CN105779406B

CN105779406B - A kind of crocetin glycosyl transferase and its encoding gene and application

Info

Publication number: CN105779406B
Application number: CN201610355942.0A
Authority: CN
Inventors: 赖成霞
Original assignee: INSTITUTE OF CASH CROPS XINJIANG ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: INSTITUTE OF CASH CROPS XINJIANG ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2016-05-25
Filing date: 2016-05-25
Publication date: 2019-06-18
Anticipated expiration: 2036-05-25
Also published as: CN105779406A

Abstract

The invention discloses a kind of crocetin glycosyl transferase and its encoding gene and applications.Crocetin glycosyl transferase provided by the present invention is following a1) or a2) or a3) or a4): a1) amino acid sequence is protein shown in sequence 2 in sequence table；A2) the fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；A3) amino acid sequence is protein shown in sequence 4 in sequence table；A4) by a1) or a2) or a3) shown in substitution and/or deletion and/or addition obtained protein with crocetin glycosyl transferase activity of the protein by one or several amino acid residues.It is demonstrated experimentally that crocetin glycosyl transferase provided by the present invention, has using crocetin as the glycosyl transferase activity of substrate, it can sequentially glycosylate crocetin and form crocin glycosides 5 and crocin glycosides 3, there is significant application value.

Description

A kind of crocetin glycosyl transferase and its encoding gene and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of crocetin glycosyl transferase and its encoding gene with answer With.

Background technique

Safflower is called safron, west safflower, is perennial irides safron (Crocus sativus L.) Drying column cap, be a kind of rare Chinese medicine in China.The total glycosides of safflower is the pigment compound extracted from safflower, master Wanting ingredient is crocin (crocus), crocetin (crocetin) and its derivative, and wherein crocin is crocetin A series of ester glycosides being combined into different sugar, are one of main actives of safflower, have platelet aggregation-against, anti-blood The multiple pharmacological effects such as bolt forms, resists myocardial ischemia, anticancer, and almost non-toxic side effect.

Existing research shows that crocetin glycosyl transferase has the function of that glycosylating crocetin forms crocin, Such as Morgan in 2004 separates discovery UGTCs2 gene (GeneID:AY36026) from safflower style, prokaryotic expression Crude protein has the function of that glycosylating crocetin forms crocin, therefore is crocetin glycosyl by UGTCs2 gene annotation The encoding gene of transferase；For another example Mai Nagatoshi in 2012 separates discovery UGT75L6 gene from cape jasmine cell (GeneID:AB555731) and UGT94E5 gene (GeneID:555739), the protein crude extract administration of UGT75L6 gene prokaryotic Have the function of that sequence glycosylates crocetin and forms crocin glycosides 5, crocin glycosides 3 and crocin glycosides 4, UGT94E5 The function that there is the protein crude extract administration of gene prokaryotic glycosylation crocin glycosides 5 to form crocin glycosides 2 and crocin glycosides 1 Can, therefore, UGT75L6 gene and UGT94E5 gene are also annotated as the encoding gene of crocetin glycosyl transferase.But it cuts To currently, can not still obtain the crocetin glycosyl transferase of high-purity.

Summary of the invention

The technical problem to be solved by the present invention is to how obtain the crocetin glycosyl transferase of high-purity.

In order to solve the above technical problems, present invention firstly provides a kind of protein.

Protein provided by the present invention, entitled protein UGTCs4, the protein UGTCs4 can be following a1) or A2) or a3) or a4):

A1) amino acid sequence is protein shown in sequence 2 in sequence table；

A2) the fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；

A3) amino acid sequence is protein shown in sequence 4 in sequence table；

A4) by a1) or a2) or a3) shown in protein pass through one or several amino acid residues substitution and/or missing And/or the protein with crocetin glycosyl transferase activity that addition obtains.

Wherein, sequence 2 can be made of 459 amino acid residues in sequence table；Sequence 4 can be by 492 amino in sequence table Sour residue composition.

In order to make a1) in protein convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or Carboxyl terminal connects upper label as shown in Table 1.

The sequence of table 1, label

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

Above-mentioned a4) in protein, the substitution and/or deletion and/or addition of one or several amino acid residues be No more than the substitution and/or deletion and/or addition of 10 amino acid residues.

Above-mentioned a4) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.

Above-mentioned a4) in the encoding gene of protein can be by will be shown in sequence 3 in sequence 1 in sequence table or sequence table The codon of one or several amino acid residues is lacked in DNA sequence dna, and/or the missense of one or several base-pairs of progress is dashed forward Become, and/or is obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.

