and AcGUS are enzymes obtained by truncating 1-39 amino acids at the nitrogen end of the beta-glucuronidase AcGUS;
The beta-glucuronidase AcGUS mutant is selected from the group consisting of: a mutant obtained by mutating the 461 st glycine of AcGUS to cysteine, a mutant obtained by mutating the 462 st glutamine of AcGUS to histidine, a mutant obtained by mutating the 575 nd isoleucine of AcGUS to lysine, or a mutant obtained by mutating the 461 st glycine of AcGUS to cysteine, the 462 st glutamine to histidine and the 575 nd isoleucine to lysine.
In a specific embodiment, the amino acid sequence of the beta-glucuronidase AcGUS is shown as SEQ ID NO. 1;
preferably, the gene sequence of the beta-glucuronidase AcGUS is shown as SEQ ID NO. 2;
preferably, the beta-glucuronidase AcGUS is an enzyme obtained by truncating the nitrogen end of the beta-glucuronidase AcGUS by 10 amino acids;
Preferably, the beta-glucuronidase AcGUS is an enzyme obtained by truncating 20 amino acids at the nitrogen end of the beta-glucuronidase AcGUS;
preferably, the beta-glucuronidase AcGUS is an enzyme obtained by truncating the nitrogen end of the beta-glucuronidase AcGUS by 30 amino acids;
Preferably, the mutants obtained by mutating the 461 st glycine of AcGUS to cysteine, the 462 st glutamine to histidine and the 575 nd isoleucine to lysine are beta-glucuronidase AcGUS M1.
Any cloning, amplification, enrichment, expression, ligation, transformation, synthesis, culture, propagation, fermentation, enrichment, production, preparation, use, inoculation, amplification, transformation, modification, alteration, marketing, offer to sell such as β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS1, and/or β -glucuronidase AcGUS2, and/or β -glucuronidase AcGUS3, and/or β -glucuronidase AcGUS3 mutant's behavior, and/or the act of transcriptionally translating such as β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS1, and/or β -glucuronidase AcGUS2, and/or β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS mutant's gene sequence to other enzymes, and/or the act of producing a mutant amino acid sequence comprising the amino acid sequence of such as β -glucuronidase AcGUS, and/or β -glucuronidase 3825, and/or β -glucuronidase 3992, and/or β -glucuronidase 963 mutant's nucleotide sequence, including the amino acid sequence of the mutant or the amino acid sequence of which is not limited to those of β -glucuronidase AcGUS, and/or β -glucuronidase 3525, and/or β -glucuronidase 5835: the actions of the active ingredients with efficacy such as glycyrrhetinic acid and/or the actions of producing medicines by using the amino acid sequences obtained by transcription and translation of the gene sequences of beta-glucuronidase AcGUS, beta-glucuronidase AcGUS1, beta-glucuronidase AcGUS, beta-glucuronidase AcGUS and/or beta-glucuronidase AcGUS mutants fall into the protection scope of the invention.
Any cloning, amplification, enrichment, expression, ligation, transformation, synthesis, culture, propagation, fermentation, enrichment, production, preparation, use, inoculation, amplification, transformation, modification, engineering, marketing, offer to sell the behavior of β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS1, and/or β -glucuronidase AcGUS2, and/or β -glucuronidase AcGUS3, and/or the behavior of the amino acid sequence of the β -glucuronidase AcGUS3 mutant, and/or the production of the amino acid sequence of the mutant such as β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS1, and/or β -glucuronidase AcGUS2, and/or β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS mutant with other enzymes, and/or the production of the amino acid sequence of the mutant such as β -glucuronidase AcGUS, and/or β -glucuronidase 3825, and/or β -glucuronidase 563, and/or β -glucuronidase 5835, but not limited to the amino acid sequence of the mutant such as β -glucuronidase AcGUS, and/or β -glucuronidase 563, and/or β -glucuronidase 5835, but is not limited to the amino acid sequence of the mutant of β -glucuronidase 563: the actions of the active ingredients with efficacy such as glycyrrhetinic acid and/or the actions of producing medicines by using amino acid sequences such as beta-glucuronidase AcGUS, and/or beta-glucuronidase AcGUS1, and/or beta-glucuronidase AcGUS2, and/or beta-glucuronidase AcGUS3, and/or beta-glucuronidase AcGUS3 mutants fall within the protection scope of the invention.
Such other enzymes include, but are not limited to: alpha-mannosidase, arabinosidase, beta-xylosidase, chitotriosidase, thioglycosidase, alpha-glucosidase, beta-galactosidase, beta-fructosidase, triose phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, beta-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, dehydratase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.
According to the actual production needs, the person skilled in the art can combine the conventional technical means or the basic common sense of the production process in the molecular biology or genetic engineering field (for example, the manual of practical molecular biology operation, the manual of molecular biology experiment operation, the manual of molecular cloning experiment, the manual of fine programming molecular biology experiment, etc.), reversely compile the amino acid sequence of the mutant of the enzyme AcGUS and/or the enzyme AcGUS1 and/or the enzyme AcGUS2 and/or the enzyme AcGUS3 and/or the enzyme AcGUS to obtain the gene sequence, design a specific amplification primer to obtain the gene sequence, connect the gene sequence with an expression vector to obtain the recombinant expression vector of the enzyme AcGUS and/or the enzyme 3825 and/or the enzyme AcGUS, and/or the enzyme AcGUS and/or the enzyme 563, and/or the enzyme AcGUS, and the recombinant expression vector of the enzyme can obtain the recombinant expression vector of the enzyme 3274, the recombinant expression vector of the enzyme 3274 and the enzyme 3278 in the enzyme or the mutant of the enzyme can be further processed by culturing the recombinant expression vector of the enzyme or the enzyme 3274 and the recombinant expression vector of the enzyme 3274 and the enzyme 3278 And/or β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS2, and/or β -glucuronidase AcGUS, and/or β -glucuronidase AcGUS3 mutants, which are clear to a person skilled in the art of technical hurdles and are easy to do.
