CN110172486A - A method of synthesis 2'-Fucosyl lactose - Google Patents

Abstract

The invention belongs to genetic engineering fields, are related to the production method of 2'-Fucosyl lactose, especially a kind of method for catalyzing and synthesizing 2'-Fucosyl lactose altogether using engineering strain and enzyme.The present invention is production bacterial strain with the Lactococcus lactis recombinant bacterium of high efficient expression hexokinase, mannose-phosphate mutase, -1 guanosine 5-monophosphate transferase of mannose, using mannose as substrate, synthesize GDP- mannose, recycle GDP- mannose 4,6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase, α 1,2- fucosyltransferase is in vitro catalyzed GDP- mannose, to synthesize 2'-Fucosyl lactose, a kind of new method is provided for industrial production 2'-Fucosyl lactose.

Description

A method of synthesis 2'-Fucosyl lactose

Technical field:

The invention belongs to genetic engineering fields, are related to the production method of 2'-Fucosyl lactose, especially a kind of to utilize base The method for catalyzing and synthesizing 2'-Fucosyl lactose altogether because of engineered strain and enzyme.

Background technique:

In recent years, with the fast development of food industry and biotechnology, the exploitation of function oligosaccharides has become international bio The important topic of technical field, oligosaccharides industry have become one and have been applied to the emerging of the industries such as food, feed, medicine, chemical industry Important industry.Not only there is viral infection resisting, strengthen immunity to adjust, reduce the physiological actions such as inflammation for human milk oligosaccharides, but also There is certain prevention effect for cancer and chronic recurrent colitis etc..Its glucide as a kind of special construction, can As the soluble recepter analog of epithelial cell, the enhancing of the nonimmune defense system of breast feeding babies is participated in.Human milk is few Its sugared content becomes the third-largest substance in human milk after lactose, lipid, and oligosaccharide content is 20-27g/L in colostrum, at It is also up to 12-14g/L in ripe cream, and content is very low in other animal milks, oligosaccharides contains only 0.7-1.2g/L in colostrum, low In as many as 20 times of people's colostrum.

In addition to rich content, human milk oligosaccharides structure is also extremely complex, according to mass spectral analysis, estimates to contain more than 900 in human milk The oligosaccharides of kind different structure, has at least isolated the different oligosaccharides of 130 various structures at present, and only contains 18 kinds of widows in cow's milk Sugar.Most of human milk oligosaccharides are in neutrality, and based on fucosylated oligosaccharide, wherein 1 α, 2- fucosylated oligosaccharide is in tested person The ratio average out to 73% of the total oligosaccharides of Zhan in cream, fucosyl residues, which are 1-15, to be differed.This content in human milk is the abundantest Fucosylated oligosaccharide do not detected but in the mature milk of the animals such as ox, sheep, goat and horse, but in its colostrum Contain more neutral oligosaccharides.Difference between different mammal milk on oligosaccharide structure, it is also possible to human milk oligosaccharides be prompted to have There is unique physiological function.In addition, human milk oligosaccharides and the like safely, effectively, do not generate anti-medicine due to function uniqueness Property, and can also resist to antibiotic generate resistance variation pathogen, with human milk oligosaccharides and the like be raw material prepare it is anti-stick Attached drug becomes the another big hot spot of current medical exploitation, is preventing and treating bacteriosis field and infant nutrition health care Aspect will play huge effect.

Do not carried out effectively yet about the function of human milk oligosaccharides and preparation research in China at present.China is to function oligosaccharides The exploitation of substance is concentrated mainly on natural plants and microbial polysaccharide, is dropped using chemical methodes such as acid, alkali, enzyme, oxidations The oligosaccharides of solution preparation low molecular weight, such as chitosan oligosaccharide, fructooligosaccharide.Human milk oligosaccharides are obtained by chemical synthesis, due to synthesis The complexity of route and the valuableness of glucosides donor, most of techniques are still unable to reach large-scale production, become chemical method synthesis of oligose It is not easy the obstacle gone beyond.

With the rapid development of the continuous parsing and technique for gene engineering of more and more glycosidases and its gene, pass through people The work building a large amount of oligosaccharide synthesis of genetic engineering bacterium have become possibility.

Summary of the invention:

The object of the present invention is to provide a kind of sides using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose Method, this method with high efficient expression hexokinase, mannose-phosphate mutase, -1 guanosine 5-monophosphate transferase of mannose Lactococcus lactis Bacterium recombinant bacterium is production bacterial strain, using mannose as substrate, synthesis GDP- mannose, and recycling GDP- mannose 4,6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase, α 1,2- fucosyltransferase are in vitro to GDP- sweet dew Sugar is catalyzed, to synthesize 2'-Fucosyl lactose, is provided for industrial production 2'-Fucosyl lactose a kind of new Method.