The nucleic acid molecules of code for said proteins UGTCs4 also belong to protection scope of the present invention.

The nucleic acid molecules of the code for said proteins UGTCs4, concretely following b1) or b2) or b3) or b4) shown in DNA molecular:

B1) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；

B2) nucleotide sequence is DNA molecular shown in sequence 3 in sequence table；

B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encoding said proteins The DNA molecular of matter UGTCs4；

B4) the nucleotide sequence hybridization limited under strict conditions with b1) or b2), and code for said proteins UGTCs4 DNA molecular.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.

Wherein, sequence 1 is made of 1377 nucleotide in sequence table, the nucleotide coding sequence table of sequence 1 in sequence table Amino acid sequence shown in middle sequence 2；Sequence 3 is made of 1476 nucleotide in sequence table, the nucleotide of sequence 3 in sequence table Amino acid sequence shown in sequence 4 in polynucleotide.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of code for said proteins UGTCs4 of the invention.Those have by manually modified The nucleotide sequence 75% of the isolated protein UGTCs4 or the nucleotide of higher identity with the present invention, as long as Code for said proteins UGTCs4 is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair The nucleotide sequence of the protein of the composition of amino acid sequence shown in the sequence 2 of bright polynucleotide or the sequence 4 of sequence table Core with 75% or higher or 80% or higher or 85% or higher or 90% or higher or 95% or higher identity Nucleotide sequence.Identity can with the naked eye or computer software is evaluated.Using computer software, two or more sequences it Between identity can be indicated with percentage (%), can be used to evaluate identity between correlated series.

The expression cassettes of nucleic acid molecules containing code for said proteins UGTCs4, recombinant vector, recombinant microorganism turn base Because cell line also belongs to protection scope of the present invention.

The recombinant vector of the nucleic acid molecules containing code for said proteins UGTCs4 can be at more grams of expression vector The recombinant plasmid that DNA molecular shown in the sequence 1 of grand site insetion sequence table obtains.

The expression vector concretely carrier pET-28a (+).

The recombinant vector of the nucleic acid molecules containing code for said proteins UGTCs4 is concretely in carrier pET-28a The recombination matter that DNA molecular shown in the sequence 1 of insetion sequence table obtains between I recognition site of I recognition site of Nde and Sal of (+) Grain pET28a-UGTCs4.

The recombinant microorganism of the nucleic acid molecules containing code for said proteins UGTCs4 can be will be any of the above-described described The recombinant vector of nucleic acid molecules containing code for said proteins UGTCs4 imports the recombinant bacterium that the microorganism that sets out obtains.

The recombinant microorganism of the nucleic acid molecules containing code for said proteins UGTCs4 is concretely by the recombination Plasmid pET28a-UGTCs4 imports the recombinant bacterium that the microorganism that sets out obtains.

The microorganism that sets out can be Escherichia coli.

The Escherichia coli concretely Escherichia coli Rosetta (DE3).

The application of the protein UGTCs4 also belongs to protection scope of the present invention.

The application of the protein UGTCs4 can be following c1) or c2):

C1) the application as crocetin glycosyl transferase；

C2) the application in the product that preparation has crocetin glycosyl transferase function.

The meaning of the crocetin glycosyl transferase are as follows: there is catalysis crocetin the enzyme of glycosylated function occurs.

The application of the protein UGTCs4 can be following d1) or d2):

D1 the application in glycosylation) occurs in catalysis crocetin；

D2 application in glycosylated product) occurs for being catalyzed crocetin in preparation.

The application of the protein UGTCs4 can be following e1) or e2):

E1) the application in production crocin glycosides 5 and/or crocin glycosides 3；

E2) application in the product for producing crocin glycosides 5 and/or crocin glycosides 3 is being prepared.

The e2) in, " production crocin glycosides 5 and/or the crocin glycosides 3 " is using crocetin as substrate 's.

It is demonstrated experimentally that protein UGTCs4 provided by the present invention is crocetin glycosyl transferase, have with safflower Acid is the glycosyl transferase activity of substrate, can sequentially glycosylate crocetin and form crocin glycosides 3 and crocin glycosides 5.Cause This, can use protein UGTCs4 production crocin glycosides 5 and/or crocin glycosides 3.Protein provided by the present invention UGTCs4 has the product of crocetin glycosyl transferase function to production crocin glycosides 5 and/or crocin glycosides 3, preparation Or catalysis crocetin occurs glycosylation and all has significant application value.

