CN111117988B - Amino acid mutant of thermophilic xylosidase and application thereof - Google Patents
Amino acid mutant of thermophilic xylosidase and application thereof Download PDFInfo
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
An amino acid mutant of thermophilic xylosidase and application thereof. The invention mutates a gene from a streptococcus thermophilus (A)Dictyoglomus thermophilum) Beta-xylosidase of DSM 3960GH39 family, and specific hydrolysis of notoginsenoside R1 by using beta-xylosidase multi-site mutant to generate ginsenoside Rg1. The multi-site mutant contains 2 mutation sites, wherein HIS284 is mutated into ASP and ALA respectively, and the mutant improves the sugar tolerance rate of the beta-xylosidase by 1.35 and 1.09 times respectively, and the mutant has the advantages of high sugar tolerance, low cost, high stability and high yieldKiThe coefficients respectively reach 4.6 mol/L and 3.7 mol/L; CYS202 is mutated into LEU and PHE, and the enzyme activities are respectively improved by 3.28 and 2.97 times.
Description
Technical Field
The invention relates to the field of genetic engineering and biological engineering, in particular to an amino acid mutant of thermophilic xylosidase and application thereof.
Background
Beta-xylosidase (EC 3.2.1.37) is a more critical exoglycosidic hydrolase in a xylan degrading enzyme system, can decompose xylooligosaccharide and xylobiose at a non-reducing end into xylose, and has been widely applied in various fields through the synergistic action with xylanase. In the energy industry, xylan in plant fiber raw materials can be converted into xylose by a xylanase system and further converted into valuable fuels or chemicals such as ethanol, furfural and the like; in the paper industry, the beta-xylosidase and xylanase have synergistic effect to effectively improve the bleaching performance; in recent years, beta-xylosidase has been developed and applied to the pharmaceutical industry for hydrolyzing xylose at the glycosyl terminal of some natural compounds (glycoside bond formed by aglycone such as steroid, terpenoid and the like and xylose), thereby converting into products with important application value. However, the enzyme has specificity and strong specificity, and the compounds with different framework structures, even the compounds with the same framework, have different xylose connection bond types (such as beta-1,2-glycosidic bond, beta-1,4-glycosidic bond) and positions, and have great difference in catalytic capability and efficiency of the beta-xylosidase. At present, the study is more and more intensive, and the study is about xylan formed by connecting hydrolyzed xylosyl through beta-1,4-glycosidic bonds. On the other hand, in the modern industry, the feedback inhibition effect of xylose greatly limits the effect of practical application of β -xylosidase. Therefore, the improvement of the tolerance of the beta-xylosidase to xylose by an enzyme engineering technology is very important.
Pseudo-ginseng (Panax notoginseng) is a plant of Araliaceae, has a long history of being used for treating diseases, and is a traditional precious medicinal material in China. Up to now more than 70 kinds of monomer saponin have been separated and identified from root block, stem leaf, flower and fruit of notoginseng. Wherein, the ginsenoside Rg1 and Rb1 have the highest content of notoginsenoside R1. From the view of molecular structure, the main structures of ginsenoside Rg1 and notoginsenoside R1 are the same, except that notoginsenoside R1 is connected with xylose on the glucosyl group at the 6 th position of Rg1 through a beta-1,2 glycosidic bond.
The common beta-xylosidase does not have the capacity of cutting off the beta-1,2-xyloside bond connected with glucosyl group at the 6-position in the structure of notoginsenoside R1 to generate Rg1.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides an amino acid mutant of thermophilic xylosidase and application thereof. The common beta-xylosidase does not have the capacity of cutting off the beta-1,2-xylosidase bond connected with glucosyl group on the 6 th position in the structure of notoginsenoside R1 to generate Rg1, and moreover, the tolerance to xylose in the enzymolysis process is poor, and the problem that the catalytic activity of the terminal product inhibiting enzyme is generated is solved. On the other hand, the anti-fatigue activity of the notoginseng extract is yet to be further improved, and an effective technology is not available at present. Aiming at the defects in the prior art, the invention obtains a novel heat-resistant sugar-resistant beta-xylosidase mutant HIS284ASP/CYS202LEU which can specifically cut off a beta-1,2-xyloside bond connected with glucosyl group in a notoginsenoside R1 structure by gene engineering technology, informatics technology and a large number of point mutation transformation and screening; the method for preparing the ginsenoside Rg1 by converting the notoginsenoside R1 by the high-efficiency enzyme method is established for the first time.
The technical scheme is as follows: amino acid mutant derived from beta-xylosidase of the Neisseria pyrrophicus (Dictyoglomyus thermophilum) DSM 3960GH39 family, characterized in that there are 2 of said mutation sites, wherein HIS284 is mutated into A and D and CYS202 is mutated into L and F.
The nucleotide sequence of the mutant is shown as SEQ ID NO. 4-7.
The amino acid mutant H284D/C202L (showing that HIS284 is mutated into D and CYS202 is mutated into L) of beta-xylosidase derived from the Neurococcus thermophilus DSM 3960GH39 family is applied to the specific hydrolysis of notoginsenoside R1 to generate ginsenoside Rg1.
The hydrolysis conditions are as follows: the reaction temperature is 25-95 ℃, the pH value is 4.0-7.0, the concentration of the notoginsenoside R1 substrate is 0.2-5 g/L, and 0.1-2U/mL beta-xylosidase is added for reaction for 0.5-12h.
Preferred hydrolysis conditions are: the reaction temperature is 75 ℃, the pH value is 6.0, the concentration of the notoginsenoside R1 substrate is 1g/L, and 1U/mL beta-xylosidase is added for reaction for 30min.
The application of the amino acid mutant H284D/C202L of the beta-xylosidase derived from the Neisseria thermophila (Dictyoglycomus thermophilus) DSM 3960GH39 family in preparing an anti-fatigue product after hydrolyzing notoginsenoside R1 is disclosed.
Has the advantages that: 1. compared with the prior art, the xylosidase provided by the invention has excellent heat resistance, and has the highest enzyme activity under the conditions of 75 ℃ and pH6.0. The enzyme has high enzyme activity at the temperature of 70-80 ℃ and the pH value of 5.0-7.0. The enzyme has good heat resistance, and the enzyme activity is basically kept unchanged after 2h of heat preservation at 75 ℃. The enzyme is suitable for degradation at a high temperature of more than 70 ℃ under a neutral condition, and has potential application value. 2. According to the invention, HIS284 is mutated into ASP and ALA respectively on the basis of Xln-DT, and the sugar resistance efficiency is improved by 1.35 and 1.09 times; CYS202 of the beta-xylosidase is mutated into LEU and PHE respectively on the basis of HIS284ASP and HIS284ALA, and the mutant enables the enzyme activity of the beta-xylosidase to be improved by 3.28 times and 2.97 times respectively. 3. The invention establishes that the notoginsenoside R1 is converted into the ginsenoside Rg1 by hydrolyzing a multi-site mutant of thermophilic bacteria recombinant beta-xylosidase, the conversion efficiency is high and reaches 100% under proper conditions, and the prepared ginsenoside Rg1 has obviously improved anti-fatigue activity compared with the ginsenoside Rg1 before conversion.
