CN103805581A - Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK - Google Patents
Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK Download PDFInfo
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Abstract
The invention discloses a beta-glycosidase mutant and a coding gene thereof, and an application thereof in producing ginsenoside CK. The protein disclosed by the invention is named as lacS-mut, and is one of defined as the following items: 1) the protein is composed of amino acid residue sequence of a sequence 4 in the sequence table; 2) the protein is derived from the protein as defined in the item 1) by substituting and/or deleting and/or adding of one or several amino acid residues in the amino acid residue sequence of the sequence 4 in the sequence table, and has beta-glycosidase activity. Experiments prove that the beta-glycosidase mutant is obtained by mutating a wild type beta-glycosidase gene; compared with the wild type, the beta-glycosidase mutant has the advantage that the activity of the beta-glycosidase mutant in synthesizing the ginsenoside CK is obviously improved, and therefore the beta-glycosidase mutant has better industrial application prospect.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of beta-glycosidase mutant and encoding gene thereof and its application in production Ginsenoside compound K.
Background technology
Ginseng is traditional traditional Chinese medical science medicinal material.According to modern pharmacological research, ginseng has enhancing body immunity, improves nutrition, anti-ageing and slow down the effects such as tired.Ginsenoside compound K, as main effectively one of activeconstituents of ginseng, is easier to be absorbed by the body, and has the medical functions such as the decline of the malignant cell of promotion, the diffusion of containment tumour or deterioration, therefore has high productive value and application prospect.But Ginsenoside compound K does not exist in natural Radix Ginseng extract, but by other ginsenoside composition (as ginsenoside Rb
1, Rb
2, Rd, Re and Rg
1deng) generate via entero-bacte metabolic conversion, therefore very low at occurring in nature content.The conversion process of ginsenoside mainly comprises removes the monose groups such as glucose from agent structure hydrolysis, the method for hydrolysis has chemical hydrolysis and biocatalytic reaction method etc.Although chemical process transformation efficiency is higher, catalysis lacks specificity, applies widely thereby limited it.And biocatalytic reaction method, utilize enzyme process to realize the synthetic research of Ginsenoside compound K fewer, mainly purifying and the property research of some zymoproteins of originated from fungus, if purifying is from the Polyglucosidase of Paecilomyces Bainier, purifying is found to have the activity of synthetic Ginsenoside compound K from the Polyglucosidase of Thermuscaldophilus etc., but in lower, the conversion process of activity, has intermediate product accumulation on the one hand; The gene order of these enzymes, all without report, cannot realize recombinant expressed on the other hand.In the middle of the research of the synthetic Ginsenoside compound K of enzyme process, not only transformation efficiency has much room for improvement, and should reduce the accumulation of even eliminating intermediate product simultaneously as far as possible, at utmost to strengthen the output of Ginsenoside compound K, reduces the difficulty of downstream separation purifying work.
Be in the news and there is the ability of synthetic Ginsenoside compound K from the beta-glycosidase (LacS, β-Glycosidase) of sulfolobus solfataricus, but transformation efficiency is limited, cannot meet the demand of scale operation.Utilize the advantage of this enzymic synthesis CK to be: 1, the reorganized expression of this enzyme, is suitable for further improving its catalytic efficiency by protein engineering means; 2, the temperature of reaction of this enzyme is higher, and thermostability is very good, contributes to complete product synthetic under high temperature production condition, improves transformation efficiency, reduces the possibility of polluting in production process; 3, the pilot process of the synthetic Ginsenoside compound K of this enzyme catalysis is by clear explaination; 4, the crystalline structure of this enzyme is in the news, for it is further transformed and provides important reference information to the analysis of enzyme mutant.
Summary of the invention
An object of the present invention is to provide a kind of beta-glycosidase mutant and encoding gene thereof.
Albumen provided by the invention, called after lacS-mut, is following 1) protein:
1) protein being formed by the amino acid residue sequence of the sequence 4 in sequence table;
2) by 4 amino acid residue sequences of the sequence in sequence table through replacement and/or disappearance and/or the interpolation of one or several amino-acid residues and have beta-glycosidase activity by 1) derivative protein.