Detailed description of the invention

Fig. 1 is the pcr amplification product of UGTCs4 gene.

Fig. 2 is the influence of expression of the various concentration IPTG to recombination crocetin glycosyl transferase.

Fig. 3 is the influence of expression of the different induction times to recombination crocetin glycosyl transferase.

Fig. 4 is the influence of expression of the different cell concentrations to recombination crocetin glycosyl transferase.

Fig. 5 is the influence of expression of the different inducing temperatures to recombination crocetin glycosyl transferase.

Fig. 6 is the influence of the expression of different inducing temperatures and induction time to recombination crocetin glycosyl transferase.

Fig. 7 is the SDS-PAGE result using ni-sepharose purification crocetin glycosyl transferase solution.

The experimental result that Fig. 8 is in 2 step 2 of embodiment 1.

The experimental result that Fig. 9 is in 2 step 2 of embodiment 2.

The experimental result that Figure 10 is in 2 step 2 of embodiment 3.

The experimental result that Figure 11 is in 2 step 2 of embodiment 4.

Figure 12 is the mass spectrogram of crocetin.

Figure 13 is the mass spectrogram of crocin glycosides 5.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

Carrier pET28a (+) is invitrogen Products；E. coli bl21 (DE3) is Tiangeng biochemical technology (north Capital) Co., Ltd's product；Nickel column is U.S. GE Products, catalog number 04003272-EE.DTT is Amersco company Product, catalog number 0281.UDPG (UDP-Glucose) is sigma Products, catalog number U4625.Safranine Spending acid is Chromedex Products, catalog number ASB00003885-010.C18 reversed-phase column (4.6mm × 250mm) is Japanese GL Sciences Inc product.High performance liquid chromatograph and diode array detector are Japanese Shimadzu Corporation product.It is super It is Xin Zhi bio tech ltd, Ningbo product that sound, which is crushed instrument,.

Safflower suspension cell in following embodiments is with reference to Chen Shuan, Wang Xiaodong etc., safflower cell suspension cultures The foundation of system and optimization biotechnology are notified to 2010. (07): the method preparation in 157-160..

LB liquid medium: being dissolved in 100mL deionized water for 1g NaCl, 0.5g yeast extract and 1g tryptone, adjusts Saving pH value is 7.0.

Sample-loading buffer is containing 2% (mass volume ratio) SDS, 10% (percent by volume) glycerol and 1% (volume basis Than) pH6.8, the 50mM Tris-HCl buffer of beta -mercaptoethanol.

Lysis buffer is pH7.4,100mmol/L Tris-HCl of NaCl containing 300mmol/L and 10mmol/L imidazoles Buffer.

Washing buffer is pH7.4, the 100mmol/L Tris-HCl buffer of the NaCl containing 300mmol/L.

Protein shown in sequence 2 by sequence table is named as protein UGTCs4, the sequence of encoding gene such as sequence table Shown in column 1.

Embodiment 1, the preparation for recombinating crocetin glycosyl transferase

One, the building of recombinant plasmid

1, the total serum IgE reverse transcriptase reverse transcription is gone out the by the total serum IgE that safflower suspension cell is extracted using Trizol method One chain cDNA.

2, artificial synthesized primers F 1:5 '-CCATATG(underscore is in restricted to GAGCAGAAAGATGAGAACGGAA-3 ' Enzyme cutting Nde I identifies sequence) and R1:5 '-CGTCGAC(underscore is restricted to TTTACAACAATGATCAATGAACTCCT-3 ' Restriction endonuclease Sal I identifies sequence).

3, for the cDNA obtained using step 1 as template, the F1 and R1 synthesized using step 2 carries out PCR amplification as primer, obtains about The pcr amplification product of 1388bp.It is detected with 1% agarose gel electrophoresis, (M is DNA Marker, swimming lane 1 to the result is shown in Figure 1 For pcr amplification product, arrow meaning is target PCR amplifications product).

Response procedures: 94 DEG C of 5min；94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 1min 30s, totally 35 recycle；72℃10min.

4, the pcr amplification product obtained in I double digestion step 3 of restriction enzyme Nde I and Sal recycles about 1386bp Segment.

5, with I double digestion carrier pET28a (+) of restriction enzyme Nde I and Sal, the carrier framework of about 5350bp is recycled.