Drawings
FIG. 1 is a schematic diagram of the enzymatic conversion of notoginsenoside R1 to generate ginsenoside Rg 1;
FIG. 2 is a graph showing the conversion efficiency of different sources of beta-xylosidase to notoginsenoside R1;
FIG. 3 is a comparison of sugar tolerance factors of the recombinant enzyme and the mutant enzyme;
FIG. 4 is a graph showing comparison of enzyme activities of a recombinase and a mutant enzyme;
FIG. 5 is HPLC chromatogram of standard sample of notoginsenoside R1 and ginsenoside Rg 1;
FIG. 6 is HPLC chromatogram before and after converting the beta-xylosidase multi-site mutant into notoginsenoside R1;
FIG. 7 is a graph comparing the anti-fatigue activity of notoginsenoside R1 and the transformed ginsenoside Rg1, illustrating the dosage: R1-L/Rg1-L:5 mg/kg. D; R1-H/Rg1-H:20 mg/kg. D.
Detailed Description
The invention provides a method for generating ginsenoside Rg1 by converting notoginsenoside R1 through mutant enzyme, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.
A series of mutant beta-xylosidase genes, the nucleotide sequences of which are shown in the following table.
Mutant genes
The series of recombinant plasmids pET-28a-Xln-DT-284A, pET-28a-Xln-DT-284D, pET-28a-Xln-DT-284A/202L, pET-28a-Xln-DT-284D/202L, pET-28a-Xln-DT-284A/202F and pET-28a-Xln-DT-284D/202F containing the above nucleotide sequences.
The preparation method of the recombinant plasmid containing the SEQ ID NO. 2-3 comprises the following construction steps: the recombinant plasmid pET-28a-Xln-DT which is constructed by experiments and carries the Xln-DT gene is used as a template, and Y284A and Y284D are mutated by inverse PCR to obtain recombinant plasmids pET-28a-Xln-DT-284A and pET-28a-Xln-DT-284D carrying the mutated gene SEQ ID NO: 2-3.
The preparation method of the recombinant plasmid containing the SEQ ID NO. 4-7 comprises the following construction steps: recombinant plasmids pET-28a-Xln-DT-284A and pET-28a-Xln-DT-284D are respectively used as templates, and further reverse PCR is used for mutating C202L and C202F to obtain recombinant plasmids pET-28a-Xln-DT-284A/202L and pET-28a-Xln-DT-284D/202L, pET-28a-Xln-DT-284A/202F and pET-28a-Xln-DT-284D/202F carrying mutant genes SEQ ID NO: 4-7.
DNA Polymerase (Prime STAR HS DNA Polymerase), deoxygen, used in the examplesRibonucleic acid (dNTP), ligase (T) 4 DNA ligase), phosphorylase (T) 4 Polynuceotide Kinase) and related buffers were purchased from TaKaRa, bio-engineering (Dalian) Ltd.
A sugar-tolerant mutant of a beta-xylosidase from the Neurospora thermophila DSM 3960GH39 family, comprising the following 2 mutation sites HIS284 (to ALA and ASP) and CYS202 (to LEU and PHE).
Application of beta-xylosidase glucose-resistant mutant HIS284ASP/CYS202LEU derived from Pyrococcus thermophilus (Dictyoglycomus thermophilus) DSM 3960GH39 family in specifically hydrolyzing notoginsenoside R1 to generate ginsenoside Rg1.
Mutants 1-2: HIS284 is mutated into ALA and ASP on the basis of Xln-DT.
The nucleotide sequence of the mutant is shown in SEQ ID NO 2-3.
Mutants 3 to 6: its CYS202 was mutated to LEU and PHE based on HIS284ALA and HIS284 ASP.
The nucleotide sequence of the mutant is shown in SEQ ID NO. 4-7.
The hydrolysis conditions were: the reaction temperature is 25-95 ℃, the pH value is 4.0-7.0, the concentration of the notoginsenoside R1 substrate is 0.2-5 g/L, and 0.1-2U/mL beta-xylosidase is added for reaction for 0.5-12h.
The hydrolysis conditions were: the reaction temperature is 75 ℃, the pH value is 6.0, the concentration of the notoginsenoside R1 substrate is 1g/L, and 1U/mL beta-xylosidase is added for reaction for 30min. A method for preparing ginsenoside Rg1 by converting notoginsenoside R1 with beta-xylosidase.
In the present invention, the manner of obtaining the β -xyloside is not limited. The beta-xylosidase can be derived from microorganisms such as bacteria and fungi, can be obtained by means of culture, fermentation, separation and purification and the like, and can also be obtained by means of gene cloning, expression, separation and purification. In the embodiment of the invention, the beta-xylosidase is derived from an escherichia coli recombinant strain of a beta-xylosidase gene of Dictyoglycous thermophilum DSM 3960.
The method screens beta-xylosidase from a variety of different families, including Aspergillus niger NL-1GH 3 family, thermotoga thermomarum DSM 5069GH 3 family, thermotoga petrophila DSM13995 GH3 family, dictyoglyces thermophilum DSM 3960GH39 family, thermoanaerobacterium thermosaccharolyticum DSM 571GH 120 family, and the like. And determining that beta-xylosidase derived from Dictyoglossus thermophilum DSM 3960GH39 family can specifically hydrolyze notoginsenoside R1 to generate ginsenoside Rg1.
Example 1 screening of the beta-xylosidase of the present invention
1.1 cloning, plasmid construction and preparation of recombinase of GH3 family beta-xylosidase gene derived from Aspergillus niger NL-1
1.1.1 culture of Aspergillus niger NL-1
Aspergillus niger NL-1 was stored in this laboratory and its liquid medium formulation was: 3g/L glucose, 0.1g/L dipotassium phosphate trihydrate, 0.05g/L potassium chloride, 0.05g/L magnesium sulfate heptahydrate, 0.001g/L ferrous sulfate heptahydrate and 0.02g/L sodium nitrate. The strain is activated by PDA culture medium, then the activated Aspergillus niger NL-1 spore is re-suspended by sterile normal saline, inoculated in 50mL liquid seed shake flask culture medium, and cultured for 72h under constant temperature shaking of 28 ℃ and 150rpm, and the thallus is collected.