Group 2 examples, recombinant expression vectors of the invention
The present set of examples provides a recombinant expression vector. The present set of embodiments all share the following common features: an expression vector comprising the gene sequence of β -glucuronidase as set forth in any one of examples in group 1.
In further embodiments, the expression vector is selected from pET28a, pGAPZ α A, pPIC9K, pPICZ α;
Preferably, the recombinant expression vector is selected from recombinant expression vector pGAPZαA-AcGUS, and/or recombinant expression vector pGAPZαA-AcGUS3, and/or recombinant expression vector pGAPZαA-AcGUS M1.
Any cloning, amplifying, enriching, expressing, ligating, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, offering to sell the recombinant expression vector, and/or combining the beta-glucuronidase expressed by the recombinant expression vector with other enzymes, and/or producing preparations of beta-glucuronidase expressed by the recombinant expression vector, including but not limited to: the actions of active ingredients such as glycyrrhetinic acid and/or the actions of producing medicines by using beta-glucuronidase expressed by the recombinant expression vector fall into the protection scope of the invention.
Such other enzymes include, but are not limited to: alpha-mannosidase, arabinosidase, beta-xylosidase, chitotriosidase, thioglycosidase, alpha-glucosidase, beta-galactosidase, beta-fructosidase, triose phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, beta-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, dehydratase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.
The person skilled in the art can use the recombinant expression vector to express the beta-glucuronidase of the present invention or transform the recombinant expression vector into competent cells to obtain a transformant (e.g., engineering bacterium) capable of expressing the beta-glucuronidase of the present invention according to actual production needs in combination with conventional technical means or basic common sense of production processes in the fields of molecular biology or genetic engineering (e.g., guidelines for practical molecular biology, guidelines for experimental techniques of molecular cloning, guidelines for experimental techniques of fine programming of molecular biology, etc.), and propagate and culture under conditions suitable for the growth of the transformant to efficiently produce the beta-glucuronidase of the present invention, which is not a technical obstacle for the person skilled in the art and can be easily achieved.
Group 3 example, engineering bacteria of the present invention
The embodiment of the group provides engineering bacteria. The present set of embodiments all share the following common features: the engineering bacteria are selected from Pichia pastoris strain AcGUS, pichia pastoris strain AcGUS, pichia pastoris strain AcGUS M1 and Pichia pastoris strain dG-GA1;
The pichia pastoris strain AcGUS can express the beta-glucuronidase AcGUS according to any one of the embodiments of group 2;
The pichia pastoris strain AcGUS can express the beta-glucuronidase AcGUS3 according to any one of the embodiments of group 2;
the pichia pastoris strain AcGUS M1 can express the beta-glucuronidase AcGUS M1 of any one of the embodiments of group 2;
the pichia pastoris strain dG-GA1 can simultaneously express the beta-glucuronidase AcGUS M1 and the aspergillus terreus (Aspergillus terreus) strain Li-20-derived beta-glucuronidase AtGUS of any one of the embodiment of the group 2.
In some embodiments, the pichia pastoris strain AcGUS contains the recombinant expression vector pgapzαA-AcGUS of any one of the embodiments of group 2;
preferably, the pichia pastoris strain AcGUS contains the recombinant expression vector pgapzαA-AcGUS3 according to any one of the embodiments of group 2;
Preferably, the pichia pastoris strain AcGUS M1 contains the recombinant expression vector pgapzαa-AcGUS M1 according to any one of the embodiments of group 2;
preferably, the pichia pastoris strain dG-GA1 comprises the recombinant expression vector pGAPZ a-AcGUS M1 according to any one of the embodiments in group 2, the recombinant expression vector pGAPZ A-AcGUS M1-NrsR modified and the recombinant expression vector pGAPZ A-AtGUS linked to the β -glucuronidase AtGUS gene sequence derived from aspergillus terreus (Aspergillus terreus) strain Li-20;
Preferably, the modification means: the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS M1 was replaced by NrsR resistance.
Any cloning, amplification, enrichment, expression, ligation, transformation, synthesis, culture, propagation, fermentation, enrichment, production, preparation, use, inoculation, amplification, transformation, modification, transformation, marketing, promise for marketing such as the behavior of the engineered bacterium, and/or for combining the beta-glucuronidase expressed by the engineered bacterium with other enzymes, and/or for producing the beta-glucuronidase expressed by the transformant (e.g., engineered bacterium) includes, but is not limited to: the behavior of active ingredients with efficacy such as glycyrrhetinic acid and/or the behavior of producing medicines by using beta-glucuronidase expressed by the engineering bacteria fall into the protection scope of the invention.
Such other enzymes include, but are not limited to: alpha-mannosidase, arabinosidase, beta-xylosidase, chitotriosidase, thioglycosidase, alpha-glucosidase, beta-galactosidase, beta-fructosidase, triose phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, beta-lactamase, superoxide dismutase, glycogen phosphorylase, hexokinase, lactate dehydrogenase, dehydratase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, amylase, lipase, phosphatase.
The person skilled in the art can culture, propagate, ferment, enrich, produce, prepare, use, inoculate the engineering bacteria to express, secrete, produce, obtain beta-glucuronidase according to the actual production needs, in combination with the conventional technical means or the basic common sense of the production process in the field of molecular biology or genetic engineering (for example, the manual of practical molecular biology operation, the manual of molecular biology experimental technique experiment, the manual of molecular cloning experiment, the manual of fine programming molecular biology experiment, etc.), which is not a technical obstacle for the person skilled in the art and can easily do.