6, the segment that step 4 obtains is connect with the carrier framework that step 5 obtains, obtains recombinant plasmid pET28a- UGTCs4。

According to sequencing result, structure is carried out to recombinant plasmid pET28a-UGTCs4 and is described as follows: by carrier pET28a (+) Nde I to identify that the DNA small fragment between sequence and Sal I identify sequence replaces with nucleotide sequence be in sequence table shown in sequence 1 DNA molecular.In recombinant plasmid pET28a-UGTCs4, on DNA molecular shown in the sequence 1 of sequence table and carrier framework The coded sequence of His-tag label (being made of 6 histidine residues) merges, and merges base shown in the sequence 3 of formation sequence table Cause, with the protein UGTCs4 of His-tag label shown in the sequence 4 of expressed sequence table, the albumen with His-tag label Matter UGTCs4 is to recombinate crocetin glycosyl transferase.The expection molecular weight of protein shown in the sequence 4 of sequence table is 54.8KD。

Two, groping for the optimum condition of the expression of crocetin glycosyl transferase is recombinated

1, influence of the various concentration IPTG to the expression of recombination crocetin glycosyl transferase

1. the recombinant plasmid pET28a-UGTCs4 of step 1 building is imported Escherichia coli Rosetta (DE3), weight is obtained Group bacterium, is named as Rosetta (DE3)-pET28a-UGTCs4 for the recombinant bacterium.

2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture bacterium solution 10.3mL is taken, with 1:100 (volume Than) it is inoculated in LB liquid medium of the 30mL containing 50 μ g/mL kanamycins, 37 DEG C, 180rpm shaken cultivation to OD₆₀₀Value 0.3~ 0.4, obtain culture bacterium solution 2.

3. after completing step 2., taking culture bacterium solution 2, centrifugation, the thallus before obtaining IPTG induction.

4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system Degree is 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM or 700 μM, then 37 DEG C, 120rpm shaken cultivation 2.5h, thalline were collected by centrifugation, the thallus after obtaining IPTG induction.

5. the thallus after thallus and IPTG induction before taking IPTG to induce respectively, is added LB liquid medium to OD₆₀₀Value is adjusted Whole to consistent, 25 DEG C, 9000rpm centrifugation 1min abandon supernatant, collect thallus.

6. the thallus for taking step 5. to obtain, is added sample-loading buffer, boiling water bath boils 13min；Then 25 DEG C, 9000rpm It is centrifuged 10min, supernatant is collected and carries out SDS-PAGE.

Experimental result is shown in Fig. 2, (M is albumen marker, and 1 is the thallus before IPTG induction, after 2 is 50 μM of IPTG inductions Thallus, 3 be the thallus after 100 μM of IPTG inductions, and 4 be the thallus after 200 μM of IPTG inductions, after 5 is 300 μM of IPTG inductions Thallus, 6 be the thallus after 400 μM of IPTG induction, and 7 be the thallus after 500 μM of IPTG inductions, and 8 be 600 μM of IPTG inductions Thallus afterwards, 9 be the thallus after 700 μM of IPTG inductions, and arrow meaning is target protein).The result shows that recombination crocetin The best IPTG induced concentration of the expression of glycosyl transferase is 50 μM~600 μM；When IPTG induced concentration is 700 μM, recombination The expression of crocetin glycosyl transferase has a degree of inhibition.

2, influence of the difference IPTG induction time to the expression of recombination crocetin glycosyl transferase

1. in step 1 1..

2. in step 1 2..

3. in step 1 3..

4. after completing step 2., after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG exists Concentration in induction system is 200 μM, then 37 DEG C, 120rpm shaken cultivation, incubation time 30min, 1h, 1.5h, 2h, 2.5h, 3h or 4h, thalline were collected by centrifugation, the thallus after obtaining IPTG induction.

5. in step 1 5..

6. in step 1 6..

Experimental result is shown in Fig. 3, (M is albumen marker, and 1 is the thallus before IPTG induction, after 2 induce 30min for IPTG Thallus, 3 induce the thallus after 1h for IPTG, and 4 induce the thallus after 1.5h for IPTG, and 5 induce the thallus after 2h for IPTG, and 6 are IPTG induces the thallus after 2.5h, and 7 induce the thallus after 3h for IPTG, and 8 induce the thallus after 4h for IPTG, and arrow meaning is mesh Mark albumen).The result shows that the best IPTG induction time of the expression of recombination crocetin glycosyl transferase is 30min~4h.