1.1.2 extraction of genomic DNA from Aspergillus niger NL-1
(1) The activated Aspergillus niger strain is inoculated on a liquid culture medium and cultured for 2 days at the temperature of 28-30 ℃.
(2) Filtering to collect mycelium, washing with sterile water for 3-4 times until the culture medium is completely cleaned, draining, wrapping with tinfoil, and freezing in liquid nitrogen.
(3) 2g of mycelium is put into a precooled mortar, and a proper amount of liquid nitrogen is added for grinding until the mycelium is ground into powder.
(4) Placing the fully ground mycelium into an EP tube, adding CTAB extraction solution (2% mercaptoethanol) preheated to 65 ℃, shaking and uniformly mixing, and carrying out warm bath at 65 ℃ for 45-60min.
(5) An equal volume of a phenol/chloroform/isoamyl alcohol (25.
(6) The upper aqueous phase was aspirated and extracted repeatedly with a phenol/chloroform/isoamyl alcohol (25.
(7) Simultaneously, 0.5 volume times of ice-cold 5M LiCl and 0.6 volume times of isopropanol were added, mixed well, left at room temperature for 20min, centrifuged at 12,000rpm for 10min, and the supernatant was discarded.
(8) Then, 1mL of 70% ethanol was added to thoroughly suspend the precipitate, the precipitate was left to stand for 10min at 12,000rpm, and the supernatant was discarded. Repeating the steps once, air-drying and recovering the precipitate.
(9) Redissolved in 50. Mu.L of TE buffer containing 50. Mu.g/mL of RNAase and incubated at 37 ℃ for 1h.
(10) Adding 0.1 volume time of 3mol/L sodium acetate (pH 5.2) and 3 volume times of absolute ethyl alcohol, mixing uniformly, and keeping the temperature at-20 ℃ for 1h.
(11) Centrifuge at 12,000rpm,4 ℃ for 30min and carefully pour off the supernatant.
(12) Adding 500 μ L70% ethanol, standing at room temperature for 10min, centrifuging at 12,000rpm for 30min at 4 deg.C.
(13) And (5) discarding the supernatant, and air-drying on a superclean bench.
(14) Add 50. Mu.L of TE buffer for resuspension and store at-20 ℃.
1.1.3 obtaining of Aspergillus niger NL-1 xylosidase xlnD
Carrying out homology analysis by referring to an Aspergillus niger xylosidase gene published on NCBI, and designing 2 upstream primers of an upstream primer and a downstream primer of the Aspergillus niger xylosidase gene xlnD according to a conserved sequence: CCCGAA TTCCAG GCC AAC ACC AGC TAC GTC and downstream primer: CCCTCT AGA CTA CTC CTT CCC CGG CCA CTT. The restriction sites EcoR I and Xba I are underlined. Carrying out PCR amplification by using the synthesized primer, wherein the amplification condition is 94 ℃ and 5min;30 cycles (94 ℃,30s, 59 ℃,30s, 72 ℃,3min; 72 ℃ for 10min; the reaction was stopped and the temperature was maintained at 4 ℃. The PCR amplification product was purified by a gel recovery kit. The Aspergillus niger NL-1-derived β -xylosidase gene was obtained.
Carrying out double enzyme digestion on the beta-xylosidase gene and the pPICZ alpha A which are obtained from Aspergillus niger NL-1 sources by using EcoR I and Xba I respectively, tapping and recovering the glue respectively, connecting overnight at 16 ℃ after concentrating, coating the conversion solution on an LLB plate containing Zencin (the final concentration is 25 mu g/mL), carrying out inversion culture at 37 ℃ for 12h, selecting positive clones, sequencing, and obtaining a recombinant expression plasmid named as pPICZ alpha A-xlnD.
1.1.4 preparation of recombinant enzymes
The recombinant plasmid pPICZ alpha A-xlnD is extracted, linearized by BxtX I and introduced into Pichia pastoris GS115 (Novagen) by an electrical transformation method, and positive clones are screened. Inoculating into YPD medium, activating, transferring into BMGY medium, activating, and collecting OD 600 Transferring 2.0-3.0 thallus into BMMY culture medium, and performing induced expression of beta-xylosidase in shaker at 30 deg.C and 180 rpm. And supplementing sterile methanol into the induced bacterial liquid every 24h according to the volume ratio of 0.6%, culturing for 15 days, centrifuging and taking the supernatant to obtain a crude enzyme solution. And (3) purifying the recombinant protein: (1) Adding ammonium sulfate with a final concentration of 80% to the crude enzyme solution to precipitate the protein, centrifuging and discarding the supernatant, and dissolving the precipitated protein with a Tris-HCl buffer solution with a pH of 7.5 50mM; (2) Dialyzing with pH 7.5 50mM Tris-HCl buffer at 4 ℃ for 8h four times to remove the salt solution; (3) Adding the enzyme solution after dialysis into a packed DEAE SFF column, and performing gradient elution by using NaCl with the concentration of 20-300 mM; (4) The enzyme solution eluted with NaCl of an appropriate concentration was dialyzed with a PB buffer of pH 6.5 10mM at 4 ℃ four times for 8 hours each time to remove the salt solution to obtain pure enzyme.
2.2 Thermotoga thermomarum DSM 5069-derived beta-xylosidase clone and recombinase preparation
1.2.1 culture of Thermotoga thermomarum DSM 5069
Thermotoga thermarum DSM 5069 was purchased from the German collection of microorganisms. The serial numbers are: DSM 5069. The formula of the culture medium is as follows: 5g/L soluble starch, 1g/L yeast powder and 1.5g/L KH 2 PO 4 ，4.2g/L Na 2 HPO 4 ·12H 2 O，3.4g/L NaCl，1g/L MgSO 4 ·7H 2 O,0.76g/L EDTA,1mL/L microelement, 0.5g/L Na 2 S·9H 2 O,0.5g/L Cysteine HCl,1mg/L resazurin, pH adjusted to 7.0, boiling with nitrogen, removing oxygen, and sterilizing the medium in anaerobic condition. Microelement (1000 ×) formulation: feCl 3 2.0g/L；H 3 BO 3 0.05g/L；ZnCl 2 0.05g/L；CuCl 2 ·2H 2 O 0.03g/L；MnCl 2 ·4H 2 O 0.05g/L；(NH 4 ) 2 MoO 4 0.05g/L；AlK(SO 4 ) 2 ·2H 2 O is 0.05g/L. ) Inoculating with 0.5% inoculating amount by syringe, static culturing at 82 deg.C for 24 hr, and collecting cells.