Group 4 examples, leavening agents of the present invention
The present set of examples provides a starter. The present set of embodiments all share the following common features: the starter comprises a starter active ingredient comprising a beta-glucuronidase according to any one of the embodiments of group1, and/or a recombinant expression vector according to any one of the embodiments of group2, and/or an engineering bacterium according to any one of the embodiments of group 3.
In a further embodiment, the one starter culture further comprises: auxiliary materials.
In a more specific embodiment, the adjuvant is selected from: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.
According to the invention, the technical means (for example, encyclopedia of preparation technology, technology of pharmaceutical preparation, technical research and application of microbial agents and the like) in the field of preparation are combined for different requirements in practical production and application, and the pharmaceutically acceptable auxiliary materials can be selected and formulated by the person skilled in the art, so that the engineering bacteria of the invention can be prepared into different dosage forms, such as powder, tablets, suppositories, gels, sprays, granules and the like.
In a specific embodiment, the dosage form of the fermenting agent is selected from the group consisting of: one or more of powder, tablet, liquid and capsule.
Example 5 group of the method for Mass production of Glycyrrhetinic acid according to the present invention
The present set of embodiments provides a method for mass production of glycyrrhetinic acid. The present set of embodiments all share the following common features: fermenting the substrate with a beta-glucuronidase according to any one of the embodiments of group 1, and/or a recombinant expression vector according to any one of the embodiments of group 2, and/or an engineered bacterium according to any one of the embodiments of group 3.
In a specific embodiment, the substrate is selected from the group consisting of: 18 alpha-GL or 18 beta-GL;
Preferably, the glycyrrhetinic acid is selected from the group consisting of: 18 a-GA or 18 β -GA;
Preferably, the conditions of the fermentation production include: 42.5 ℃, pH5.5, stirring rotation speed 300rpm, and charging concentration of 20g/L.
Experimental example 1 amplification of beta-glucuronidase AcGUS Gene
Gene amplification primers AcGUS-F and AcGUS-R were designed specifically based on the β -glucuronidase AcGUS sequence. Then, a cDNA library of the Aspergillus flavus CLH-22 strain is constructed, wherein the Aspergillus flavus CLH-22 strain is subjected to RNA extraction by using an RNA rapid extraction kit, and then the extracted RNA is subjected to reverse transcription by using an RNA reverse transcription reagent PRIMESCRIPT ™ RT Master Mix, and reverse transcription reaction conditions are as follows: the reaction is carried out for 15min at 37 ℃, the enzyme is inactivated for 5s at 85 ℃ and is preserved at 4 ℃, and then the cDNA library of the aspergillus flavus CLH-22 is obtained. Further, a cDNA library of the A.pyrogallol CLH-22 strain is used as a PCR template, and PCR amplification is carried out by a Nuo-wei Phanta system, and the PCR system is as follows: 2X Phanta Max Buffer. Mu.L of each of AcGUS-F and AcGUS-R primers, 2. Mu.L of dNTP Mix, 1. Mu.L of cDNA library, 1. Mu.L of Phanta Max Super-FIDELITY DNA Polymerase, and 50. Mu.L of sterile water were used. PCR reaction procedure: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at Tm-5℃for 15s, extension at 72℃for 2kb/min, cycling for 35 times, extension at 72℃for 5min, and preservation at 16℃to obtain AcGUS PCR products.
The following are the PCR primer sequences used in the experimental examples:
TABLE 1 PCR amplification primer sequence Listing
Primer name Sequence (5-3')
AcGUS-F CGGGGTACCATGAAGCTCCTACAAGGACTCTCGCTACTC（SEQ ID NO.3）
AcGUS-R AAGGAAAAAAGCGGCCGCTCACTCACTGCCCAACTGCGTCCAC（SEQ ID NO.4）
pGAP-F gtccctatttcaatcaattgaa（SEQ ID NO.5）
3AOX1 GCAAATGGCATTCTGACATCC（SEQ ID NO.6）
Construction of cloning vector of Experimental example 2, acGUS and screening of Positive clones
(1) Restriction and purification of Gene fragments
The PCR product described in experimental example 1 was subjected to 1% agarose gel electrophoresis, and the result of the electrophoresis is shown in FIG. 2, and the PCR fragment length is consistent with AcGUS, which indicates that the target gene fragment was obtained initially. Further, kpnI and NotI were digested simultaneously, and reacted at a constant temperature of 37℃for 2 hours, followed by purification of the digested fragments using GeneJET gel recovery kit. Carrying out T4 enzyme-linked reaction on the purified AcGUS gene fragment and a cloning vector pGAPZ alpha A, and carrying out an enzyme-linked reaction system: 10X T4 DNA Ligase Buffer 1 mu L, pGAPZ. Alpha. A plasmid 1. Mu.L, the gene fragment of interest (about 500 ng), T4 DNA LIGASE. Mu.L, sterile water was filled to 10. Mu.L. And (5) carrying out constant-temperature connection for 2 hours at 22 ℃ after instantaneous centrifugation, thus obtaining a connection product.
(2) Large intestine transformation
Mu.L of ligation product was added to E.coli Top10 competence and ice-bathed for 30min. Then, the mixture was subjected to a water bath heat shock at 42℃for 90 seconds, and after another 2 minutes of ice bath, 500. Mu.L of LB liquid medium was added, and the mixture was placed on a shaking table at 200rpm at 37℃for incubation for 1 hour. After incubation, the bacterial solution was centrifuged at 5000rpm for 3min, 500. Mu.L of supernatant was removed, and the solution was spread evenly on LB solid plates containing 50mg/L bleomycin, and incubated overnight at 37 ℃.