3, influence of the different cell concentrations to the expression of recombination crocetin glycosyl transferase

1. in step 1 1..

2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture bacterium solution 10.3mL is taken, with 1:100 (volume Than) it is inoculated in LB liquid medium of the 30mL containing 50 μ g/mL kanamycins, 37 DEG C, 180rpm shaken cultivation obtain culture bacterium solution 2, cultivate the OD of bacterium solution 2₆₀₀Value is 0.32,0.41,0.54,0.63 or 0.76.

3. in step 1 3..

4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system Degree is 200 μM, and then 37 DEG C, 120rpm shaken cultivation 2.5h, thalline were collected by centrifugation, the thallus after obtaining IPTG induction.

5. in step 1 5..

6. in step 1 6..

Experimental result is shown in Fig. 4, (M is albumen marker, and 1 is the thallus before IPTG induction, and 2 be OD₆₀₀The culture that value is 0.32 Thallus after bacterium solution 2IPTG induction, 3 be OD₆₀₀Value is the thallus after 0.41 culture bacterium solution 2IPTG induction, and 4 be OD₆₀₀Value is Thallus after 0.54 culture bacterium solution 2IPTG induction, 5 be OD₆₀₀Thallus after the culture bacterium solution 2IPTG induction that value is 0.63,6 For OD₆₀₀Value is the thallus after 0.76 culture bacterium solution 2IPTG induction, and arrow meaning is target protein).The result shows that recombination hiding The OD of the best cell concentration of the expression of safflower acid glycosyl transferase₆₀₀Value is 0.3~0.8.

4, influence of the different inducing temperatures to the expression of recombination crocetin glycosyl transferase

(1) influence of 28 DEG C of the inducing temperature expression to recombination crocetin glycosyl transferase

1. in step 1 1..

2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture bacterium solution 10.3mL is taken, with 1:100 (volume Than) it is inoculated in LB liquid medium of the 30mL containing 50 μ g/mL kanamycins, 28 DEG C, 120rpm shaken cultivation to OD₆₀₀Value 0.3~ 0.4, obtain culture bacterium solution 2.

3. in step 1 3..

4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system Degree is 200 μM, and then 28 DEG C, 120rpm shaken cultivation, bacterium is collected by centrifugation in incubation time 3h, 4h, 5h, 6h, 7h, 8h or 9h Body, the thallus after obtaining IPTG induction.

5. in step 1 5..

6. in step 1 6..

Experimental result is shown in Fig. 5, (M is protein low molecular weight marker, and 1 induces for the thallus before IPTG induction, 2 for IPTG Thallus after 3h, 3 induce the thallus after 4h for IPTG, and 4 induce the thallus after 5h for IPTG, and 5 induce the thallus after 6h for IPTG, 6 induce the thallus after 7h for IPTG, and 7 induce the thallus after 8h for IPTG, and 8 induce the thallus after 9h for IPTG, and arrow meaning is Target protein).The result shows that recombination crocetin glycosyl transferase is primarily present in inclusion body, it is seen that recombination crocetin sugar The temperature of the expression of based transferase can not be 28 DEG C.

(2) influence of 37 DEG C of the inducing temperature expression to recombination crocetin glycosyl transferase

According to the method for step (1), 37 DEG C are replaced with by 28 DEG C, other steps are constant, as a result recombinate crocetin sugar Based transferase is also primarily present in inclusion body, it is seen that the temperature of the expression of recombination crocetin glycosyl transferase can not be 37 DEG C.

(3) influence of 18 DEG C of the inducing temperature expression to recombination crocetin glycosyl transferase

1. in step 1 1..

2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture bacterium solution 10.3mL is taken, with 1:100 (volume Than) 30mL is inoculated in containing 50 μ g/mL kanamycins LB liquid mediums, 18 DEG C, 120rpm shaken cultivation to OD₆₀₀Value 0.3~ 0.4, obtain culture bacterium solution 2.

3. in step 1 3..

4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system Degree is 200 μM, then 18 DEG C, 120rpm shaken cultivation, incubation time 6h, 8h, 10h, 12h, 14h, 16h or 18h, and centrifugation is received Collect thallus, the thallus after obtaining IPTG induction.

5. in step 1 5..

6. in step 1 6..