Preferably, the hexokinase comes from E. coli K12, nucleotide sequence such as sequence table SEQ ID Shown in NO.1；

Preferably, the mannose-phosphate mutase comes from E. coli K12, nucleotide sequence such as sequence table Shown in SEQ ID NO.2；

Preferably, the mannose-1-phosphate guanosine transferase comes from E. coli K12, and nucleotide sequence is such as Shown in sequence table SEQ ID NO.3；

Preferably, the host cell of the Lactococcus lactis recombinant bacterium is Lactococcus lactis NZ3900；

Preferably, the expression vector of the Lactococcus lactis recombinant bacterium is plasmid pNZ8149；

Preferably, the GDP- mannose 4,6- dehydratase and GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- is also Protoenzyme comes from E. coli K12, and nucleotide sequence is respectively as shown in sequence table SEQ ID NO.4 and 5；

Preferably, the α 1,2- fucosyltransferase come from helicobacter pylori (Helicobacter pylori) HPAG1, nucleotide sequence is as shown in sequence table SEQ ID NO.6.

The method that the present invention produces 2'-Fucosyl lactose is specific as follows:

(1) cell fermentation: being production bacterial strain with above-mentioned Lactococcus lactis recombinant bacterium, the fermentation to containing mannose being substrate Culture medium ferments, and synthesizes GDP- mannose；

The concentration of the mannose substrate is 20mg/mL；

(2) cell and mixed enzyme fermentation synthesize human milk fucosyllactose: utilizing GDP- mannose 4,6- dehydratase, GDP-4- Fermentation obtains in ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase, α 1,2- fucosyltransferase catalytic step (1) The lactose of product GDP- mannose and external addition, synthesizes 2'-Fucosyl lactose；GDP- mannose in every mL reaction system 4,6- dehydratase: GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase: α 1,2- fucosyltransferase: GDP- Mannose: lactose=2U:2U:3U:1mg:2mg；

Preferably, the fermentation medium is M9 culture medium, composition are as follows: Na₂HPO₄12.8g KH₂PO 3g, NaCl 0.5g, NH₄Cl 1g, 10mL (1moL/L) MgSO₄, 0.5mL (1moL/L) CaCl₂, 2% glucose, sterile water is settled to 1L。

Preferably, the fermentation condition of step (1) are as follows: 30-37 DEG C of fermentation temperature, shaking speed 160-220r/min, fermentation Time 36-48h；

Preferably, step (2) carries out the syncatalytic condition of enzyme are as follows: 30-40 DEG C of catalytic temperature, reaction time 6-10h；

After 6-10h ferments, the yield of 2'-Fucosyl lactose reaches 1.0-2.5 μ g/mL.

The advantages and positive effects of the present invention are:

1, the present invention constructs NZ3900/ with genetic engineering recombinant technique based on Lactococcus lactis and Escherichia coli Tetra- kinds of genetic engineering bacteriums of pNZ8149-glk-manB-manC, BL21/pET-gmd, BL21/pET-wcaG and BL21/pET-futc Strain realizes the high efficient expression of enzyme in metabolic pathway, and applies fermentation technique, and substrate mannose is made to be converted into GDP- mannose, with Supernatant and lactose containing GDP- mannose are substrate, pass through GDP- mannose 4,6- dehydratase, GDP-4- ketone -6- deoxidation Mannose 3,5- mutarotase/4- reductase, α 1,2- fucosyltransferase carry out catalyzing and synthesizing 2'-Fucosyl lactose, this is One feasible way of offer of industrialized production human milk oligosaccharides, this production method reduce production cost, improve production Efficiency not only has economic benefit, also has certain social benefit.

2, the present invention synthesizes GDP- mannose using recombinant bacterial strain NZ3900/pNZ8149-glk-manB-manC fermentation, GDP- mannose is the important preceding substrate for synthesizing 2'-Fucosyl lactose, and a large amount of accumulation of GDP- mannose are conducive to 2 '-rocks A large amount of synthesis of algae glycosyl lactose, this not only to produce human milk oligosaccharides in batches, while being also native oligosaccharides drug Production provide raw material, have stronger fundamental research value and economic results in society, the prospect of marketing it is wide.

Detailed description of the invention:

The PCR of Fig. 1 target gene glk, manB and manC are verified；

Wherein, M:1kb DNA marker；1:glk；2:manB；3:manC；

The amplification PCR of Fig. 2 target gene gmd and wcaG are verified

Wherein, 1-3:wcaG；4:1kb DNA marker；5-7:gmd；

The amplification PCR of Fig. 3 target gene futc is verified

Wherein, M:1kb DNA marker；1-2:futc；

Fig. 4 recombinant plasmid pET-gmd/wcaG/futc constructs schematic diagram；

Fig. 5 recombinant plasmid pNZ8149-glk-manB-manC constructs schematic diagram；

Fig. 6 present invention catalyzes and synthesizes the flow chart of 2'-Fucosyl lactose using recombinant bacterial strain fermentation and enzyme altogether；

Fig. 7 a recombinant plasmid pET-wcaG and pET-gmd digestion and PCR verify electrophoretogram

Wherein, M:1kb DNAmark；1:pET-22b/HindⅢ；I+Hind III of 2:pET-wcaG/Nco；3:pET-gmd/ Nco Ⅰ+HindⅢ；4:pET-gmd/PCR；5:pET-wcaG/PCR；

Fig. 7 b recombinant plasmid pET-futc digestion and PCR verify electrophoretogram

Wherein, M:1kb DNAladder；3:pET-futc/BamHI/HindIII；4:pET-futc/BamHI；1-2: pET-futc/PCR；

