CN103805581B - 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, particularly relate to a kind of beta-glycosidase mutant and encoding gene and its thereof and producing the application in 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 medical functions such as promoting malignant cell decline, 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 by monose groups such as glucose from agent structure hydrolysis removing, and the method for hydrolysis has chemical hydrolysis and biocatalytic reaction method etc.Although chemical process transformation efficiency is higher, catalysis lacks specificity, thus limits it and apply widely.And biocatalytic reaction method, utilize enzyme process realize Ginsenoside compound K synthesis research fewer, the mainly Purification and 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 synthesis Ginsenoside compound K from the Polyglucosidase etc. of Thermuscaldophilus, but on the one hand has intermediate product to accumulate in lower, the conversion process of activity; The gene order of these enzymes is all without report on the other hand, cannot realize recombinant expressed.In the middle of the research of enzymatic clarification Ginsenoside compound K, not only transformation efficiency has much room for improvement, and should reduce the accumulation 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 purification work.
Beta-glycosidase (LacS, β-Glycosidase) from sulfolobus solfataricus has been in the news and has had the ability of synthesis Ginsenoside compound K, 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, be suitable for improving its catalytic efficiency further by protein engineering means; 2, the temperature of reaction of this enzyme is higher, and thermostability is very good, contributes to completing Product formation under high temperature production condition, improves transformation efficiency, reduces the possibility polluted in production process; 3, the pilot process of this Enzyme catalyzed synthesis Ginsenoside compound K is by clear explaination; 4, the crystalline structure of this enzyme is in the news, transforms further and provide important reference information to the analysis of enzyme mutant for it.
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 be made up of the amino acid residue sequence of the sequence 4 in sequence table;
2) by sequence 4 amino acid residue sequence in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation 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 one or several amino-acid residue described and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
The gene of above-mentioned albumen of encoding also is 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) DNA sequence dna limited with (1) is under strict conditions hybridized and is encoded and has the DNA molecular of beta-glycosidase activated protein;
(3) DNA sequence dna limited 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 encode and have the DNA molecular of beta-glycosidase activated protein.
Above-mentioned stringent condition is at 6 × SSC, and in the solution of 0.5% SDS, hybridize at 65 DEG C, then use 2 × SSC, 0.1% SDS and 1 × SSC, 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 containing said gene, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.
Above-mentioned recombinant vectors is insert in expression vector by the encoding gene of above-mentioned albumen, obtain the recombinant vectors of expressing above-mentioned albumen, be specially the recombinant vectors will obtained between NheI and the XhoI recognition site of the DNA molecular insertion vector pET28a shown in the sequence 3 in sequence table.
Above-mentioned albumen, said gene or above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium are also being the scope of protection of the invention as the application in beta-glycosidase.
Above-mentioned albumen is also the scope of protection of the invention producing the application 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) ferment above-mentioned recombinant bacterium, collected by centrifugation supernatant liquor after collection fermented liquid, collected by centrifugation thalline, broken thalline;
2) by described supernatant liquor and ginsenoside substrate Rb
1react in MC damping fluid, obtain Ginsenoside compound K.
In aforesaid method, 1) described fermentation is carried out under IPTG induction;
2) described MC damping fluid to be pH value be 5.5 MC damping fluid; Described pH value be 5.5 MC damping fluid specifically by final concentration be 200mM Sodium phosphate dibasic, final concentration is that 100mM citric acid and water form.
Experiment of the present invention proves, the present invention suddenlys change wild-type beta-glycosidase genes, and obtain beta-glycosidase mutant, compared with wild-type, it is significantly increased on synthesis Ginsenoside compound K vigor, therefore has good prospects for commercial application.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., 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 with it for template, with 5 '-atggctagcatgtactcatttccaaatagc-3 ' (forward) and 5 '-gtgctcgagttagtgccttaatggctttac-3 ' (reverse) for primer carries out pcr amplification.Pcr amplification condition is as follows: first 95 DEG C of denaturation 4min, then 95 DEG C of 45s, 55 DEG C of 30s, 72 DEG C of 1min30s, totally 30 circulations; Last 72 DEG C extend 10min.
Reclaim above-mentioned PCR reaction product, carry out agarose gel electrophoresis detection, result obtains the fragment of about 1470bp size.