Experimental result is shown in Fig. 6, (M is albumen marker, and 1 is the thallus before IPTG induction, and 2 induce the bacterium after 6h for IPTG Body, 3 induce the thallus after 8h for IPTG, and 4 induce the thallus after 10h for IPTG, and 5 induce the thallus after 12h for IPTG, and 6 are IPTG induces the thallus after 14h, and 7 induce the thallus after 16h for IPTG, and 8 induce the thallus after 18h for IPTG, and arrow meaning is Target protein).The result shows that 18 DEG C, the inducible expression for recombinating crocetin glycosyl transferase of IPTG induction 6h~18h.

Three, the expression and purification of crocetin glycosyl transferase is recombinated

1. recombinant plasmid pET28a-UGTCs4 is imported Escherichia coli Rosetta (DE3), recombinant bacterium is obtained, by the recombination Bacterium is named as Rosetta (DE3)-pET28a-UGTCs4.

3. after completing step 2., taking culture bacterium solution 2,5000rpm is centrifuged 10min, collects thallus, before obtaining IPTG induction Thallus.

4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system Degree is 200 μM, and then 18 DEG C, 120rpm shaken cultivation 16h, 5000rpm centrifugation 10min collect thallus, after obtaining IPTG induction Thallus.

5. the thallus after taking IPTG to induce, washed once with lysis buffer.

6. taking into the thallus of step 5., lysis buffer is added with 1:10 (volume ratio), -20 DEG C is put into and freezes, obtain Freeze bacterium solution.

7. take into step 6. freeze bacterium solution, rapidly thaw after, set on ice on Ultrasonic Cell Disruptor ultrasonication (ultrasound Wave power 100W, cyclic program are as follows: broken 6s stops 4s, totally 120 circulations), then 4 DEG C, 13000rpm centrifugation 30min are collected Supernatant, the bacterial cell disruption supernatant after being named as IPTG induction.

According to 5. to 7. the step of, the thallus after IPTG is induced replace with IPTG induction before thallus, obtain IPTG and lure The bacterial cell disruption supernatant of leading.

8. first balancing nickel column (flow velocity 1mL/min) with the lysis buffer of 5~10 column volumes, then loading step is 7. Bacterial cell disruption supernatant (flow velocity 1mL/min) after obtained IPTG induction, then successively with 5 column volumes containing 20mmol/L The washing buffer of imidazoles, 5 column volumes the imidazoles containing 80mmol/L washing buffer and 5 column volumes contain 100mmol/ The washing buffer of L imidazoles rinses nickel column, to remove most of foreign protein, finally with the miaow containing 250mmol/L of 5 column volumes The washing buffer of azoles rinses nickel column and collected solution after column, as recombination crocetin glycosyl transferase solution.

Bacterial cell disruption supernatant after IPTG is induced, the bacterial cell disruption supernatant before IPTG induction and step 8. in respectively wash The elution collection liquid of de- step carries out SDS-PAGE.

Experimental result is shown in Fig. 7, (M is protein molecular weight Marker, and 1 is the bacterial cell disruption supernatant before IPTG induction, and 2 are Bacterial cell disruption supernatant after IPTG induction, the 3 elution collection liquids eluted for the washing buffer of the imidazoles containing 20mmol/L, The 4 elution collection liquids eluted for the washing buffer of the imidazoles containing 80mmol/L, 5 is slow for the washing of the imidazoles containing 100mmol/L The elution collection liquid that fliud flushing is eluted, the 6 elution collection liquids eluted for the washing buffer of the imidazoles containing 250mmol/L). The result shows that recombination crocetin glycosyl transferase solution shows that unimodal molecular weight band, corresponding molecular weight are 54.8KDa, with It is expected that molecular weight is consistent.

The content of crocetin glycosyl transferase is recombinated in bacterial cell disruption supernatant after 7. IPTG induction that step obtains Crocetin glycosyl transferase is recombinated in the recombination crocetin glycosyl transferase solution 8. obtained for 0.03 μ g/ μ L, step Content is 2.13 μ g/ μ L.Recombination crocetin glycosyl transfer in bacterial cell disruption supernatant after 7. IPTG induction that step obtains The calculation method of the content of enzyme are as follows: the total protein content of liquid-phase system is detected, then by each band in protein electrophoresis It carries out gray scale scanning and calculates recombination crocetin glycosyl transferase ratio shared in total protein, recombination safflower is calculated The content of sour glycosyl transferase.Crocetin glycosyl is recombinated in 8. recombination crocetin glycosyl transferase solution that step obtains to turn The content for moving enzyme is measured according to Bradford method.