Fig. 8 recombinant plasmid pNZ-gBC digestion and PCR verify electrophoretogram

Wherein, M:1kb DNA ladder；1-2:pNZ-gBC/KpnI/PstI；1-3:pNZ-gBC/PCR；

Fig. 9 expresses the SDS-PAGE analysis of albumen

A: recombinant bacterial strain BL21/pET-gmd and BL21/pET-wcaG express the SDS-PAGE analysis of albumen

Wherein, M:protein marker；1:BL21/pET-gmd；2:BL21/pET-wcaG；

3:BL21/pET-22b；

The SDS-PAGE analysis of b: recombinant bacterial strain BL21/pET-futc expression albumen

Wherein, M:Marker；1:BL21/pET-22b；2:BL21/pET-futc；

The SDS-PAGE analysis of c: recombinant bacterial strain NZ3900/pNZ8149-glk-manB-manC expression albumen

Wherein, M:Marker；1-2:NZ3900/pNZ8149-glk-manB-manC；3:NZ3900/pNZ8149；

Figure 10 product HPLC detection

Wherein, A- marks the HPLC figure of product 2'-Fucosyl lactose；The HPLC of B- blank control reaction solution schemes；C- sample is anti- The HPLC of liquid is answered to scheme；

Figure 11 GDP- fucose mark product and fermentation liquid liquid chromatogram

Wherein, A:GDP- fucose mark product HPLC chromatogram；B: recombination bacillus coli BL21/pET-22b fermentation liquid HPLC chromatogram.

Specific embodiment:

In order to which the objects, technical solutions and advantages of this patent are more clearly understood, below in conjunction with specific embodiment, to this Patent is further elaborated.It should be appreciated that specific embodiment described herein is only to explain this patent, and do not have to It is of the invention in limiting.

The method according to the present invention for catalyzing and synthesizing 2'-Fucosyl lactose altogether using recombinant bacterial strain fermentation and enzyme, it is first The NICE expression system for utilizing Lactococcus lactis before this, to hexokinase, mannose-phosphate mutase, mannose-1-phosphate bird Glycosides acyltransferase (Glk, ManB, ManC) carries out inducing expression, and catalysis substrate mannose synthesizes GDP- mannose；Secondly, sharp With the expression system of Escherichia coli, respectively to GDP- mannose 4,6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotation Enzyme/4- reductase, α 1,2- fucosyltransferase (Gmd, WcaG, Futc) carry out inducing expression and obtain corresponding enzyme.With cream Bacterial cell disruption liquid centrifugation supernatant after sour Lactococcus fermentation synthesis GDP- mannose is substrate, and suitable lactose is added, GDP- fucose is first synthesized under the action of Gmd, WcaG, then under the catalytic action of Futc, synthesize 2'-Fucosyl lactose.This Invention is as shown in Figure 6 using the flow chart that recombinant bacterial strain fermentation and enzyme catalyze and synthesize 2'-Fucosyl lactose altogether.

Using the hexokinase gene glk in various sources in the prior art, mannose-phosphate mutase gene manB, sweet Reveal 4,6- dehydrase gene gmd, GDP-4- ketone -6- deoxidation of sugar -1- guanosine 5-monophosphate acyltransferase gene manC, GDP- mannose The present invention can be achieved in mannose 3,5- mutarotase/4- reductase gene wcaG, α 1,2- fucosyl transferase gene futc Shown in effect.

Hexokinase gene glk, mannose-phosphate mutase gene manB, mannose-used in the embodiment of the present invention 1- guanosine 5-monophosphate acyltransferase gene manC, GDP- mannose 4,6- dehydrase gene gmd, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotases/4- reductase gene wcaG, derive from E.coli K12；Genbank number is respectively ECK2384, ECUMN_2374, ECIAI39_0987, ECK2047, ECK2046, nucleotide sequence is respectively such as sequence table SEQ ID NO.1-5 It is shown；

α 1,2- fucosyl transferase gene futc used in the embodiment of the present invention comes from helicobacter pylori (Helicobacter pylori) HPAG1, Genbank number are HELPY_0091, nucleotide sequence such as sequence table SEQ ID Shown in NO.6；

The embodiment of the present invention expresses hexokinase Glk, mannose-phosphate mutase ManB, mannose-1-phosphate guanosine acyl The host of transferase ManC is Lactococcus lactis NZ3900, expression vector pNZ8149；The embodiment of the present invention expresses GDP- sweet dew The place of sugared 4,6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase, α 1,2- fucosyltransferase It is main, it is e. coli bl21, expression vector pET-22b.

Culture medium used in the present invention is as follows:

M9 culture medium, composition are as follows: Na₂HPO₄12.8g KH₂PO 3g, NaCl 0.5g, NH₄Cl 1g, 10mL (1moL/L) MgSO₄, 0.5mL (1moL/L) CaCl₂, 2% glucose, sterile water is settled to 1L.

GM₁₇Culture medium (g/L): lactose 20, glucose 40, yeast powder 5, ammonium sulfate 5, disodium hydrogen phosphate 4, potassium dihydrogen phosphate 0.5, pH 6.8.