By the PCR primer NheI of above-mentioned acquisition and XhoI double digestion, digestion products and pET28a carrier (the Novagen company of cutting through same enzyme, numbering 69864-3) to connect with T4 ligase enzyme (purchased from precious biotech firm) 16 DEG C and spend the night, connect product conversion intestinal bacteria MC1061 competent cell, obtain recombinant bacterium.
Extract the plasmid sequence verification of recombinant bacterium, sequencing analysis, the unnamed gene of this PCR primer is lacS, its nucleotides sequence is classified as the sequence 1 in sequence table, pass through comparison, it is the gene of the beta-glycosidase lacS of sulfolobus solfataricus, and the protein designations of this genes encoding is LacS, and the aminoacid sequence of this albumen is the sequence 2 in sequence table.By the plasmid called after pET28a-lacS containing this PCR primer, this plasmid is that the sequence 1 in sequence table is inserted the carrier obtained between NheI and the XhoI double enzyme site of pET28a carrier.
2, sudden change obtains beta-glycosidase mutant
On the basis of beta-glycosidase lacS gene, utilize the method for fallibility PCR, build libraries of random mutants.Mainly by changing the concentration of mn ion in PCR amplification system, improve the mispairing rate of Taq archaeal dna polymerase, thus introduce sudden change in gene order.In this research, carry out taking turns mutated library and build, obtain the PCR primer 2 of the PCR primer 1 and sudden change suddenlyd change.
By PCR primer 2 NheI and the XhoI double digestion respectively of the PCR primer 1 of said mutation and sudden change, digestion products is connected with the pET28a carrier cut through same enzyme respectively, and connection product transforms BL21(DE3 respectively) competent cell in, obtain mutated library.Screening recombinant bacterium, screening method is to be chosen by the single colony clone that contain mutant with sterile toothpick, into dividing, 800 ul LB substratum (Tryptones 10 g/L are housed between aseptic technique, NaCl 10 g/L, yeast extract 5g/L, add microbiotic kantlex to 50 μ g/mL) after 37 DEG C of shaking tables cultivate 2h, add inductor IPTG to final concentration 0.4mM, induce about 16h at 30 DEG C 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 DEG C 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 DEG C, pH5.5), with dinitrosalicylic acid system (DNS method) detection reaction by product reducing sugar content, embodies the ability transforming and generate Ginsenoside compound K indirectly.
For generating the more recombinant bacterium of reducing sugar, extract plasmid and send to order-checking, the gene of the PCR primer in this plasmid of result has the Nucleotide shown in sequence 3 in sequence table, this unnamed gene is lacS-mut, the protein designations of this genes encoding is lacS-mut, and 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 nucleotide sequence with wild-type beta-glycosidase lacS:
The aminoacid sequence (sequence 4) of lacS-mut is that from N ' end, the 218th α-amino-isovaleric acid Val sports glycine Gly by the aminoacid sequence of wild-type beta-glycosidase lacS (in sequence table sequence 2), and the nucleotide sequence (sequence 3) of lacS-mut gene is that ' end, 652-654 position GTT sports GGT from 5 by the nucleotide sequence of wild-type beta-glycosidase lacS gene (in sequence table sequence 1).Illustrate that lacS-mut is the mutant of lacS.
By the plasmid called after pET28a-lacS-mut containing lacS-mut, this plasmid is that the sequence 3 in sequence table is inserted the carrier obtained between NheI and the XhoI double enzyme site of pET28a, the recombinant bacterium called after BL21(DE3 by containing 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 inserted the pET28a-lacS-mut obtained between NheI and the XhoI double enzyme site of pET28a; Again pET28a-lacS-mut is imported 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
BL21(DE3 by above-mentioned acquisition)/pET28a-lacS-mut single colony inoculation to containing kantlex (final concentration is 50 μ g/ml) LB liquid nutrient medium in, cultivate 12h for 37 DEG C, collect fermented liquid, by fermented liquid according to 1%(volumn concentration) inoculum size be forwarded in the fresh LB liquid nutrient medium of 100ml, 37 DEG C are cultured to OD
600reach 0.6, then add aseptic IPTG in the medium, make IPTG final concentration in the medium be that 0.4mM carries out induction fermentation at 30 DEG C.Terminate fermentation after 16h, centrifugal for fermented liquid 4000g 10min is collected thalline.