Embodiment 2, the glycosyl transferase that recombination crocetin glycosyl transferase under the conditions of differential responses is detected using HPLC Activity

One, it dialyses

Under the conditions of 4 DEG C, the recombination crocetin glycosyl transferase solution that embodiment 1 obtains is subjected to dialysis desalination, then It replaces pH7.5, in 5mM Tris-HCl buffer, finally carries out ultrafiltration concentration egg using the super filter tube that molecular weight is 30MW It is white, obtain the protein sample for the recombination crocetin glycosyl transferase that purity is 98%.The protein sample is subjected to 25 ammonia of N-terminal The sequencing of base acid residue, the results showed that, in the sequence such as sequence table of 25 amino acid of the albumen n end sequence 4 from N-terminal the 1st to Shown in 25.

Two, the glycosyl transferase activity of recombination crocetin glycosyl transferase under the conditions of differential responses is detected using HPLC

1, influence of the differential responses time to the glycosyl transferase activity of recombination crocetin glycosyl transferase

(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH7.5, 50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of MgCl₂, 10 μM of safflowers The recombination crocetin glycosyl transferase that acid and 16 μ g step 1 obtain.

(2) by step (1) prepare system be placed in 37 DEG C under the conditions of, then respectively react 0min, 30min, 1h, 1.5h, 2h, 2.5h, 3h or 4h.

(3) after completing step (2), 400 μ L hplc grade methanols is added and terminate reaction, then 13000rpm is centrifuged 10min, receives Collect supernatant and through 0.45 μm of organic membrane filtration, obtains the to be measured of the external enzymatic reaction of recombination crocetin glycosyl transferase Sample.

(4) sample to be tested and hiding of the external enzymatic reaction for the recombination crocetin glycosyl transferase for taking step (3) to obtain Safflower acid standard solution (dissolving crocetin to its concentration with methanol is 10 μM), carries out the detection of HPLC-MS.Using being furnished with The high performance liquid chromatograph and UV-vis detector of C18 reversed-phase column (4.6mm × 150mm) carry out HPLC detection.Mobile phase by Methanol (A) and water (B) composition, flow velocity 1mL/min use following condition of gradient elution: in 0~20min, first in mobile phase The volumn concentration of alcohol at the uniform velocity increases to 50% by 20%, and the volumn concentration of water is at the uniform velocity down to 50% by 80%, carries out line Property gradient elution；In 20~50min, the volumn concentration of methanol at the uniform velocity increases to 70% by 50% in mobile phase, the body of water Product percentage composition is at the uniform velocity down to 30% by 50%, carries out linear gradient elution；In 50~55min, with by first alcohol and water according to The mobile phase 1 that the volume ratio of 7:3 is mixed to get is eluted；In 55~60min, eluted with methanol；In 60~65min Interior, the volumn concentration of methanol is at the uniform velocity down to 20% by 100% in mobile phase, and the volumn concentration of water is at the uniform velocity risen by 0% To 80%, linear gradient elution is carried out；In 65~70min, with the stream being mixed to get by first alcohol and water according to the volume ratio of 2:8 Dynamic phase 2 is eluted.Detection wavelength is 440nm.(methanol dissolves crocetin to Simultaneous Quantitative Analysis crocetin standard solution It is 10 μM to its concentration).Mass Spectrometer Method condition are as follows: scanning of the mass spectrum range 100-2000 (m/z)；Carrier gas: high pure nitrogen, carrier gas stream Speed: 40arb, secondary air speed: 2arb, blowback gas velocity: 1arb, capillary temperature: 275 DEG C, capillary voltage: 100V, spray Mist voltage: 3.2V.Experiment in triplicate, repeats to set 10 triangular flasks every time.

Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in Fig. 8 (CK is crocetin standard items, and abscissa is retention time), under the chromatographic condition, the retention time of crocetin is 35min, the retention time of crocin glycosides 5 are 25min.Experimental result is shown in Figure 12, safflowers for the high resolution mass spectrum of crocetin Experimental result is shown in Figure 13 for the high resolution mass spectrum of plain glycosides 5.The result shows that sugar of the reaction time to recombination crocetin glycosyl transferase Based transferase activity influence is more significant, and with the extension of reaction time, crocetin concentration and crocin glycosides 3 gradually subtract Few, crocin glycosides 5 gradually increases.Therefore, there is recombination crocetin glycosyl transferase sequence glycosylation crocetin to be formed The function of crocin glycosides 3 and crocin glycosides 5, recombination crocetin glycosyl transferase prepared by embodiment 1 have with safranine Spending acid is the glycosyl transferase activity of substrate.