Sequence 4 in above-mentioned sequence table is made up of 489 amino acid, the replacement of described one or several amino-acid residue and/or disappearance and/or be added to the replacement and/or disappearance and/or the interpolation that are no more than 10 amino-acid residues.
The gene of above-mentioned albumen of encoding is also the scope of protection of the invention.
Said gene is the DNA molecular of following (1) or (2) or (3):
(1) DNA molecular shown in sequence 3 in sequence table;
(2) the DNA sequence dna hybridization limiting with (1) under stringent condition and coding have the DNA molecular of beta-glycosidase activated protein;
(3) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have 99% homology and coding and have a DNA molecular of beta-glycosidase activated protein.
Above-mentioned stringent condition is at 6 × SSC, and in the solution of 0.5% SDS, at 65 ℃, hybridization, then uses 2 × SSC, 0.1% SDS and 1 × SSC, and 0.1% SDS respectively washes film once.
Above-mentioned sequence 3 forms by 1470 Nucleotide, and the coding region of described sequence 3 is from 5 ' end 1-1470 position Nucleotide.
Recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contains said gene is also the scope of protection of the invention.
Above-mentioned recombinant vectors is that the encoding gene of above-mentioned albumen is inserted in expression vector, obtain expressing the recombinant vectors of above-mentioned albumen, be specially the recombinant vectors obtaining between the NheI of the DNA molecular insertion vector pET28a shown in the sequence in sequence table 3 and XhoI recognition site.
Above-mentioned albumen, said gene or above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention in the application as in beta-glycosidase.
Above-mentioned albumen is also the scope of protection of the invention in the application of producing in Ginsenoside compound K.
Another object of the present invention is to provide a kind of method of producing Ginsenoside compound K.
Method provided by the invention, comprises the steps:
1) the above-mentioned recombinant bacterium that ferments, collects centrifugal collection supernatant liquor after fermented liquid, centrifugal collection thalline, broken thalline;
2) by described supernatant liquor and ginsenoside substrate Rb
1in MC damping fluid, react, obtain Ginsenoside compound K.
In aforesaid method, 1) described fermentation is carried out under IPTG induction;
2) described MC damping fluid is that pH value is 5.5 MC damping fluid; The MC damping fluid that described pH value is 5.5 is specifically that 200mM Sodium phosphate dibasic, final concentration are that 100mM citric acid and water form by final concentration.
The present invention of experiment showed, of the present invention wild-type beta-glycosidase gene that suddenlys change, obtains beta-glycosidase mutant, and compared with wild-type, it is significantly increased on synthetic Ginsenoside compound K vigor, therefore has good prospects for commercial application.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, beta-glycosidase mutant and encoding gene thereof
1, the acquisition of beta-glycosidase and encoding gene thereof
Extract sulfolobus solfataricus (Sulfolobus solfataricus) (ATCC 35091, DSM 1616) genomic dna and take it as template, take 5 '-atggctagcatgtactcatttccaaatagc-3 ' (forward) and 5 '-gtgctcgagttagtgccttaatggctttac-3 ' (reverse) as primer carries out pcr amplification.Pcr amplification condition is as follows: first 95 ℃ of denaturation 4min, then 95 ℃ of 45s, 55 ℃ of 30s, 72 ℃ of 1min30s, totally 30 circulations; Last 72 ℃ are extended 10min.
Reclaim above-mentioned PCR reaction product, carry out agarose gel electrophoresis detection, result obtains the fragment of about 1470bp size.
By NheI and XhoI double digestion for the PCR product of above-mentioned acquisition, enzyme is cut product and pET28a carrier (the Novagen company of cutting through same enzyme, numbering 69864-3) spend the night with 16 ℃ of connections of T4 ligase enzyme (purchased from precious biotech firm), connect product and transform intestinal bacteria MC1061 competent cell, obtain recombinant bacterium.