(3) Screening of E.coli Positive recombinants
Recombinant was identified using colony PCR and plasmid sequencing. E. coli colony PCR system: 2 XM 5 TAQ HIFI PCR Mix 7.5. Mu.L, pGAP-F and 3AOX1 primers each 1. Mu.L, template LB plate single colony, and 15. Mu.L with sterile water. PCR reaction procedure: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, circulation for 30 times, extension at 72 ℃ for 7min, and preservation at 16 ℃. The correct recombinant is verified by colony PCR and sent to DNA sequencing, and the construction success of pGAPZalpha A-AcGUS plasmid is determined, and the AcGUS gene sequence is shown as SEQ ID NO. 2.
Experimental example 3 construction of Pichia pastoris AcGUS engineering bacteria and enzyme Activity verification
(1) Construction of Pichia pastoris AcGUS Strain
Carrying out linearization treatment on AcGUS by adopting endonuclease BlnI, wherein a linearization system is as follows: 10X QuickCut Buffer 2. Mu.L, blnI 1. Mu.L, total plasmid 1. Mu.g, and 20. Mu.L was supplemented with sterile water. After instantaneous centrifugation, the reaction is carried out for 1h at a constant temperature of 37 ℃. The linearized plasmid was purified using GeneJET gel recovery kit. The pGAPZalpha A-AcGUS linearization plasmid is transferred into Pichia pastoris GS115 by adopting the common electrotransfer method of Pichia pastoris, and is evenly coated on a YPD solid plate containing 100mg/L bleomycin, and is cultured for 2-4 days at 30 ℃.
Colony PCR was used for identification of Pichia transformants. 6-12 single colonies were selected from YPD plates, 50. Mu.L of yeast lysate was added, boiled in boiling water for 30min, and 150. Mu.L of sterile water was added to PCR tubes to obtain lysate templates. Colony PCR system: 2 XM 5 TAQ HIFI PCR Mix 7.5. Mu.L, pGAP-F and 3AOX1 primer each 1. Mu.L, lysate template 1. Mu.L, and 15. Mu.L was supplemented with sterile water. PCR reaction procedure: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, circulation for 30 times, extension at 72 ℃ for 10min, and preservation at 16 ℃. The PCR product is verified by 1% agarose gel electrophoresis, whether the length of the strip meets the requirement is judged, and the success of the construction of the transformant is determined.
(2) Enzyme activity verification of AcGUS transformants
And (3) verifying the enzyme activity of eukaryotic expression of pichia pastoris AcGUS. Firstly, picking AcGUS transformants which are successfully verified into 4g/L glycyrrhizic acid-YPD verification solution, culturing for 2 days at a temperature of 30 ℃ under a shaking table, and detecting whether AcGUS strain has GL hydrolysis activity by adopting high performance liquid chromatography. The liquid phase detection method comprises the following steps: the mobile phase is methanol/glacial acetic acid aqueous solution (6%o) =84:16, the chromatographic column is Kromasil 100-3.5-C18, the sample injection amount is 5 mu L, the flow rate is 0.8mL/min, and the detection wavelength is 254nm. FIG. 3 shows the liquid phase detection during substrate conversion. The results showed that AcGUS exhibited excellent GL hydrolysis activity while the accumulation of the intermediate GAMG was extremely low, indicating that AcGUS is a GUS enzyme with a higher preference for GAMG hydrolysis. Further, the results of the verification by using GL and GAMG buffer substrates (pH 5.5) respectively revealed that the rate of AcGUS metabolic substrates GAMG was about 2 times that of GL, which was consistent with the phenotype of the wild strain Aspergillus calidoustus CLH-22 (preservation number: CGMCC No. 40213) in the verification. Shows AcGUS is the correct target gene and can construct the eukaryotic heterologous expression system of Pichia pastoris.
Construction and verification of mutants of Experimental examples 4 and AcGUS
In order to improve the production efficiency of GA as much as possible, the inhibition effect caused by the accumulation of an intermediate GAMG is reduced, and a feasible method is provided for the industrial preparation of GA. In the experimental example, acGUS is designed into more efficient beta-glucuronidase by combining enzyme engineering to screen mutant enzyme with improved activity.
(1) Construction of AcGUS prokaryotic expression of E.coli
First, acGUS was ligated with pET-28a vector by the usual Gibson assembly method to obtain recombinant vector pET-28a-AcGUS. Then, large intestine transformation was performed (the same procedure as in Experimental example 2) and recombinant plasmids were introduced into competent E.coli BL21 (DE 3) and JM109 (DE 3), respectively, and positive clone selection was performed on LB plates containing 50mg/L kanamycin resistance, and correct recombinant DNA sequencing was confirmed by colony PCR, and the recombinant E.coli was successfully constructed.
(2) AcGUS nitrogen end analysis and truncate construction and verification
The structure simulation of AcGUS is carried out by adopting AlphaFold < 2 >, and the structure analysis is carried out on the nitrogen end flexible Loop, so that the Loop is considered to have large flexibility and poor structural stability, and therefore, whether the nitrogen end of AcGUS is truncated or not can improve the enzyme activity of heterologous expression is examined. The recombinant nitrogen end truncates in the heterologous expression of escherichia coli and pichia pastoris respectively truncate 10, 20, 30 and 39 amino acids at the nitrogen end of the gene by taking pET-28a-AcGUS and pGAPZ alpha A-AcGUS as templates, and correspondingly obtain AcGUS1, acGUS2, acGUS3 and AcGUS4 of prokaryotic or eukaryotic expression.