1.2.2 extraction of genomic DNA
(1) The cells were collected by static culture of Thermotoga thermomarum DSM 5069 24h and centrifugation at 6,000rpm for 10min with 30mL of bacterial suspension.
(2) The cells were resuspended in 9.5mL of TE buffer, 0.5mL of 10% Sodium Dodecyl Sulfate (SDS) and 50. Mu.L of proteinase K (20 mg/mL) were added thereto, mixed well, and incubated at 37 ℃ for 1 hour.
(3) 1.8mL of 5mol/L NaCl,1.5mL of hexadecyltriethylammonium bromide (CTAB)/NaCl, mixing, and incubating at 65 ℃ for 20min.
(4) Adding equal volume of chloroform/isoamyl alcohol, mixing, and centrifuging at 6,000rpm for 10min.
(5) To prevent genomic DNA fragmentation due to shear forces, the supernatant was transferred to another centrifuge tube with a wide-mouthed pipette, mixed with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuged at 6,000rpm for 10min.
(6) In another centrifuge tube, 0.6 volume of isopropanol was added and gently shaken until white filamentous DNA precipitate was clearly visible.
(7) The DNA was wound with a pipette and washed in 70% alcohol.
(8) The DNA was scraped from the pipette with a sterile toothpick and transferred to a 1.5mL centrifuge tube.
(9) Air-dried at room temperature, and dissolved in 500. Mu.L of TE buffer.
(10) 50. Mu.L of the DNA was assayed by a nucleic acid protein detector.
1.2.3 construction of recombinant plasmid pET-20b-xynB3
Designing primers according to GH3 family beta-xylosidase gene derived from Thermotoga thermarum DSM 5069, wherein an upstream primer comprises the following components: CATGCCA TGGATC TTT ACA AGA ATC CAA A and the downstream primer: CC (challenge collapsar)CTC GAGCTC GAT CTT TGT ATT TGT GA. The restriction sites NcoI and XhoI are underlined. Using the extracted genome DNA of Thermotoga thermarum DSM 5069 as template, and using the DNA libraryThe obtained primer is used for PCR amplification, and the amplification condition is 94 ℃ for 3min;30 cycles (94 ℃,30s, 55 ℃,30s, 72 ℃,2min20 s); 72 ℃ for 10min; the reaction was stopped and the temperature was maintained at 4 ℃. And purifying the PCR amplification product by using a gel recovery kit. Obtaining GH3 family beta-xylosidase gene from Thermotoga thermarum DSM 5069.
Obtaining GH3 family beta-xylosidase gene and pET-20b from Thermotoga thermomarum DSM 5069, performing double enzyme digestion by NcoI and XhoI respectively, tapping and recovering the gel respectively, concentrating, connecting at 16 ℃ overnight, transforming the connection product into escherichia coli Top10F' competent cells, coating the transformation product on an LB solid culture medium containing Amp (100 mu g/mL) for overnight culture at 37 ℃, inoculating a plurality of single bacteria to an LB liquid culture medium containing Amp (100 mu g/mL) for culture for 8-10h, collecting bacteria to extract plasmids, performing enzyme digestion verification to remove idle-load plasmids, and performing nucleic acid sequence determination on the recombinant plasmids to obtain a correct recombinant expression vector pET-20b-xynB3.
1.2.4 preparation of recombinant enzymes
Transforming a recombinant plasmid pET-20b-xynB3 into an escherichia coli BL21 (DE 3) host bacterium (Novagen), culturing overnight at 37 ℃ on an LB plate containing Amp (100 mu g/mL), picking a transformant into 200mL of LB culture medium (100 mu g/mL Amp), carrying out shaking culture at 37 ℃ and 180rpm until OD600 is 0.6, adding an isopropyl beta-D-thiogalactopyranoside (IPTG) inducer with a final concentration of 0.5mM, carrying out induction culture at 30 ℃ for 8h, centrifuging the culture solution at 4 ℃ and 13,000rpm for 15min by using a high-speed refrigerated centrifuge, collecting thalli, removing supernatant, adding sterile water, carrying out ultrasonic cell disruption, carrying out heat treatment at 70 ℃ for 30min, centrifuging the culture solution at 4 ℃ and 13,000rpm for 15min by using the high-speed refrigerated centrifuge, and obtaining the supernatant which is GH3 family recombinant beta-xylosidase derived from Thermota thermomarum DSM 5069.
1.3 beta-xylosidase clone of GH3 family derived from Thermotoga petrophila DSM13995 and recombinase preparation
1.3.1 culture of Thermotoga petrophila DSM13995
Thermotoga petrophila DSM13995 was purchased from the German collection of microorganisms. The serial numbers are: DSM 13995. The formula of the culture medium is as follows: 10g/L of starch and 5g/L of tryptone，3g/L yeast extract，5g/L meat extract，10g/L2-morpholinoethanesulfonic,10mg/L FeSO 4 ·7H 2 O,1mg/L resazurin, and the pH was adjusted to 7.2. Inoculating with 0.5% inoculating amount by syringe, static culturing at 85 deg.C for 24 hr, and collecting cells.
1.3.2 extraction of genomic DNA
(1) The Thermotoga petrophila DSM13995 was cultured by standing for about 24 hours, 30mL of the lysate was centrifuged at 4,000g for 10min to collect cells.
(2) The cells were resuspended in 9.5mL of TE buffer, 0.5mL of 10% Sodium Dodecyl Sulfate (SDS) and 50. Mu.L of proteinase K (20 mg/mL) were added thereto, mixed well, and incubated at 37 ℃ for 1 hour.
(3) 1.8mL of 5mol/L NaCl and 1.5mL of hexadecyltriethylammonium bromide (CTAB)/NaCl were added, mixed well, and incubated at 65 ℃ for 20min.
(4) Adding equal volume of chloroform/isoamyl alcohol, mixing, and centrifuging at 6,000g for 10min.
(5) To prevent genomic DNA fragmentation due to shear force, the supernatant was transferred to another centrifuge tube with a wide-mouthed pipette, mixed with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuged at 6,000g for 10min.
(6) In another centrifuge tube, 0.6 volume of isopropanol was added and gently shaken until white filamentous DNA precipitate was clearly visible.
(7) The DNA was wound with a pipette and washed in 70% alcohol.
(8) The DNA was scraped from the pipette with a sterile toothpick and transferred to a 1.5mL centrifuge tube.
(9) Air-dried at room temperature, and dissolved in 500. Mu.L of TE buffer.
(10) 50. Mu.L of the DNA was assayed by a nucleic acid protein detector.