Extract the plasmid sequence verification of recombinant bacterium, sequencing analysis, the unnamed gene of this PCR product is lacS, its nucleotides sequence is classified the sequence 1 in sequence table as, by comparison, it is the gene of the beta-glycosidase lacS of sulfolobus solfataricus, the albumen called after LacS of this genes encoding, and the aminoacid sequence of this albumen is the sequence 2 in sequence table.By the plasmid called after pET28a-lacS that contains this PCR product, this plasmid is that the sequence in sequence table 1 is inserted to the carrier obtaining between the NheI of pET28a carrier and XhoI double enzyme site.
2, sudden change obtains beta-glycosidase mutant
On the basis of beta-glycosidase lacS gene, utilize the method for fallibility PCR, build random mutation library.Be mainly the concentration by changing mn ion in pcr amplification system, improve the mispairing rate of Taq archaeal dna polymerase, thereby in gene order, introduce sudden change.In this research, carry out taking turns sudden change library construction, obtain the PCR product 1 of sudden change and the PCR product 2 of sudden change.
By the PCR product of the PCR product of said mutation 1 and sudden change 2 NheI and XhoI double digestions respectively, enzyme is cut product and is connected with the pET28a carrier of cutting through same enzyme respectively, and connection product transforms respectively BL21(DE3) competent cell in, obtain the library that suddenlys change.Screening recombinant bacterium, screening method is with aseptic toothpick, the single colony clone that is comprising mutant to be chosen into dividing 800 ul LB substratum (Tryptones 10 g/L are housed between aseptic technique, NaCl 10 g/L, yeast extract 5g/L, adding microbiotic kantlex to 50 μ g/mL) 37 ℃ of shaking tables cultivate after 2h, add inductor IPTG to final concentration 0.4mM, induce about 16h at 30 ℃ of shaking tables.Centrifugal, throw aside supernatant liquor, collect thalline, freeze-thaw method cell wall breaking, and add N,O-Diacetylmuramidase (10mg/ml, Tris-HCl damping fluid, pH8.0) at 37 ℃ of reaction 1h, centrifugal, supernatant liquor is the crude enzyme liquid after broken wall.With crude enzyme liquid and ginsenoside substrate Rb
1reaction (condition is 85 ℃, pH5.5), with dinitrosalicylic acid method (DNS method) detection reaction by product reducing sugar content, embodies the ability that generates Ginsenoside compound K that transforms indirectly.
For generating the more recombinant bacterium of reducing sugar, extract plasmid and send to order-checking, the gene of the PCR product in this plasmid of result has the Nucleotide shown in sequence 3 in sequence table, this unnamed gene is lacS-mut, the albumen called after lacS-mut of this genes encoding, the aminoacid sequence of this albumen is the sequence 4 in sequence table.
The amino acid of above-mentioned lacS-mut is compared with nucleotide sequence with the amino acid of wild-type beta-glycosidase lacS with nucleotide sequence:
The aminoacid sequence (sequence 4) of lacS-mut is for by the aminoacid sequence of wild-type beta-glycosidase lacS (sequence 2 in sequence table), from N ' end, the 218th α-amino-isovaleric acid Val sports glycine Gly, and the nucleotide sequence (sequence 3) of lacS-mut gene is for by the nucleotide sequence of wild-type beta-glycosidase lacS gene (sequence 1 in sequence table), ' end, 652-654 position GTT sports GGT from 5.Illustrate that lacS-mut is the mutant of lacS.
By the plasmid called after pET28a-lacS-mut that contains lacS-mut, this plasmid is that the sequence in sequence table 3 is inserted to the carrier obtaining between the NheI of pET28a and XhoI double enzyme site, by the recombinant bacterium called after BL21(DE3 that contains this plasmid)/pET28a-lacS-mut.
Above-mentioned BL21(DE3)/pET28a-lacS-mut also can be prepared as follows: sequence 3 in artificial synthesized sequence table, the DNA molecular shown in sequence in sequence table 3 is inserted to the pET28a-lacS-mut obtaining between the NheI of pET28a and XhoI double enzyme site; Again pET28a-lacS-mut is imported to BL21(DE3) competent cell in, obtain BL21(DE3)/pET28a-lacS-mut.