Thalline is resuspended in binding buffer liquid that 50ml concentration is 50mM (Tris-hydrochloric acid, pH value is 8, containing 300mM NaCl and 10mM imidazoles), and ultrasonication (200W, work 3s suspends 3s, work 100 times), centrifugal (15,700g, 4 DEG C, 30min) remove cell debris, collect supernatant liquor.
2, purifying
By above-mentioned supernatant liquor after 0.22 μm of membrane filtration, by the condition of purifying be:
Wash buffer: 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 to be pH value be 5.5 MC damping fluid, its by final concentration be 200mM Sodium phosphate dibasic, final concentration is that 100mM citric acid and water form; The molecular weight cut-off 8000-14000 of dialysis tubing) after, obtain purifying enzyme liquid (sudden change).
The purity of the method validations such as test and SDS-PAGE, the confirmation purified components of living with enzyme; 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, beta-glycosidase Activity determination and the application of producing in Ginsenoside compound K
By 10 μ l purifying enzyme liquid (sudden change) obtained above, 10 μ l ginsenoside substrate Rb
1(pH value is the MC damping fluid of 5.5, its by final concentration be 200mM Sodium phosphate dibasic, final concentration is that 100mM citric acid and water form), 85 DEG C 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, C18 post (Waters Symmetry, 250mm × 4.6mm, 5 μm), column temperature is 35 DEG C, 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 determined wavelength is 203nm.With purifying enzyme liquid (wild) for 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; Explanation obtains 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, the ability of mutant generation CK is 2-5 times of wild-type.
Claims (11)
1. an albumen, the protein be made up of the aminoacid sequence shown in sequence in sequence table 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 as shown in sequence in sequence table 3.
4. the recombinant vectors containing gene described in Claims 2 or 3.
5. recombinant vectors as claimed in claim 4, is characterized in that:
Described recombinant vectors, for being inserted in expression vector by the encoding gene of albumen described in claim 1, obtains the recombinant vectors of expressing albumen described in claim 1.
6. the expression cassette containing gene described in Claims 2 or 3.
7. the transgenic cell line containing gene described in Claims 2 or 3.
8. the recombinant bacterium containing gene described in Claims 2 or 3.
9. albumen described in claim 1 is as the application in beta-glycosidase.
10. recombinant bacterium described in transgenic cell line described in expression cassette, claim 7 described in recombinant vectors, claim 6 described in gene, claim 4 described in albumen, Claims 2 or 3 described in claim 1 or claim 8 is producing the application in Ginsenoside compound K.
11. 1 kinds of methods of producing Ginsenoside compound K, comprise the steps:
1) ferment recombinant bacterium described in claim 8, collected by centrifugation supernatant liquor after collection fermented liquid, collected by centrifugation thalline, broken thalline; Described fermentation is carried out under IPTG induction;
2) by described supernatant liquor and ginsenoside substrate Rb
1react in MC damping fluid, obtain Ginsenoside compound K; Described MC damping fluid to be pH value be 5.5 MC damping fluid; Described pH value be 5.5 MC damping fluid specifically by final concentration be 200mM Sodium phosphate dibasic, final concentration is that 100mM citric acid and water form.
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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 |
| KR102655938B1 (en) * | 2016-09-28 | 2024-04-11 | (주)아모레퍼시픽 | Composition for producing ginsenoside compound K comprising thermo satble alpha-L-arabinofuranosidase enzyme and preparation method of ginsenoside compound K |
| CN108220308B (en) * | 2016-12-09 | 2020-12-01 | 中粮集团有限公司 | Beta-galactosidase, gene and carrier thereof, bacterial strain, product containing beta-galactosidase and method for converting lactose and fructose into lactulose |
| CN110004099B (en) * | 2018-01-04 | 2021-06-15 | 孙敬方 | Fermentation production method of salidroside |
| 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 |
| CN117210442B (en) * | 2023-05-30 | 2024-05-28 | 西北大学 | Beta-glycosidase SS-BGL mutant for modifying ginsenoside and application thereof |
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Effective date of registration: 20160314 Address after: 065400 Langfang environmental protection industrial park, Xianghe County, Hebei, China Patentee after: Xianghe Tianyi Yi Biological Technology Co., Ltd. Address before: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Patentee before: Institute of Microbiology, Chinese Academy of Sciences |