1.3.3 construction of recombinant plasmid pET-28a-xln3
Designing a primer according to a GH3 family beta-xylosidase gene derived from Thermotoga petrophila DSM13995, wherein an upstream primer comprises the following components: CATGCCA TGGAAC TGT ACA GGG ATC CTT CG and downstream primer: CCGCTC GAGCTC CTC GCA GGC TTC CGT GAA. The restriction sites NcoI and XhoI are underlined. Using the extracted genome DNA of Thermotogatephrilla DSM13995 as template, and using synthesized primerPerforming PCR amplification under the condition of 94 ℃ for 3min;30 cycles (94 ℃,30s, 55 ℃, 72 ℃,2min, 20 s;72 ℃ for 10min; the reaction was stopped and the temperature was maintained at 4 ℃. The PCR amplification product was purified by a gel recovery kit. Obtaining GH3 family beta-xylosidase gene derived from Thermotoga petrophila DSM 13995.
Obtaining GH3 family beta-xylosidase gene and pET-28a derived from Thermotoga petrophila DSM13995, performing double enzyme digestion by NcoI and XhoI respectively, tapping and recovering the gel respectively, concentrating the gel, connecting the gel at 16 ℃ overnight, transforming the connection product into escherichia coli Top10F' competent cells, coating the transformation product on an LB solid culture medium containing Kana (100 mu g/mL) for overnight culture at 37 ℃, inoculating a plurality of single bacteria to an LB liquid culture medium containing Kana (100 mu g/mL) for culture for 8-10h, collecting bacteria extraction plasmids, performing enzyme digestion verification to remove idle-load plasmids, and performing nucleic acid sequence determination on the recombinant plasmids to obtain the correct recombinant expression vector pET-28a-xln3.
1.3.4 preparation of recombinant enzymes
The recombinant plasmid pET-28a-xln3 is transformed into a host bacterium (Novagen) of Escherichia coli BL21 (DE 3), the host bacterium is cultured on an LB plate containing Kana (100 mu g/mL) at 37 ℃ overnight, and the transformant is picked up into 200mL of LB medium (100 mu g/mL Kana) at 37 ℃ and is cultured with shaking at 180rpm until OD is achieved 600 At 0.6, isopropyl beta-D-thiogalactopyranoside (IPTG) inducer is added to the mixture to a final concentration of 0.5mM, induction culture is carried out for 8h at 30 ℃, the culture solution is centrifuged for 15min at 13,000rpm by a high-speed refrigerated centrifuge, thalli are collected, supernatant is removed, sterile water is added, cells are crushed by ultrasonic waves, heat treatment is carried out for 30min at 70 ℃, and then purification is carried out by an Ni-NTA affinity chromatography column to finally obtain purified recombinant beta-xylosidase of GH3 family from Thermotoga petrophila DSM 13995.
1.4Thermoanaerobacterium thermosaccharolyticum DSM 571-derived beta-xylosidase clone and recombinase preparation
1.4.1 cultivation of Thermoanaerobacterium thermosaccharolyticum DSM 571
Thermoanaerobacterium thermosaccharolyticum DSM 571 is available from the German Collection of microorganisms. The serial numbers are: DSM 571. The culture medium is preparedThe method comprises the following steps: 5g/L soluble starch, 1g/L yeast powder and 1.5g/L KH 2 PO 4 ，4.2g/L Na 2 HPO 4 ·12H 2 O，3.4g/L NaCl，1g/L MgSO 4 ·7H 2 O,0.76g/L EDTA,1mL/L microelement, 0.5g/L Na 2 S·9H 2 O,0.5g/L Cysteine HCl,1mg/L resazurin, pH adjusted to 7.0, boiling with nitrogen, removing oxygen, and sterilizing the medium in anaerobic condition. Trace elements (1000 ×) formulation: feCl 3 2.0g/L；H 3 BO 3 0.05g/L；ZnCl 2 0.05g/L；CuCl 2 ·2H 2 O 0.03g/L；MnCl 2 ·4H 2 O 0.05g/L；(NH 4 ) 2 MoO 4 0.05g/L；AlKSO 4 ·2H 2 O is 0.05g/L. Inoculating with 0.5% inoculating amount by syringe, static culturing at 82 deg.C for 24 hr, and collecting cells.
1.4.2 extraction of genomic DNA
(1) Standing at 69 deg.C for about 8 hr, and centrifuging 30mL of bacterial liquid 6 at 000rpm for 10min to collect cells.
(2) The cells were resuspended in 9.5mL of TE buffer, 0.5mL of 10% Sodium Dodecyl Sulfate (SDS) and 50. Mu.L of proteinase K (20 mg/mL) were added thereto, mixed well, and incubated at 37 ℃ for 1 hour.
(3) 1.8mL of 5mol/L NaCl,1.5mL of hexadecyltriethylammonium bromide (CTAB)/NaCl, mixing, and incubating at 65 ℃ for 20min.
(4) Adding equal volume of chloroform/isoamyl alcohol, mixing, and centrifuging at 6,000rpm for 10min.
(5) To prevent genomic DNA fragmentation due to shear forces, the supernatant was transferred to another centrifuge tube using a wide-mouthed pipette, mixed with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuged at 10,000rpm for 10min.
(6) In another centrifuge tube, 0.6 volume of isopropanol was added and gently shaken until white filamentous DNA precipitate was clearly visible.
(7) The DNA was wound with a pipette and washed in 70% alcohol.
(8) The DNA was scraped from the pipette with a sterile toothpick and transferred to a 1.5mL centrifuge tube.
(9) Air-dried at room temperature, and dissolved in 500. Mu.L of TE buffer.
(10) 50. Mu.L of the DNA was assayed by a nucleic acid protein detector.
1.4.3 construction of recombinant plasmid pET-20b-xyl
Designing a primer according to a GH120 family beta-xylosidase gene derived from Thermoanaerobacterium thermosaccharolyticum DSM 571, wherein an upstream primer: CCCCAT ATGGAA TAT CAT GTA GCG AA and the downstream primer: CCCCTC GAGCCA AAC TTT AAT ATA ATT ATC G. The restriction sites NdeI and XhoI are underlined. Using the extracted genome DNA of Thermoanaerobacterium thermosaccharolyticum DSM 571 as a template, and performing PCR amplification by using a synthesized primer, wherein the amplification condition is 95 ℃ and 5min;25 cycles (95 ℃,30s, 57 ℃,30s, 72 ℃,2 min; 72 ℃ for 10min; the reaction was stopped and the temperature was maintained at 4 ℃. And purifying the PCR amplification product by using a gel recovery kit. The GH120 family beta-xylosidase gene derived from Thermoanaerobacterium thermosaccharolyticum DSM 571 is obtained.