Then, comparative analysis verification of GL transformation and accumulation of intermediate GAMG was performed on eukaryotic expression of pichia pastoris AcGUS, acGUS1, acGUS2, acGUS and AcGUS. Firstly, five transformants which are successfully verified are respectively picked from a scribing plate into a test tube containing 5mL of YPD liquid, transferred into a 250mL shaking bottle containing 4g/L glycyrrhizic acid-YPD with equal quantity according to OD 600 = 0.1, continuously cultured for 4 days at a temperature of 30 ℃ under a shaking table, sampled once at intervals of 12 hours, and detected by adopting high performance liquid chromatography. As shown in FIG. 4, acGUS has 7 times higher enzyme activity than AcGUS in the aspect of GA production, and has better GL hydrolysis activity. In terms of accumulation of intermediate GAMG, the amount of intermediate GAMG accumulation of AcGUS is small, 3.74 times lower than AcGUS. Further, analysis of pure enzymes by measuring Mie kinetics with GL and GAMG as substrates, respectively, showed that AcGUS catalyzes GL with a k cat/Km of 2.94 times AcGUS and GAMG with a k cat/Km of 9.06 times AcGUS, which results indicate a significant improvement in AcGUS affinity and catalytic efficiency.
(3) AcGUS mutant construction and validation
By analyzing the three-dimensional structure, sequence alignment analysis, molecular docking and other enzyme engineering methods of AcGUS, the G461, Q462 and I575 with the distance within the pocket 5A of AcGUS are selected as saturation mutation sites (Table 2). And subjecting the sites having a positive effect to combinatorial mutation. Here, based on the AcGUS truncations, the mutated PCR product was obtained by plasmid loop P, loop P system, using pET-28a-AcGUS plasmid as template: 2X PHANTA FLASH MASTER Mix 25. Mu.L, 1. Mu.L each of the upstream and downstream primers, 1-2. Mu.L of plasmid template, and 50. Mu.L of sterile water. PCR reaction procedure: pre-denaturation at 98℃for 30s, denaturation at 98℃for 10s, annealing at Tm-5℃for 5s, extension at 72℃for 50s, cycling for 35 times, extension at 72℃for 1min, and storage at 16 ℃. Then, the PCR product was digested with Dpn I enzyme for 2 hours, followed by large intestine transformation to introduce competent JM109 (DE 3). Subsequently, verification was performed by colony PCR, colony PCR system: 2 XM 5 TAQ HIFI PCR Mix 7.5. Mu.L, 1. Mu.L each of T7 and T7 term universal primers, template LB plate single colony, and 15. Mu.L with sterile water. PCR reaction procedure: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, circulation for 30 times, extension at 72 ℃ for 7min, and preservation at 16 ℃. And (3) verifying correct recombinant DNA sequencing by colony PCR, and determining that the mutant plasmid is constructed successfully.
Table 2. AcGUS3 saturated mutation primer sequence Listing
Primer name Sequence (5-3')
AcGUS3-G461-F TAATGTCNNKCAGGCGACGTATGAGACGGATAAGATTTCGGATTTGTTTG（SEQ ID NO.7）
AcGUS3-G461-R TCGCCTGMNNGACATTAACAAACGCAACCGGCCTCGCCGCCGGATCAG（SEQ ID NO.8）
AcGUS3-Q462-F TGTCGGGNNKGCGACGTATGAGACGGATAAGATTTCGGATT（SEQ ID NO.9）
AcGUS3-Q462-R TACGTCGCMNNCCCGACATTAACAAACGCAACCGGCCTC（SEQ ID NO.10）
AcGUS3-Q462N-F TGTCTGTNNKGCGACGTATGAGACGGATAAGATTTCGGATT（SEQ ID NO.11）
AcGUS3-Q462N-R TACGTCGCMNNACAGACATTAACAAACGCAACCGGCCTC（SEQ ID NO.12）
AcGUS3-I575-F GGGGATTNNKCGTGTTGATGGGAATAAGAAGGGGGTGTT（SEQ ID NO.13）
AcGUS3-I575-R TCAACACGMNNAATCCCCATCGAGGTCTGAAAGTCTG（SEQ ID NO.14）
To rapidly compare the activity of the mutants, a validation comparison was performed using crude enzyme solution. First, the constructed recombinant E.coli was inoculated into 5mL of LB medium containing 50mg/L kanamycin resistance, and cultured at 37℃for 12 hours. Then, the medium was transferred to 40mL of LB medium at a transfer rate of 1%, and when OD 600 reached 0.6-0.8, IPTG was added for induction. After further incubation at 16℃for 18h, 2mL of cells were collected by centrifugation at 12000rpm (4 ℃) and washed twice with 50mM HAc-NaAc buffer (pH 5.5). Next, 1mL of 50mM HAc-NaAc buffer (pH 5.5) and an appropriate amount of crushing beads were added, and the crushing treatment was performed by a homogenizer. 200. Mu.L of the crude enzyme supernatant and 800. Mu.L of 50mM HAc-NaAc buffer (pH 5.5) containing 5g/L GL or GAMG were mixed, reacted at 42.5℃for 5 hours and 0.5 hours, respectively, and then sampled and analyzed by High Performance Liquid Chromatography (HPLC).
The results in fig. 5 show that G461, Q462, and I575 are mutation sites with positive effect, where single point mutations AcGUS, G461C、AcGUS3Q462H, and AcGUS, I575K increase the hydrolytic activity of the GAMG substrate by 106.71%, 108.72%, and 126.41%, respectively, compared to unmutated AcGUS3 (WT), indicating that a pocket engineering based mutation strategy achieves an effective mutation site.