2, influence of the different pH value to the glycosyl transferase activity of recombination crocetin glycosyl transferase

(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH6.8, 50mM Tris-HCl buffer, pH7.5,50mM Tris-HCl buffer, pH8.0,50mM Tris-HCl buffer or PH8.8,50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of MgCl₂、10μM The recombination crocetin glycosyl transferase that crocetin and 16 μ g step 1 obtain.

(2) under the conditions of system prepared by step (1) being placed in 37 DEG C, 30min is reacted.

(3) with (3) in step 1.

(4) with (4) in step 1.

Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in Fig. 9 (CK is crocetin standard items, and abscissa is retention time), under the chromatographic condition, the retention time of crocetin is 35min.The result shows that different pH value are more significant on the glycosyl transferase activity influence of recombination crocetin glycosyl transferase, When pH value is 7.5, the glycosyl transferase activity highest of crocetin glycosyl transferase is recombinated.

3, influence of the different metal ions to the glycosyl transferase activity of recombination crocetin glycosyl transferase

(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH7.5, 50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of chlorates, 10 μM of safflowers The recombination crocetin glycosyl transferase that acid and 16 μ g step 1 obtain.Chlorate is MgCl₂、CaCl₂、MnCl₂、CuCl₂、 NaCl or ZnCl₂。

(3) with (3) in step 1.

(4) with (4) in step 1.

Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in figures Under the chromatographic condition, the retention time of crocetin is 10 (CK is crocetin standard items, and abscissa is retention time) 35min.The result shows that different metal ions influence more to show on the glycosyl transferase activity of recombination crocetin glycosyl transferase It writes: Cu²⁺(with CuCl₂Form be added), Zn²⁺(with ZnCl₂Form is added) or Mn²⁺(with MnCl₂Form is added) to recombination safranine The glycosyl transferase activity of sour glycosyl transferase is spent to have apparent inhibiting effect；Mg²⁺(with MgCl₂Form be added), Ca²⁺(with Ca Cl₂Form is added) or Na⁺(being added in the form of NaCl) has the glycosyl transferase activity of recombination crocetin glycosyl transferase bright Aobvious facilitation can promote to recombinate crocetin glycosyl transferase sequence glycosylation crocetin formation 3 He of crocin glycosides Crocin glycosides 5.

4, influence of the different concentration of substrate to the glycosyl transferase activity of recombination crocetin glycosyl transferase

(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH7.5, 50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of MgCl₂, crocetin and The recombination crocetin glycosyl transferase that 16 μ g step 1 obtain.Concentration of the crocetin in the system is 10 μM, 20 μM, 30 μM, 40 μM, 50 μM or 60 μM.

(3) with (3) in step 1.

(4) with (4) in step 1.

Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in figures Under the chromatographic condition, the retention time of crocetin is 11 (CK is crocetin standard items, and abscissa is retention time) 35min.The result shows that different concentration of substrate influence more to show on the glycosyl transferase activity of recombination crocetin glycosyl transferase It writes: when concentration of substrate is 10 μM, recombinating the glycosyl transferase activity highest of crocetin glycosyl transferase；With concentration of substrate Increase, crocin glycosides 3 constantly accumulates, and crocin glycosides 5 is constantly reduced.

Claims

1. protein, amino acid sequence is protein shown in sequence 2 in sequence table.

2. encoding the nucleic acid molecules of protein described in claim 1.

3. nucleic acid molecules as claimed in claim 2, it is characterised in that: the nucleic acid molecules is shown in sequences 1 in sequence table DNA molecular.

4. expression cassette, recombinant vector, recombinant microorganism containing nucleic acid molecules described in Claims 2 or 3.

5. recombinant vector as claimed in claim 4, it is characterised in that: the recombinant vector is in the polyclonal position of expression vector The recombinant plasmid that DNA molecular shown in the sequence 1 of point insetion sequence table obtains.

6. recombinant microorganism as claimed in claim 4, it is characterised in that: the recombinant microorganism is will be described in claim 5 Recombinant plasmid imports the recombinant bacterium that the microorganism that sets out obtains.

7. the application of protein described in claim 1 is following e1) or e2):

8. the use as claimed in claim 7, it is characterised in that: the production crocin glycosides 5 and/or crocin glycosides 3 are Using crocetin as substrate.