Obtaining GH120 family beta-xylosidase gene and pET-20b from Thermoanaerobacterium thermosaccharolyticum DSM 571, performing double enzyme digestion by NdeI and XhoI respectively, tapping and recovering the gel respectively, concentrating, connecting at 16 ℃ overnight, transforming the connection product into escherichia coli Top10F' competent cells, coating the transformation product on LB solid culture medium containing Amp (100 mu g/mL) for overnight culture at 37 ℃, inoculating a plurality of single bacteria to LB liquid culture medium containing Amp (100 mu g/mL) for culture for 8-10h, collecting bacteria to extract plasmids, performing enzyme digestion verification to remove the unloaded plasmids, and performing nucleic acid sequence determination on the recombinant plasmids to obtain the correct recombinant expression vector pET-20b-xyl.
1.4.4 preparation of recombinant enzymes
The recombinant plasmid pET-20b-xyl is transformed into a host bacterium (Novagen) of Escherichia coli BL21 (DE 3), the host bacterium is cultured on an LB plate containing Amp (100 mu g/mL) at 37 ℃ overnight, and the transformant is picked up into 200mL of LB medium (100 mu g/mL Amp) at 37 ℃ and cultured with shaking at 180rpm until OD is achieved 600 At 0.6, adding isopropyl beta-D-thiogalactopyranoside (IPTG) inducer with final concentration of 0.5mM, performing induction culture at 30 deg.C for 8h, and centrifuging the culture solution at 4 deg.C with high-speed refrigerated centrifuge to obtain 13,000rpAnd (5) centrifuging for 15min, collecting thalli, removing supernatant, adding sterile water, and breaking cells by ultrasonic waves. Since the recombinant plasmid pET-20b-xyl contained a His-tag, it was purified by His Bind Purification Kit (Novagen). The crude enzyme is firstly heat treated for 1h at 75 ℃ to remove heat-labile foreign protein, and then purified by a Ni-NTA affinity chromatography column, and finally the purified GH120 family beta-xylosidase from Thermoanaerobacterium thermosaccharolyticum DSM 571 is obtained.
1.5 Dictyoglycomus thermophilum DSM 3960-derived beta-xylosidase cloning and recombinase preparation
1.5.1 extraction of genomic DNA from Dictyoglycomus thermophilum DSM 3960
Adopting 10g of fresh dictyosphaea thermophilum DSM 3960 (purchased from German culture collection center of microorganisms) to suspend in 9.5mL of TE buffer solution, adding 0.5mL of 10% Sodium Dodecyl Sulfate (SDS) and 50 μ L of proteinase K (20 mg/mL), uniformly mixing, and preserving heat at 37 ℃ for 1h; adding 1.8mL of 5mol/L NaCl and 1.5mL of hexadecyltriethylammonium bromide (CTAB)/NaCl, uniformly mixing, and incubating at 65 ℃ for 20min; adding equal volume of chloroform/isoamyl alcohol, mixing, centrifuging at 6,000g for 10min; transferring the supernatant into another centrifuge tube with a coarse-mouthed pipette, adding equal volume of phenol/chloroform/isoamylol, mixing, and centrifuging for 10min at 6,000g; adding isopropanol with 0.6 times volume, and gently shaking until white filiform DNA precipitate is clearly visible; DNA was wound on it with a pipette and washed in 70% alcohol; scraping the DNA from a suction tube by using a sterile toothpick, and transferring the DNA into a 1.5mL centrifuge tube; air-drying at room temperature, and dissolving with 500 μ L TE buffer solution; 50. Mu.L of the DNA was assayed by a nucleic acid protein detector.
1.5.2 preparation of beta-xylosidase xln-DT
Can be prepared by the following method or obtained by artificial synthesis.
PCR amplification was performed using the following primer pair, using the total DNA of tennis thermophilus Dictyoglomycotus thermophilum DSM 3960 (1.1 preparation) as template: the upstream primer is CGC GGA TCC ATG AAC CAT ATA AAG ATT GAA A; downstream primer CCG CTC GAG ATA TCC ACC TGG TAT TTT GCT ATC. The cleavage sites BamHI and XhoI are underlined. The PCR system is as follows: dictyoglomyus thermophilum DSM 3960 genome1.0. Mu.L of DNA, 2.0. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 4.0. Mu.L of dNTP mix, 10 XEx Taq buffer (Mg) 2+ free)5.0μL，MgCl 2 4.0. Mu.L, ex Taq enzyme (5U/. Mu.L) 0.5. Mu.L, and ultrapure water 31.5. Mu.L. And (3) PCR reaction conditions: 94 ℃ for 3min;30 cycles (94 ℃,30s, 60 ℃,30s, 72 ℃,1min30 s); 72 ℃ for 10min; the reaction was stopped and the temperature was maintained at 4 ℃.
The PCR product was checked for yield and specificity by electrophoresis on a 1% agarose gel and purified using a PCR product recovery kit.
1.5.3 construction and validation of recombinant clone and expression vector pET-20b-xln-DT
The purified PCR product and pET-20b (Novagen) were digested with BamHI and XhoI, respectively, and the digested PCR and the vector-sized fragment were recovered by 1% agarose gel electrophoresis. Concentrating the target fragment and the carrier after tapping recovery, connecting overnight at 16 ℃, converting the connecting product into escherichia coli Top10F' competent cells, coating the competent cells on a culture medium of 100 mu g/mL Amp, culturing for 12h at 37 ℃, screening positive clones, and carrying out sequence analysis; selecting the clone with correct sequence to extract plasmid, and obtaining the recombinant plasmid pET-28a-xln-DT containing the heat-resistant glycosidase gene.
Preparation and purification of 1.5.4 recombinase
The recombinant plasmid pET-28a-xln-DT is transformed into Escherichia coli BL21 (DE 3) host bacteria (Novagen), cultured for 12h at 37 ℃ on an LB plate containing Amp (100 mu g/mL), picked into 200mL LB medium (100 mu g/mL Amp), cultured at 37 ℃ with shaking at 200rpm to OD 600 When the concentration is 0.6, adding an isopropyl beta-D-thiogalactopyranoside (IPTG) inducer with the final concentration of 0.5mM, carrying out induction culture at 28 ℃ for 8h, centrifuging the culture solution at 13,000rpm by using a high-speed refrigerated centrifuge at 4 ℃ for 15min, collecting thalli, removing supernatant, adding sterile water, carrying out ultrasonic cell disruption, and centrifuging to obtain supernatant. Since the recombinant plasmid pET20b-xln-DT contains a His-tag, it was purified by His Bind Purification Kit (Novagen). The crude enzyme is firstly subjected to heat treatment at 75 ℃ for 1h to remove heat-labile foreign protein, and then is purified by using a Ni-NTA affinity chromatography column, and finally the purified beta-xylosidase of GH39 family derived from Dictyoglycomus thermophilum DSM 3960 is obtained. Identification of purity and molecular weight of purified enzymesThe determination was carried out by SDS-PAGE, and the result is shown in FIG. 3, and the molecular weight is about 55kDa, which is close to the theoretical value.