The building of embodiment 1 Recombinant Lactococcus lactis and recombination bacillus coli

Gene according to the present invention can be obtained by genomic PCR amplification, can also pass through complete sequence according to gene order Synthesis obtains, below by taking PCR amplification as an example:

1, the amplification of target gene

(1) extract Escherichia coli (E.coli K12) genomic DNA, design following primer be orderly used to amplification hexose swash Enzyme gene glk, mannose-phosphate mutase gene manB, mannose-1-phosphate guanosine acyltransferase gene manC, GDP- sweet dew Sugared 4,6- dehydrase gene gmd, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase gene wcaG:

(2) helicobacter pylori (Helicobacter pylori) HPAG1 genomic DNA is extracted, following primer is designed and uses In amplification α 1,2- fucosyl transferase gene futc:

Primer	Sequence 5 ' -3 '	Restriction enzyme site	SEQ ID
				futc1	GCTCTAGAATGGCTTTTAAAAGTGTGCAA	XbaI	NO.17
futc2	GGGGTACCTTAAGCGTTATACTTTTGGGATTT	KpnI	NO.18

It (3) is template respectively to target gene (hexokinase base using E. coli K12 and HPAG1 chromosomal DNA Because of glk, mannose-phosphate mutase gene manB, mannose-1-phosphate guanosine acyltransferase gene manC, GDP- mannose 4, 6- dehydrase gene gmd, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase gene wcaG, α 1,2- fucose Based transferase gene futc) PCR amplification is carried out, amplification condition is as follows, verifies as shown in Figure 1, Figure 2, Fig. 3

A. the PCR amplification system of 20 μ L is used:

B. it is used for the PCR condition of amplifying target genes:

2, the building of recombinant plasmid pET-gmd, pET-wcaG, pET-futc, pNZ8149-glk-manB-manC

It is as shown in Figure 4 that recombinant plasmid pET-gmd/wcaG/futc constructs schematic diagram；Recombinant plasmid pNZ8149-glk- It is as shown in Figure 5 that manB-manC constructs schematic diagram；

Pcr amplification product in plasmid pET-22b, pNZ8149, step 1 is subjected to double enzymes with corresponding restriction enzyme It cuts, carries out gel verifying, purification and recovery is carried out to the segment after above-mentioned digestion with DNA purification kit (TaKaBa company).It obtains The pET-22b, pNZ8149 linearly purified, genetic fragment.By linear carrier pET-22b, pNZ8149 of acquisition, gene piece Section is attached, and obtains recombinant plasmid pET-gmd, pET-wcaG, pET-futc, pNZ8149-glk-manB-manC.

The connection of carrier and target gene after digestion, select 10 μ L linked systems (I 5 μ L of Solution, 1 μ L of carrier, 4 μ L of segment), 16 DEG C of reaction 8h.

Gained connection mixture is transferred to e. coli jm109 using heat shock method, carries out the amplification of purpose plasmid, is weighed Group plasmid pET-gmd, pET-wcaG, pET-futc, pNZ8149-glk-manB-manC.

3, the building and verifying of recombinant bacterial strain BL21/pET-gmd, BL21/pET-wcaG, BL21/pET-futc

(1) competent cell of e. coli bl21 is prepared

(2) recombinant plasmid connected the conversion of competent cell: is separately added into BL21 competent cell PET-gmd, pET-wcaG, pET-futc mixed liquor, gently piping and druming is uniform, ice bath 20min；Thermal shock 90s, fast in 42 DEG C of water-baths In speed insertion ice, ice bath 10min；300 μ LLB resuscitation fluids are added into EP pipe in superclean bench gently to shake up, then fix 37 DEG C of shake culture 1h on to the spring(-supported) mount of shaking table；Thalline were collected by centrifugation, is applied to the LA containing ampicillin (100 μ g/mL) On plate, 37 DEG C of inversion overnight incubations (16-20h)；

(3) verifying of recombination bacillus coli transformant: single bacterium colony is picked from the plate, is inoculated in containing ampicillin In (100 μ g/mL) LB liquid medium, in 37 DEG C of culture 12-18h, Plasmid DNA are then extracted in a small amount, with corresponding restricted Restriction endonuclease carries out double digestion identification, and verifying is correct, such as Fig. 7 a and Fig. 7 b, obtains engineering strain BL21/pET-gmd respectively, BL21/ pET-wcaG, BL21/pET-futc.

4, the building and verifying of engineering strain Lactococcus lactis NZ3900/pNZ8149-glk-manB-manC

(1) the competence preparation of Lactococcus lactis NZ3900；

(2) Lactococcus lactis NZ3900's is electroporated: weight being added into the competent cell of Lactococcus lactis NZ3900 Group plasmid pNZ8149-glk-manB-manC, piping and druming mix, the liquid after mixing are transferred in the electric revolving cup being pre-chilled in advance；It beats Electric shock instrument is opened, parameter: 2.0kV, 200 Ω is set, 25 μ F carry out electric pulse；After electric shock, electric revolving cup is taken out, 800 μ are added L GM₁₇Bacterium solution is transferred in EP pipe by MC recovery media, cultivates 3h in 30 DEG C, 160r/min；12000r/min is centrifuged 2min, Supernatant is abandoned, 50 μ L GM are taken₁₇Thallus is resuspended in culture medium, is coated on the plate containing chloramphenicol, 30 DEG C of static gas wave refrigerator 72h are grown Yellow positive bacterium colony be possible positive transformant.