Adopting uses the same method proceeds to BL21(DE3 by plasmid pET28a-lacS) competent cell in, obtain recombinant bacterium BL21(DE3)/pET28a-lacS.
Embodiment 2, lacS-mut have beta-glycosidase activity
One, the acquisition of beta-glycosidase mutant
1, abduction delivering
By the BL21(DE3 of above-mentioned acquisition) single colony inoculation of/pET28a-lacS-mut is to the LB liquid nutrient medium that contains kantlex (final concentration is 50 μ g/ml), cultivate 12h for 37 ℃, collect fermented liquid, by fermented liquid according to 1%(volumn concentration) inoculum size be forwarded in the LB liquid nutrient medium that 100ml is fresh, 37 ℃ are cultured to OD
600reach 0.6, then in substratum, add aseptic IPTG, making the final concentration of IPTG in substratum is that 0.4mM induces fermentation at 30 ℃.After 16h, finish fermentation, centrifugal fermented liquid 4000g 10min is collected to thalline.
Thalline is resuspended in to the binding buffer liquid that 50ml concentration is 50mM (Tris-hydrochloric acid, pH value is 8, contain 300mM NaCl and 10mM imidazoles), and ultrasonication (200W, work 3s suspends 3s, work 100 times), centrifugal (15,700g, 4 ℃, 30min) remove cell debris, collect supernatant liquor.
2, purifying
By above-mentioned supernatant liquor after 0.22 μ m membrane filtration, by the condition of purifying be:
Rinsing damping fluid: 50 mM pH8 Tris-HCl, 300mM NaCl, 20 mM imidazoles;
Elution buffer: 50 mM pH8 Tris-HCl, 300mM NaCl, 250 mM imidazoles.
Collect effluent liquid, through dialysis treatment, (dialyzate is that pH value is 5.5 MC damping fluid, and it is that 200mM Sodium phosphate dibasic, final concentration are that 100mM citric acid and water form by final concentration; The molecular weight cut-off 8000-14000 of dialysis tubing) after, purifying enzyme liquid (sudden change) obtained.
With method validations such as enzyme test alive and SDS-PAGE, confirm the purity of purified components; With Bradford determination of protein concentration method test purifying protein concentration.
Adopting and use the same method recombinant bacterium BL21(DE3)/pET28a-lacS carries out abduction delivering, purifying, obtains purifying enzyme liquid (wild).
Two, the application in the active detection of beta-glycosidase and production Ginsenoside compound K
By 10 μ l purifying enzyme liquid obtained above (sudden change), 10 μ l ginsenoside substrate Rb
1(the MC damping fluid that pH value is 5.5, it is that 200mM Sodium phosphate dibasic, final concentration are that 100mM citric acid and water form by final concentration), 85 ℃ of reaction 1h times, reaction final vacuum dry sample also dissolves again with methyl alcohol, and high pressure liquid chromatography detects.High pressure liquid chromatography testing conditions is that (m), column temperature is 35 ℃ to 5 μ to C18 post for Waters Symmetry, 250mm × 4.6mm, and moving phase is acetonitrile.0-20min, 70-60% water, 30-40% acetonitrile; 2-35min, 60-0% water, 40-100% acetonitrile; 35-40min, 100% acetonitrile; 40-45min, 70% water, 30% acetonitrile.Flow velocity is 1.0ml/min, and detection wavelength is 203nm.Take purifying enzyme liquid (wild) as contrast.Standard substance ginsenoside Rb
1(Beijing lark waffle learn a skill company limited, production code member ASB-00007190-010) and Ginsenoside compound K (Chengdu Purification Technology Development Co., Ltd., production code member G4038) reference.
The retention time of result standard product Ginsenoside compound K is 32.402min, and the retention time of purifying enzyme liquid (wild) reaction after product and purifying enzyme liquid (sudden change) reaction after product is respectively 32.397min and 32.400min; Illustrate and obtain Ginsenoside compound K.