Therefore, by further taking AcGUS G461C mutant as an original strain and carrying out iterative saturation mutation with I575, constructing a saturation mutation library of G461C and I575 by taking AcGUS3-I575-F (SEQ ID NO. 13) and AcGUS-I575-R (SEQ ID NO. 14) as primers, screening the same strategy as that of single-point saturation mutation verified in example 4, obtaining a mutant strain AcGUS3 G461C/I575K, AcGUS3G461C/I575K with further provided hydrolytic capacity, wherein the activity of hydrolyzing GL is improved by 124.29% and the activity of hydrolyzing GAMG is improved by 118.72% compared with that of non-mutated AcGUS 3.
Further, by using AcGUS.sup.3 G461C/I575K mutant as an original strain and performing iterative saturation mutation with Q462, constructing a saturated mutation library of G461C/I575K and Q462 by using AcGUS.sup.3-Q462N-F (SEQ ID NO. 11) and AcGUS-Q462N-R (SEQ ID NO. 12) as primers, and obtaining a mutant strain AcGUS.sup. 3 G461C/Q462H/I575K with further provided hydrolytic capacity by the same screening strategy as that of the single-point saturated mutation verified and example 4, wherein the enzyme activity is respectively improved by 138.3% (18 beta-GL group) and 136.6% (18 beta-GAMG group) compared with control AcGUS. Here, the optimal combination mutant AcGUS, G461C/Q462H/I575K was renamed AcGUS3M1.
In addition, further kinetic parameters were measured for activity characterization, which indicated that mutant AcGUS M1 catalyzes 6.1-fold of GL for k cat/Km compared to wild-type AcGUS, and that k cat/Km for GAMG compared to AcGUS is 11.02-fold, indicating further improvement in affinity and catalytic efficiency. The significant improvement of AcGUS M1 enzyme activity provides more powerful support for the construction of the beta-glucuronidase combined engineering bacteria in the next step.
Experimental example 5 construction of Pichia pastoris AcGUS M1
First, the recombinant vector pGAPZαA-AcGUS M1 was obtained by ligating the AcGUS M1 gene with the pGAPZαA vector by the Gibson assembly method using the pET-28a-AcGUS M1 plasmid obtained by the verification in Experimental example 4 as a template. Then, the recombinant plasmid is introduced into competent Top10 by a heat shock method, positive clone screening is carried out on an LB plate containing 100mg/L bleomycin resistance, correct recombinant DNA sequencing is carried out by colony PCR verification, and the recombinant pGAPZalpha A-AcGUS M1 is successfully constructed. Further referring to the Pichia pastoris construction and verification method in experimental example 3, pichia pastoris AcGUS M1 strain was obtained.
Experimental example 6 construction of beta-glucuronidase Combined engineering bacteria
(1) Construction of pGAPZαA-AcGUS3M1-NrsR vector
In order to facilitate the screening and construction of the subsequent beta-glucuronidase combined engineering bacteria, the pGAPZA-AcGUS M1 plasmid in the example 5 is replaced by the bleomycin resistance fragment in the pGAPZA-AcGUS M1 plasmid according to the operation instruction of the NEB company Gibson seamless connection kit, so as to obtain the new resistant pGAPZA-AcGUS M1-NrsR recombinant plasmid.
(2) Construction and verification of dG-GA1 combined engineering bacteria
The recombinant plasmid pGAPZalpha A-AcGUS M1-NrsR is linearized by adopting endonuclease BlnI, and the linearization system is as follows: 10X QuickCut Buffer 2. Mu.L, blnI 1. Mu.L, total plasmid 1. Mu.g, and 20. Mu.L was supplemented with sterile water. After instantaneous centrifugation, the reaction is carried out for 1h at a constant temperature of 37 ℃. The linearized plasmid was purified using GeneJET gel recovery kit. Transferring the pGAPZalpha A-AcGUS M1-NrsR recombinant plasmid linearization plasmid into Pichia pastoris AtGUS by adopting an electrotransfer method, uniformly coating the plasmid on a YPD solid plate containing 100mg/L bleomycin and 100mg/L nociceptin, and culturing for 2-3 days at a constant temperature of 30 ℃.
Single colonies of the above resistant plates were picked, inoculated into 5mL of YPD liquid medium containing 100mg/L bleomycin and 100mg/L nourseothricin, and cultured for 2 days at 30℃and in a shaker at 200 rpm. Then, the medium was transferred to YPD medium containing 4g/L GL at a transfer rate of 1%, and the medium was further cultured for 2 days, and samples were taken every 6 hours, and HPLC detection was performed. The detection shows that the intermediate GAMG is always at the duty ratio lower than 4%, which indicates that the construction of the pichia pastoris dG-GA1 combined engineering bacteria is successful.
The recombinant expression vectors contained in the engineering bacterium dG-GA1 are pGAPZalpha A-AtGUS and pGAPZalpha A-AcGUS M1-NrsR, wherein NrsR refers to that the resistance gene in the original plasmid is replaced by NrsR resistance.
Experimental example 7 comparison of enzyme Activity of Pichia pastoris engineering bacteria different for GA production
To meet the actual production requirements, the enzyme activity comparison form of Pichia pastoris for producing GA adopts crude enzyme liquid to react with GL with higher concentration. Here, examples for analysis and comparison include AtGUS-mix described in patent CN109628427B previously developed by the inventors, acGUS3M1 and dG-GA1 according to the present invention, and other comparative examples of the subject group to which the inventors are directed: the sequences of AuGUS and AtGUS are reported in AtGUS and AuGUS（"Properties and structures of β-glucuronidases with different transformation types of glycyrrhizin" of patent CN 109628427B).