(3) verifying of Recombinant Lactococcus lactis transformant: picking from the plate single bacterium colony, is inoculated in containing chloramphenicol (100 μ g/mL) liquid GM₁₇In culture medium, in 30 DEG C of culture 12-18h, Plasmid DNA is then extracted in a small amount, in restricted accordingly Enzyme cutting carries out double digestion identification, and verifying is correct, such as Fig. 8, obtains Recombinant Lactococcus lactis NZ3900/ pNZ8149-glk-manB- manC。

Embodiment 2, the analysis of the inducing expression of recombinant bacterial strain

1, recombination bacillus coli inducing expression is analyzed

(1) engineering strain BL21/pET-gmd, BL21/pET-wcaG, BL21/ that picking embodiment 1-3 is obtained PET-futc single colonie is transferred to respectively in 5mL LB liquid medium, 37 DEG C, 220r/min overnight incubation；

(2) bacterium solution is transferred in 50mL M9 fluid nutrient medium, controls the dense OD of starter bacteria₆₀₀=0.1, continue to cultivate；

(3) when bacterial strain concentration reaches OD₆₀₀When being 0.4, the IPTG of 40 μ L0.1mol/L is added, induces 4h at 16 DEG C；

(4) 6000r/min is centrifuged 10min, collects Escherichia coli；

(5) it is washed bacterial strain 2 times with PBS buffer solution, using sonicated cells instrument come smudge cells, 4 DEG C, 12000r/min It is centrifuged 10min, collects supernatant, sampling carries out SDS-PAGE analysis, as a result sees Fig. 9 a and 9b.

2, Recombinant Lactococcus lactis inducing expression is analyzed

(1) the Recombinant Lactococcus lactis NZ3900/pNZ8149-glk-manB-manC single bacterium that picking embodiment 1-4 is obtained It falls and is inoculated into 5mL GM₁₇In fluid nutrient medium, 30 DEG C, 160r/min overnight incubation；

(2) bacterium solution is inoculated into 50mL GM₁₇In fluid nutrient medium, the dense OD of initial bacterium is controlled₆₀₀=0.1, continue to cultivate；

(3) reach OD when bacterium is dense₆₀₀When being 0.4, the Nisin of 40 μ L0.1mg/mL is added, induces 4h at 30 DEG C；

(4) 6000r/min is centrifuged 10min, collects Lactococcus lactis；

(5) it is washed cell 2 times with PBS buffer solution, and is suspended with the lysozyme of 400 μ L, in 37 DEG C of warm bath 3h, and with ultrasonic Smudge cells instrument smudge cells, 4 DEG C, 12000r/min is centrifuged 10min, collects supernatant, and sampling carries out SDS-PAGE analysis, as a result See Fig. 9 c.

As seen from the figure, occurs band at about 35kDa, 50kDa, 63kDa, 41kDa, 37kDa, 33kDa, this and hexose Kinases (Glk), mannose-phosphate mutase (ManB), mannose-1-phosphate guanosine transferase (ManC), GDP- mannose 4, 6- dehydratase (Gmd), GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase (WcaG), α 1,2- fucosido turn It is consistent to move enzyme (Futc) molecular size range, this shows that target gene glk, manB and manC, gmd, wcaG, futc success are real Now express.

The fermentation of 3 recombinant strain of embodiment and enzyme catalyze and synthesize 2'-Fucosyl lactose altogether

(1) picking Recombinant Lactococcus lactis NZ3900/pNZ-glk-manB-manC single colonie is inoculated into 5mL GM₁₇Liquid In culture medium, 30-37 DEG C, 160-220r/min is incubated overnight.Bacterium solution is inoculated into 50mL M9 fermentation medium, is controlled The dense OD of beginning bacterium₆₀₀=0.1, the OD to bacterial strain₆₀₀When=0.4, the substrate mannose of 20mg/mL is added, while Nisin is added and does Inducer, makes its final concentration of 35ng/mL, and 35 DEG C, after 200r/min fermentation 40h.Supernatant is collected in smudge cells, centrifugation；

(2) GDP- mannose 4,6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase and α 1, The preparation of 2- fucosyltransferase: recombination bacillus coli BL21/pET-glk, BL21/pET-manB and BL21/ are picked them separately PET-manC single colonie is inoculated into 5mL LB liquid medium, and 37 DEG C, 220r/min is incubated overnight.Bacterium solution is inoculated with respectively Into 50mL M9 fermentation medium, the dense OD of starter bacteria is controlled₆₀₀=0.1, the OD to bacterial strain₆₀₀When=0.3, it is separately added into induction Agent IPTG, makes its final concentration of 0.5mmoL/L, and 20 DEG C, after 200r/min induction 25h.Supernatant is collected in smudge cells, centrifugation.

(3) Enzyme catalyzed synthesis 2'-Fucosyl lactose step is utilized: in the EP pipe of 1.5mL, with thin in step (1) It is substrate that born of the same parents, which are crushed liquid centrifugation supernatant, while suitable lactose is added；Contain GDP- mannose 4 using what is obtained in step (2), 6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase and α 1,2- fucosyltransferase supernatant To catalyze and synthesize 2'-Fucosyl lactose.Synthetic reaction: 1mgGDP- mannose: 2U GDP- mannose is carried out by following system 4,6- dehydratase: 2U GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase: 3U α 1,2- fucosyltransferase: The lactose of 2mg.Under the conditions of 35 DEG C, 6h is reacted.