Example 2 determination of mutation site of recombinant beta-xylosidase Xln-DT
Research shows that the sugar tolerance and expression capacity of beta-xylosidase are closely related to the key sites around the active pocket inside the protein structure. The three-dimensional structure of beta-xylosidase Xln-DT is obtained through homologous modeling, sugar-resistant mutation sites are designed based on the theory, the mutation energy of the virtual mutation sites is analyzed by using a bioinformatics software Discovery Studio, the mutation sites with high xylose affinity are screened as research objects, and the mutation sites are finally determined to be H284 and C202.
Design of mutation primer: the mutation points are designed by changing partial amino acids of the beta-xylosidase Xln-DT molecule by utilizing bioinformatics analysis and homologous modeling comparison. Obtaining mutant sequences by utilizing a reverse PCR technology, and designing positive and negative oligonucleotide sequences at each mutant site. The mutation site is yeast preferred codon, and the mutation primers are shown in the following table.
TABLE 1 site-directed mutagenesis Table
Example 3 construction of a series of recombinant plasmids carrying a mutant Gene
The recombinant plasmid pET-28a-Xln-D is used as a template, and HIS284ALA and HIS284ASP are further mutated by reverse PCR to obtain recombinant plasmids pET-28a-Xln-DT-284A and pET-28a-Xln-DT-284D carrying mutant genes SEQ ID NO of 2-3. Recombinant plasmids pET-28a-Xln-DT-284A and pET-28a-Xln-DT-284D are used as templates, and further reverse PCR is used for mutating C202L and C202F to obtain recombinant plasmids pET-28a-Xln-DT-284A/202L, pET-28a-Xln-DT-284D/202L, pET-28a-Xln-DT-284A/202F and pET-28a-Xln-DT-284D/202F carrying mutant genes SEQ ID NO: 4-7.
TABLE 2 preparation of reverse PCR reaction solution (50. Mu.L in total)
TABLE 3 inverse PCR reaction conditions
The PCR fragment 5' end phosphorylation reaction was performed at 37 ℃ for 1 hour as shown in the following Table.
TABLE 4 preparation of phosphorylation reaction solution
After phosphorylation at 37 ℃ 1. Mu. L T4 ligase was added and ligation was performed for 3h at 16 ℃. And transforming plasmid DNA, selecting mutants, and identifying through sequencing.
Example 4 analysis of xylose tolerance coefficients of recombinant enzymes
In a reaction system added with xylose of different concentrations, enzyme activity values of the recombinase and the mutant enzyme are measured according to a standard method, and relative enzyme activity values of the recombinase and the mutant enzyme under different xylose concentrations are calculated and obtained by taking the activity values of the recombinase and the mutant enzyme measured when xylose is not added as 100%.
The enzyme activity was determined as follows: p-nitrophenol-beta-xylopyranoside (pNPX) is used as a substrate, the p-nitrophenol obtained by hydrolysis and sodium carbonate are subjected to a color reaction, and the absorbance of the product is measured at the wavelength of 405 nm. The reaction system of 100 mul comprises 90 mul of buffer solution of 100mmol/L with the optimum pH value and 5 mul of substrate of 20mmol/L, after uniform mixing and preheating, 5 mul of diluted enzyme solution is added, the reaction is carried out for 10min at the optimum temperature, and then 0.3mL of 1mol/L Na is added 2 CO 3 The reaction is stopped, and the mixture is evenly mixed and then is measured by an enzyme-labeling instrument under the condition of 405 nm. A control with enzyme solution and no substrate and a control with substrate and no enzyme solution were also performed.
One enzyme activity unit (U) is defined as: the amount of enzyme required to release 1. Mu. Mol of p-nitrophenol (pNP) upon hydrolysis of p-nitrophenol-beta-xylopyranoside (pNPX) within 1 min.
Enzyme activity was calculated against the standard curve:
enzyme activity (U/mL) = c × V 1 /(t×V 2 )×N
c: the p-nitrophenol content (mu mol/mL) after the enzyme reaction is calculated by a p-nitrophenol standard equation;
V 1 : total reaction system volume (mL);
t: enzyme and substrate reaction time (min);
V 2 : volume of enzyme solution (mL) at the time of enzyme reaction;
n: and (4) diluting enzyme solution by multiple times.
Example 5 comparison of the ability of recombinant β -xylosidase from different sources to convert notoginsenoside R1
The concentration of notoginsenoside R1 is 1g/L, 5 beta-xylosidase from different sources are converted under respective optimum temperature and optimum pH conditions, 1U/mL enzyme is added for reaction for 2h, and detection is carried out by HPLC. The results show that after 2h of reaction, the conversion of notoginsenoside R1 by beta-xylosidase derived from Aspergillus niger NL-1, thermotoga thermarum DSM 5069, thermotoga petrophilum DSM13995 and Thermoanaerobacterium therococcharolyticum DSM 571 was almost 0, while the conversion of notoginsenoside R1 by beta-xylosidase mutants derived from Dictyoglyces therophilum DSMZ 3960GH39 family produced ginsenoside Rg1, see FIG. 2.
Example 6 transformation of beta-xylosidase Xln-DT 284D into notoginsenoside R1 and HPLC detection spectra before and after reaction
HPLC liquid phase detection of Notoginseng radix saponin R1 and ginsenoside Rg1 standard products is shown in FIG. 5. The peak emergence time of notoginsenoside R1 and ginsenoside Rg1 is 10.022min and 10.949min respectively.
The recombinant beta-xylosidase Xln-DT mutant 284D is applied to the transformation of notoginsenoside R1, and the transformation system is as follows: the concentration of the notoginsenoside R1 is 1g/L, the pH value is 6.0 50mmol/L citric acid-disodium hydrogen phosphate buffer solution, 1U/mL beta-xylosidase Xln-DT is added to react for 30min at 75 ℃, and detection is carried out by HPLC, wherein the method and conditions are the same as those of HPLC liquid phase detection conditions of standard products of notoginsenoside R1 and ginsenoside Rg1.
The results show that: beta-xylosidase Xln-DT mutant 284D has significant conversion capacity to notoginsenoside R1, and is almost completely converted into ginsenoside Rg1 after reaction for 0.5 hour, with the molar conversion rate close to 100% (fig. 6).