First, single colonies of the above strain were picked from the resistance plate and inoculated into 5mL of YPD medium containing 100mg/L zeocin, and cultured in a shaker at 30℃and 200rpm for 48 hours. Then, the culture was continued for 48 hours by transferring the culture medium to YPD medium having a liquid content of 100mL at an amount of OD 600 =0.1. Then, 200. Mu.L of the crude enzyme supernatant was mixed with 800. Mu.L of 10g/L GL reaction buffer (pH 5.5), reacted at 40℃for 1 hour, and then sampled for HPLC detection.
The results in FIG. 6 show that the combined engineering bacterium dG-GA1 performs best at higher concentration of GL substrate, has highest GA production efficiency, and can keep extremely low accumulation of intermediate GAMG. While comparative example AuGUS had no enzyme activity, indicating that it could not be expressed heterologously, consistent with the reported results that AuGUS could only be expressed in the wild strain. In addition AtGUS and Atgusmix have higher GAMG accumulation and far weaker conversion efficiency than dG-GA1. Notably, in the present invention, mutant AcGUS M1 under expression of the putative safety producer pichia pastoris was still 13.18-fold more active than the AcGUS wild type, again indicating the effectiveness of the resulting mutant in rational design as described in experimental example 4, not only for prokaryotic expression in e.coli, but also for successful expression in eukaryotic pichia pastoris. Therefore, the combined engineering bacterium dG-GA1 was considered as an optimal GA-producing strain in comparison of enzyme activities.
Experimental examples 8, 5L fermentation tank amplification preparation of 18 alpha-GA
The ability of dG-GA1 to produce 18. Alpha. -GA was verified in a 5L fermenter. dG-GA1 was inoculated from a streaked YPD plate into a 5mL YPD liquid medium tube and activated for 36 hours at 30℃in a 200rpm shaker. The seed solution was transferred to shake flasks containing 100mL YPD liquid medium at OD 600 = 0.1 and incubated for 24h at 30℃in a 200rpm shaker. Then transferred to a 5L fermenter containing 3L YPD liquid medium. The culture conditions were set at a fermentation temperature of 30℃at pH5.5 at a rotation speed of 200rpm. The fed-batch fermentation is adopted, the duration of the fermentation stage is about 60 hours, and glucose is fed in two times. The growth OD 600 of the strain is monitored in real time in the fermentation process. The reaction conditions of the substrate catalysis process are set to 42.5 ℃, the pH value is 5.5, the stirring rotation speed is 300rpm, the feeding concentration of 18 alpha-GL is 20g/L each time, and the feeding is carried out for 4 times. The concentration changes of the substrates 18α -GL, the intermediates GAMG and 18α -GA of the reaction system were examined in real time at this stage to accurately evaluate the catalytic effect of dG-GA1, and the results are shown in FIG. 7A. The result shows that in the fed-batch process of a 5L fermentation tank, the conversion process takes 20 hours, the final concentration of 18 alpha-GA reaches 41.09g/L, the conversion rate is up to 96.57%, and the method has good industrial application potential in the aspect of 18 alpha-GL conversion.
Experimental example 9, 1000L fermentation tank amplification preparation of 18 beta-GA
To further verify the large-scale production capacity of dG-GA1, while characterizing the capacity to convert 18 β -GL, a verification was performed in a 1000L fermenter. In the fermentation stage, pichia dG-GA1 combined engineering bacteria were first picked from streaked YPD plates, single colonies were inoculated into a liquid medium containing 100mL of YPD, and cultured at 30℃for about 24 hours. The inoculum was then sequentially scaled up in 10L, 100L and 1000L fermentors containing YPD medium, and the whole fermentation was maintained at 30℃and at pH5.5 and at 200 rpm stirring speed. Wherein seed solutions of both the 10L fermenter and the 100L fermenter were cultured for about 24 hours. In addition, the culture was cultured in a 1000L fermenter for about 48 hours, and then 20g/L glucose was fed every 12 hours, and sugar was fed twice. In the 1000L fermentation culture process, the condition of the strain growth OD 600 is monitored in real time. In the substrate catalysis stage, the reaction conditions were set at 42.5℃and pH5.5 and at a stirring speed of 300rpm, the 18. Beta. -GL charge concentration was 20g/L each time, and the concentration changes of the substrate 18. Beta. -GL, the intermediate product GAMG and 18. Beta. -GA were monitored in real time, and the results were shown in FIG. 7B.
The results showed that the whole substrate catalytic stage took 24h times for a total of 6 substrate 18 β -GL feeds. At 24h, the GA concentration was 48.73g/L, and the GL conversion rate reached 97.26%. Meanwhile, in the whole amplification test process, the accumulation amount of 18 beta-GAMG in a reaction system is always lower than 4%, and the GAMG concentration in 24 hours is lower than 1.43g/L. Meets the requirements of solving the engineering problems, and the efficiency and the yield are the highest in the currently reported processes.
From the results, the dG-GA1 combined engineering bacteria have excellent comprehensive performance, and the obvious effect of AcGUS and mutants thereof on hydrolyzing GAMG and the effectiveness of the beta-glucuronidase combination strategy are proved. The invention further shows that the method has the advantages of high catalytic efficiency, short fermentation period, environment-friendly process and the like, and has the prospect of industrialized mass production of glycyrrhetinic acid.