Enzyme unit (U): 30-40 DEG C, under the conditions of pH7.0,1 μm of ol substrate GDP- mannose of catalysis reacts institute in 1min The enzyme amount needed.

Reaction solution processing: boiling reaction solution using boiling water bath, terminates reaction, and centrifugation takes supernatant to carry out HPLC analysis, as a result See Figure 10: occurring and Figure 10 (A) same time in the HPLC figure of the example reaction liquid represented by Figure 10 (C) in 21min or so Peak, and without there is corresponding peak in the HPLC figure of the blank control reaction solution represented by Figure 10 (B), thus may determine that, This method can synthesize 2'-Fucosyl lactose.It is 1.25 μ g/mL with the yield that external standard method calculates fucosylated oligosaccharide.

HPLC chromatogram condition are as follows: chromatographic column: 5 μ 250mm*4.6mm of Carbohydrate ES, detector: evaporation light detection Device；Mobile phase: 70% acetonitrile (acetonitrile: water)；Column temperature: 30 DEG C；Sample volume: 20 μ L；Flow velocity: 1.0mL/min.

4 comparative example of embodiment

The recombination bacillus coli BL21/pET-22b single colonie of picking load empty plasmid is inoculated into 5mL GM₁₇Fluid nutrient medium In, 30 DEG C, 160r/min is incubated overnight.Bacterium solution is inoculated into 50mL M9 fermentation medium, the dense OD of starter bacteria is controlled₆₀₀= 0.1, the OD to bacterial strain₆₀₀When=0.4, the substrate mannose of 20mg/mL is added, while inducer IPTG is added, makes its final concentration For 35ng/mL, 16 DEG C, after 160r/min fermentation for 24 hours.Supernatant is collected in centrifugation, and supernatant is taken to carry out HPLC-ELSD analysis.As a result such as Shown in Figure 11.Do not occur in the HPLC-ELSD figure of recombination bacillus coli BL21/pET-22b fermentation liquid represented by Figure 11 B The corresponding peak of GDP- fucose, therefore can be determined that the recombination bacillus coli BL21/pET-22b of building cannot synthesize GDP- rock Algae sugar.

It is by current published genome it is found that sweet comprising hexokinase Glk, phosphoric acid on e. coli bl21 genome Reveal sugared mutase ManB, mannose-1-phosphate guanosine acyltransferase ManC, GDP- mannose 4,6- dehydratase Gmd, GDP-4- ketone In 6-de-oxy-L-mannose 3,5- mutarotase/4- reductase WcaG encoding gene, and gene order and e. coli k12 Corresponding gene is identical, and the present embodiment cannot be using mannose as substrate by verifying discovery recombination bacillus coli BL21/pET-22b GDP- fucose is synthesized, it is sweet by hexokinase Glk, phosphoric acid by substrate of mannose to illustrate that Escherichia coli itself are not had Reveal sugared mutase ManB, mannose-1-phosphate guanosine acyltransferase ManC, GDP- mannose 4,6- dehydratase Gmd synthesizes GDP- Mannose, and then pass through GDP- mannose 4,6- dehydratase Gmd, GDP-4- ketone -6- deoxidation sweet dew by substrate of GDP- mannose Sugared 3,5- mutarotase/4- reductase WcaG synthesis GDP- fucose metabolic pathway.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously The limitation to the scope of the patents therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art, Under the premise of not departing from this patent design, the respective embodiments described above can also make several deformations, combination and improve, these all belong to In the protection scope of this patent.Therefore, the protection scope of this patent should be subject to the claims.

Sequence table

<110>University Of Science and Technology Of Tianjin

<120>a kind of method for synthesizing 2'- fucosyllactose

<130> 1

<141> 2019-05-14

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1033

<212> DNA

<213>E. coli K12 ()

<400> 1

atgtgggcgg caccaacgca cgtcttgctc tgtgtgatat tgccagtggt gaaatctcgc 60

aggctaagac ctattcaggg cttgattacc ccagcctcga agcggtcatt cgcgtttatc 120

ttgaagaaca taaggtcgag gtgaaagacg gctgtattgc catcgcttgc ccaattaccg 180

gtgactgggt ggcgatgacc aaccatacct gggcgttctc aattgccgaa atgaaaaaga 240

atctcggttt tagccatctg gaaattatta acgattttac cgctgtatcg atggcgatcc 300

cgatgctgaa aaaagagcat ctgattcagt ttggtggcgc agaaccggtc gaaggtaagc 360

ctattgcggt ttacggtgcc ggaacggggc ttggggttgc gcatctggtc catgtcgata 420

agcgttgggt aagcttgcca ggcgaaggcg gtcacgttga ttttgcgccg aatagtgaag 480

aagaggccat tatcctcgaa atattgcgtg cggaaattgg tcatgtttcg gcggagcgcg 540

tgctttctgg ccctgggctg gtgaatttgt atcgcgcaat tgtgaaagct gacaaccgcc 600

tgccagaaaa tctcaagcca aaggatatta ccgaacgcgc gctggctgac agctgcaccg 660

attgccgccg cgcattgtcg ctgttttgcg tcattatggg ccgttttggc ggcaatctgg 720

cgctcaatct cgggacattt ggcggcgtgt ttattgcggg cggtatcgtg ccgcgcttcc 780

ttgagttctt caaacctccg gtttccgtgc cgcatttgaa gataaagggc gctttaaaga 840

atatgtccat gatattccgg tgtatctcat cgtccatgac aatccgggcc ttctcggttc 900

cggtgcacat ttacgccaga ccttaggtca cattctgtaa ggatccgaat tcgagctccg 960

tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta 1020

acaaagcccg aaa 1033

<210> 2

<211> 1412

<212> DNA

<213>E. coli K12 ()