Example 7 comparison of anti-fatigue Activity before and after conversion of notoginsenoside R1
The antifatigue activities of notoginsenoside R1 and ginsenoside Rg1 are shown in FIG. 7. The body weight of five groups of mice steadily increased after the continuous administration for 14 days, which indicates that the drug has no toxic effect on the mice. After 14 days of administration, the mice have obviously prolonged exhaustive swimming time after 12.5 percent of the weight of the mice, wherein the low dose (5 mg/kg. D) and the high dose group (20 mg/kg. D) of Rg1 are respectively improved by 14.5 percent and 16.4 percent. After exercise, the hemoglobin concentration and the liver glycogen and muscle glycogen contents of the mice in the administration group are obviously higher than those of the mice in the control group. The ginsenoside Rg1 and the notoginsenoside R1 both have certain anti-fatigue effect, and the effect of the Rg1 is superior to that of the R1.
In conclusion, the invention carries out multi-site mutation on the heat-resistant beta-xylosidase cloned from Dictyoglossus thermophilum DSM 3960 genome, and utilizes the mutant to efficiently convert notoginsenoside R1 to generate ginsenoside Rg1. The invention has the technical advantages that: (1) The sugar tolerance coefficient of the obtained novel beta-xylosidase mutant is improved by 1.35 times, and K is i 4.602mol/L is achieved, and the enzyme activity is improved by 3.28 times; (2) The mutant can specifically hydrolyze terminal beta-1,2-glycosidic bond, has high optimal action temperature, and is favorable for improving the solubility of a substrate; (3) The enzymatic conversion is safe and efficient, the time is short, and the conversion rate is high; and (4) the converted product has stronger anti-fatigue biological activity.
Sequence listing
<110> Nanjing university of forestry
<120> thermophilic xylosidase amino acid mutant and application thereof
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atctgtggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacc atcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 2
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atctgtggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacg ctcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 3
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atctgtggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacg atcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 4
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atcctcggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacg ctcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 5
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atcctcggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacg atcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 6
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atcttcggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacg ctcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 7
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgaaccata taaagattga aaaaggcaaa tatgttggtg tttttcctga caattggaag 60
ttttgcgtag gaagtggaag aataggtctt gctctccaaa aagaatatat agatgccctc 120
tcttttgtaa aaaggcatat agattttaaa tatctaagag cccacggatt actccatgac 180
gatgtgggaa tttacagaga agacatagta gacggaaaaa caatcccttt ttataatttc 240
acctacattg acagaatata cgattccttt ttagaaattg gaataagacc tttcgttgaa 300
attggattta tgccctcaaa acttgcctct ggagatcaaa cagtatttta ctggagaggc 360
aatgttactc cccctaaaga ttataaagaa tgggaaaagt taataaaaaa tgtggtgaaa 420
catttcatag atagatatgg agaaaaagag gttactcaat ggccttttga gatttggaat 480
gagcccaatt taaccgtatt ttggaaagat gcaaatcagg cagaatactt caaattatac 540
gaggttacag taaaagcaat aaaagaggta aatgaaaaca taaaggttgg aggacctgca 600
atcttcggtg gatcagacta ttggataact gattttctaa atttctgtta taagaacaat 660
gtacctgtag actttttaac tcgacatgcc tatacaggca aacctcctat atatacccct 720
cactttgtat accaagatgt gcatcccatc gaatacatgt taaatgaatt caaaaccgtt 780
agagagatgg tgaaaaattc tccattccct aacctaccaa tacatattac tgaatttaac 840
agttcttacg atcccctctg ccccatacac gatactccct tcaatgctgc ttacttagca 900
agggtactaa gtgagggagg agactatgtt gactccttct cttattggac ctttagtgat 960
gtgtttgagg aagcagacgt tccaagatca ctcttccatg gtggatttgg tcttgtagca 1020
ttccacaata ttccaaaacc agttttccat atgttcacct tctttaatgc tatgggagaa 1080
aagatcctct atagagatga ccatatctta ataaccgaaa gagaagacaa gtcagttgcc 1140
ttaattgctt ggaatgaagt catgacaaaa gaagaaaatc aagaaagaaa atatagaata 1200
gagatacccg tagattacaa agaggttttc ataaaacaaa agttaattga tgaggaatac 1260
ggaaatccat ggcgcacctg gattcaaatg ggcaggccga gatttccaag caaaaagcaa 1320
atagaaacat taagagaagt agcaactcct aaagtaacta ctttcagaaa aacagtagaa 1380
aatggacata ttactcttga atttacatta ggtaaaaatg ctgttaccct ctttgaaata 1440
agcaaggtta ttgatgaatc acatacctat ataggtctcg acgatagcaa aataccaggt 1500
ggatattaa 1509
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cccgaattcc aggccaacac cagctacgtc 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccctctagac tactccttcc ccggccactt 30
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
catgccatgg atctttacaa gaatccaaa 29
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ccctcgagct cgatctttgt atttgtga 28
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
catgccatgg aactgtacag ggatccttcg 30
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ccgctcgagc tcctcgcagg cttccgtgaa 30
<210> 14
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccccatatgg aatatcatgt agcgaa 26
<210> 15
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cccctcgagc caaactttaa tataattatc g 31
<210> 16
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cgcggatcca tgaaccatat aaagattgaa a 31
<210> 17
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ccgctcgaga tatccacctg gtattttgct atc 33
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gctcccctct gccccataca cgatact 27
<210> 19
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gatcccctct gccccataca cgatact 27
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gtaagaactg ttaaattcag taatatgtat 30
<210> 21
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ctcggtggat cagactattg gataactgat tt 32
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ttcggtggat cagactattg gataactgat tt 32
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gattgcaggt cctccaacct ttatgttttc 30
Claims (4)
1. From Pyrenophora teres (Dictyoglomus thermophilum) Amino acid mutant of the beta-xylosidase of the GH39 family of DSM 3960, characterized in that the nucleotide sequence coding for the amino acid mutant is shown in SEQ ID NO 4-7.
2. From Pyrenophora teres (Dictyoglomus thermophilum) The application of an amino acid mutant of beta-xylosidase of GH39 family of DSM 3960 in the specific hydrolysis of notoginsenoside R1 to generate ginsenoside Rg1 is characterized in that the nucleotide sequence for coding the amino acid mutant is shown as SEQ ID NO. 5.
3. Use according to claim 2, characterized in that the hydrolysis conditions are: the reaction temperature is 75 ℃, the pH value is 6.0, the substrate concentration of the notoginsenoside R1 is 1g/L, and 1U/mL of the amino acid mutant of beta-xylosidase is added for reaction for 30min.
4. The use according to claim 2, wherein the product of hydrolysis of notoginsenoside R1 is used to prepare an anti-fatigue product.
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