In purifying enzyme liquid (wild) reaction after product, the amount of Ginsenoside compound K is 0.09g/L/h;
In purifying enzyme liquid (sudden change) reaction after product, the amount of Ginsenoside compound K is 0.24g/L/h.
The above results shows, mutant generates 2-5 that the ability of CK is wild-type doubly.
Claims (9)
1. an albumen is following (a) or (b):
(a) protein being formed by the aminoacid sequence shown in sequence in sequence table 4;
(b) aminoacid sequence shown in sequence in sequence table 4 is passed through to replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and has the protein that beta-glycosidase activity is derived by sequence 4.
2. the gene of albumen described in coding claim 1.
3. gene as claimed in claim 2, is characterized in that: described gene is the DNA molecular of following (1) or (2) or (3):
(1) DNA molecular shown in sequence 3 in sequence table;
(2) the DNA sequence dna hybridization limiting with (1) under stringent condition and coding have the DNA molecular of beta-glycosidase activated protein;
(3) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have 99% homology and coding and have a DNA molecular of beta-glycosidase activated protein.
4. contain recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium of gene described in claim 2 or 3.
5. recombinant vectors as claimed in claim 4, is characterized in that:
Described recombinant vectors is that the encoding gene of albumen described in claim 1 is inserted in expression vector, obtains expressing the recombinant vectors of albumen described in claim 1.
Described in claim 1 albumen in the application as in beta-glycosidase.
Described in claim 1 described in albumen, claim 2 or 3 described in gene or claim 4 recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium in the application of producing in Ginsenoside compound K.
8. a method of producing Ginsenoside compound K, comprises the steps:
1) recombinant bacterium described in fermentation claim 4, centrifugal collection supernatant liquor after collection fermented liquid, centrifugal collection thalline, broken thalline;
2) by described supernatant liquor and ginsenoside substrate Rb
1in MC damping fluid, react, obtain Ginsenoside compound K.
9. method according to claim 8, is characterized in that:
1) described fermentation is carried out under IPTG induction;
2) described MC damping fluid is that pH value is 5.5 MC damping fluid; The MC damping fluid that described pH value is 5.5 is specifically that 200mM Sodium phosphate dibasic, final concentration are that 100mM citric acid and water form by final concentration.
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| CN106011106B (en) * | 2016-06-20 | 2017-05-10 | 云南与诺生物工程有限责任公司 | Enzyme, encoding gene thereof, application of enzyme and encoding gene, and method for preparing ginseng saponin compound K |
| CN110325648A (en) * | 2016-09-28 | 2019-10-11 | 株式会社爱茉莉太平洋 | The method for producing the composition of ginsenoside compound K and preparing ginsenoside compound K comprising high temperature α-l-arabfuranglycosidase |
| CN108220308A (en) * | 2016-12-09 | 2018-06-29 | 中粮集团有限公司 | Beta galactosidase and its gene and carrier and bacterial strain and product containing the enzyme and by lactose and the fructose converting method for lactulose |
| CN110004099A (en) * | 2018-01-04 | 2019-07-12 | 安徽正方生物科技有限公司 | A kind of fermentation method for producing of rhodioside |
| CN110004099B (en) * | 2018-01-04 | 2021-06-15 | 孙敬方 | Fermentation production method of salidroside |
| CN111304272A (en) * | 2019-12-24 | 2020-06-19 | 昆明理工大学 | Method for preparing rare ginsenoside by converting panax notoginseng stem and leaf saponin through fusarium verticillii |
| CN111304272B (en) * | 2019-12-24 | 2023-05-26 | 昆明理工大学 | Method for preparing rare ginsenoside from caulis et folium Notoginseng saponin converted from Fusarium verticillatum |
| CN116590259A (en) * | 2023-05-30 | 2023-08-15 | 西北大学 | Beta-glycosidase SS-BGL mutant for modifying ginsenoside and application thereof |
| CN116590259B (en) * | 2023-05-30 | 2023-11-03 | 西北大学 | Beta-glycosidase SS-BGL mutant for modifying ginsenoside and application thereof |
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