<400> 2

atgatattcg cgggaaatta ggcgaagaac tgaatgaaga tatcgcctgg cgcattggtc 60

gcgcctatgg cgaatttctc aaaccgaaaa ccattgtgtt aggcggtgat gtccgcctca 120

ccagcgaaac cttaaaactg gcgctggcga aaggtttaca ggatgcgggc gttgacgtgc 180

tggatattgg tatgtccggc accgaagaga tctatttcgc cacgttccat ctcggcgtgg 240

atggcggcat tgaagttacc gccagccata atccgatgga ttataacggc atgaagctgg 300

ttcgcgaggg ggctcgcccg atcagcggag ataccggact gcgcgacgtc cagcgtctgg 360

ctgaagccaa cgactttcct cccgtcgatg aaaccaaacg cggtcgctat cagcaaatca 420

acctgcgtga cgcttacgtt gatcacctgt tcggttatat caatgtcaaa aacctcacgc 480

cgctcaagct ggtgatcaac tccgggaacg gcgcagcggg tccggtggtg gacgccattg 540

aagcccgctt taaagccctc ggcgcgcccg tggaattaat caaagtgcac aacacgccgg 600

acggcaattt ccccaacggt attcctaacc cactactgcc ggaatgccgc gacgacaccc 660

gcaatgcggt catcaaacac ggcgcggata tgggcattgc ttttgatggc gattttgacc 720

gctgtttcct gtttgacgaa aaagggcagt ttattgaggg ctactacatt gtcggcctgt 780

tggcagaagc attcctcgaa aaaaatcccg gcgcgaagat catccacgat ccacgtctct 840

cctggaacac cgttgatgtg gtgactgccg caggtggcac gccggtaatg tcgaaaaccg 900

gacacgcctt tattaaagaa cgtatgcgca aggaagacgc catctatggt ggcgaaatga 960

gcgcccacca ttacttccgt gatttcgctt actgcgacag cggcatgatc ccgtggctgc 1020

tggtcgccga actggtgtgc ctgaaagata aaacgctggg cgaactggta cgcgaccgga 1080

tggcggcgtt tccggcaagc ggtgagatca acagcaaact ggcgcaaccc gttgaggcga 1140

ttaaccgcgt ggaacagcat tttagccgtg aggcgctggc ggtggatcgc accgatggca 1200

tcagcatgac ctttgccgac tggcgcttta acctgcgcac ctccaatacc gaaccggtgg 1260

tgcgcctgaa tgtggaatcg cgcggtgatg tgccgctgat ggaagcgcga acgcgaactc 1320

tgctgacgtt gctgaacgag taaaagcttg cggccgcact cgagcaccac caccaccacc 1380

actgagatcc ggctgctaac aaagcccgaa ag 1412

<210> 3

<211> 1437

<212> DNA

<213>E. coli K12 ()