Claims (14)

1. A beta-glucuronidase, characterized in that it is selected from: beta-glucuronidase AcGUS, beta-glucuronidase AcGUS, beta-glucuronidase AcGUS2, beta-glucuronidase AcGUS3 or beta-glucuronidase AcGUS mutants;
The beta-glucuronidase AcGUS is an enzyme obtained by deleting 1 st amino acid, 7 th alanine, 66 th arginine, 256 th alanine, 270 th threonine, 332 th valine, 363 rd aspartic acid and 506 th methionine of an amino acid sequence with GenBank accession number AEK 69352.1;
The beta-glucuronidase AcGUS, acGUS2 and AcGUS are obtained by respectively truncating 10, 20 and 30 amino acids at the nitrogen end of the beta-glucuronidase AcGUS; the amino acid sequence of the beta-glucuronidase AcGUS is shown as SEQ ID NO. 1;
The beta-glucuronidase AcGUS mutant is selected from the group consisting of: a mutant obtained by mutating the 461 st glycine of AcGUS to cysteine, a mutant obtained by mutating the 462 st glutamine of AcGUS to histidine, a mutant obtained by mutating the 575 nd isoleucine of AcGUS to lysine, or a mutant obtained by mutating the 461 st glycine of AcGUS to cysteine, the 462 st glutamine to histidine and the 575 nd isoleucine to lysine.
2. The beta-glucuronidase according to claim 1, wherein the gene sequence of said beta-glucuronidase AcGUS is shown in SEQ ID No. 2;
The mutants obtained by mutating the 461 st glycine of AcGUS to cysteine, the 462 st glutamine to histidine and the 575 nd isoleucine to lysine are beta-glucuronidase AcGUS M1.
3. A recombinant expression vector comprising a gene sequence of a β -glucuronidase as defined in claim 1 or 2 linked thereto.
4. A recombinant expression vector according to claim 3, wherein said expression vector is selected from pET28a, pgapzα A, pPIC K or ppiczα.
5. A recombinant expression vector according to claim 3, wherein said recombinant expression vector is selected from the group consisting of recombinant expression vector pgapzαA-AcGUS3 and recombinant expression vector pgapzαA-AcGUS M1.
6. An engineering bacterium, characterized by being selected from pichia pastoris strain AcGUS, pichia pastoris strain AcGUS M1 or pichia pastoris strain dG-GA1;
The pichia pastoris strain AcGUS can express the beta-glucuronidase AcGUS3 according to claim 1 or 2;
The pichia pastoris strain AcGUS M1 can express the beta-glucuronidase AcGUS M1 of claim 2;
The pichia pastoris strain dG-GA1 can simultaneously express the beta-glucuronidase AcGUS M1 and the beta-glucuronidase AtGUS derived from the aspergillus terreus (Aspergillus terreus) strain Li-20.
7. The engineered bacterium of claim 6, wherein the pichia pastoris strain AcGUS contains the recombinant expression vector pgapzαA-AcGUS3 of claim 5.
8. The engineered bacterium of claim 6, wherein the pichia pastoris strain AcGUS M1 contains the recombinant expression vector pgapzαa-AcGUS M1 of claim 5.
9. The engineering bacterium according to claim 6, wherein the pichia pastoris strain dG-GA1 comprises the recombinant expression vector pGAPZ A-AcGUS M1 modified recombinant expression vector pGAPZ A-AcGUS M1-NrsR and the recombinant expression vector pGAPZ A-AtGUS linked with the gene sequence of β -glucuronidase AtGUS derived from aspergillus terreus (Aspergillus terreus) strain Li-20; the improvement means: the resistance gene of the original expression vector pGAPZαA of the recombinant expression vector pGAPZαA-AcGUS M1 was replaced by NrsR resistance.
10. A starter comprising a starter active ingredient, characterized in that the starter active ingredient comprises a β -glucuronidase according to claim 1 or 2, and/or a recombinant expression vector according to any one of claims 3-5, and/or an engineering bacterium according to any one of claims 6-9.
11. A leavening agent according to claim 10 further comprising: auxiliary materials.
12. A method for mass production of glycyrrhetinic acid, characterized in that a substrate is fermented and produced by using a pichia pastoris strain AcGUS M1 or a pichia pastoris strain dG-GA1 in one of the engineering bacteria according to any one of claims 6-9; the substrate of the pichia pastoris strain AcGUS M1 is 18 beta-GL, and the substrate of the pichia pastoris strain dG-GA1 is selected from the following materials: 18 alpha-GL or 18 beta-GL.
13. The method for mass production of glycyrrhetinic acid according to claim 12 wherein when the substrate is 18 β -GL, the glycyrrhetinic acid is 18 β -GA; when the substrate is 18α -GL, the glycyrrhetinic acid is 18α -GA.
14. The method for mass production of glycyrrhetinic acid according to claim 12 wherein the fermentation production conditions comprise: 42.5 ℃, pH5.5, stirring rotation speed 300rpm, and charging concentration of 20g/L.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0297944A1 (en) * 1987-06-16 1989-01-04 Pernod-Ricard Production of beta-glucuronidase type enzyme, hydrolysis of glycyrrhizine and of beta-glycyrrhetinic acid
CN109735518A (en) * 2019-02-28 2019-05-10 北京理工大学 A kind of the beta-glucuronidase enzyme mutant and its conversion glycyrrhizic acid technique of optimal reaction pH raising

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0297944A1 (en) * 1987-06-16 1989-01-04 Pernod-Ricard Production of beta-glucuronidase type enzyme, hydrolysis of glycyrrhizine and of beta-glycyrrhetinic acid
CN109735518A (en) * 2019-02-28 2019-05-10 北京理工大学 A kind of the beta-glucuronidase enzyme mutant and its conversion glycyrrhizic acid technique of optimal reaction pH raising

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