<400> 3

atggcaggtg gctccggtag ccgcttatgg ccgctttccc gcgtacttta tcccaagcag 60

tttttatgcc tgaaaggcga tctcaccatg ctgcaaacca ccatctgccg cctgaacggc 120

gtggagtgcg aaagcccggt ggtgatttgc aatgagcagc accgctttat tgtcgcggaa 180

cagctgcgtc aactgaacaa acttaccgag aacattattc tcgaaccggc agggcgaaac 240

acggcacctg ccattgcgct ggcggcgctg gcggcaaaac gtcatagccc ggagagcgac 300

ccgttaatgc tggtattggc ggcggatcat gtgattgccg atgaagacgc gttccgtgcc 360

gccgtgcgta atgccatgcc atatgccgaa gcgggcaagc tggtgacctt cggcattgtg 420

ccggatctac cagaaaccgg ttatggctat attcgtcgcg gtgaagtgtc tgcgggtgag 480

caggatatgg tggcctttga agtggcgcag tttgtcgaaa aaccgaatct ggaaaccgct 540

caggcctatg tggcaagcgg cgaatattac tggaacagcg gtatgttcct gttccgcgcc 600

ggacgctatc tcgaagaact gaaaaaatat cgcccggata tcctcgatgc ctgtgaaaaa 660

gcgatgagcg ccgtcgatcc ggatctcaat tttattcgcg tggatgaaga agcgtttctc 720

gcctgcccgg aagagtcggt ggattacgcg gtcatggaac gtacggcaga tgctgttgtg 780

gtgccgatgg atgcgggctg gagcgatgtt ggctcctggt cttcattatg ggagatcagc 840

gcccacaccg ccgagggcaa cgtttgccac ggcgatgtga ttaatcacaa aactgaaaac 900

agctatgtgt atgctgaatc tggcctggtc accaccgtcg gggtgaaaga tctggtagtg 960

gtgcagacca aagatgcggt gctgattgcc gaccgtaacg cggtacagga tgtgaaaaaa 1020

gtggtcgagc agatcaaagc cgatggtcgc catgagcatc gggtgcatcg cgaagtgtat 1080

cgtccgtggg gcaaatatga ctctatcgac gcgggcgacc gctaccaggt gaaacgcatc 1140

accgtgaaac cgggcgaggg cttgtcggta cagatgcacc atcaccgcgc ggaacactgg 1200

gtggttgtcg cgggaacggc aaaagtcacc attgatggtg atatcaaact gcttggtgaa 1260

aacgagtcca tttatattcc gctgggggcg acgcattgcc tggaaaaccc ggggaaaatt 1320

cgctcgattt cagaccttag gtcacattct gtaaaagctt gaattgaagt gcgctccggc 1380

tcttatctcg aagaggatga tgtggtgcgt ttcgcggatc gctacggacg ggtgtaa 1437

<210> 4

<211> 1253

<212> DNA

<213>E. coli K12 ()

<400> 4

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatgg atatgtcaaa agtcgctctc atcaccggtg taaccggaca agacggttct 120

tacctggcag agtttctgct ggaaaaaggt tacgaggtgc atggtattaa gcgtcgcgca 180

tcgtcattca acaccgagcg cgtggatcac atttatcagg atccgcacac ctgcaacccg 240

aaattccatc tgcattatgg cgacctgagt gatacctcta acctgacgcg cattttgcgt 300

gaagtacagc cggatgaagt gtacaacctg ggcgcaatga gccacgttgc ggtctctttt 360

gagtcaccag aatataccgc tgacgtcgac gcgatgggta cgctgcgcct gctggaggcg 420

atccgcttcc tcggcctgga aaagaaaact cgtttctatc aggcttccac ctctgaactg 480

tatggtctgg tgcaggaaat tccgcagaaa gagaccacgc cgttctaccc gcgatctccg 540

tatgcggtcg ccaaactgta cgcctactgg atcaccgtta actaccgtga atcctacggc 600

atgtacgcct gtaacggaat tctcttcaac catgaatccc cgcgccgcgg cgaaaccttc 660

gttacccgca aaatcacccg cgcaatcgcc aacatcgccc aggggctgga gtcgtgcctg 720

tacctcggca atatggattc cctgcgtgac tggggccacg ccaaagacta cgtaaaaatg 780

cagtggatga tgctgcagca ggaacagccg gaagatttcg ttatcgcgac cggcgttcag 840

tactccgtgc gtcagttcgt ggaaatggcg gcagcacagc tgggcatcaa actgcgcttt 900

gaaggcacgg gcgttgaaga gaagggcatt gtggtttccg tcaccgggca tgacgcgccg 960

ggcgttaaac cgggtgatgt gattatcgct gttgacccgc gttacttccg tccggctgaa 1020

gttgaaacgc tgctcggcga cccgaccaaa gcgcacgaaa aactgggctg gaaaccggaa 1080

atcaccctca gagagatggt gtctgaaatg gtggctaatg acctcgaagc ggcgaaaaaa 1140

cactctctgc tgaaatctca cgggtacgac gtggcgatcg cgctggagtc ataaaagctt 1200

gcggccgcac tcgagcacca ccaccaccac caatgagatc cggctgctaa caa 1253

<210> 5

<211> 1040

<212> DNA

<213>E. coli K12 ()

<400> 5

atggcgatga gtaaacaacg agtttttatt gttggtcatc gcgggatggt cggttccgcc 60

atcaggcggc agctcgaaca gcgcggtgat gtggaactgg tattacgcac ccgcgacgag 120

ctgaacctgc tggacagccg cgccgtgcat gatttctttg ccagcgaacg tattgaccag 180

gtctatctgg cggcggcgaa agtgggcggc attgttgcca acaacaccta tccggcggat 240

ttcatctacc agaacatgat gattgagagc aacatcattc acgccgcgca tcagaacgac 300

gtgaacaaac tgctgtttct cggatcgtcc tgcatctacc cgaaactggc aaaacagccg 360

atggcagaaa gcgagttgtt gcagggcacg ctggagccga ctaacgagcc ttatgctatt 420

gccaaaatcg ccgggatcaa actgtgcgaa tcatacaacc gccagtacgg acgcgattac 480

cgctcagtca tgccgaccaa cctgtacggg ccacacgaca acttccaccc gagtaattcg 540

catgtgatcc cagcattgct gcgtcgcttc cacgaggcga cggcacagaa tgcgccggac 600

gtggtggtat ggggcagcgg tacaccgatg cgcgaatttc tgcacgtcga tgatatggcg 660

gcggcgagca ttcatgtcat ggagctggcg catgaagtct ggctggagaa cacccagccg 720

atgttgtcgc acattaacgt cggcacgggc gttgactgca ctatccgcga gctggcgcaa 780

accatcgcca aagtggtggg ttacaaaggc cgggtggttt ttgatgccag caaaccggat 840

ggcacgccgc gcaaactgct ggatgtgacg cgcctgcatc agcttggctg gtatcacgaa 900

atctcactgg aagcggggct tgccagcact taccagtggt tccttgagaa tcaagaccgc 960

tttcgggggt aaaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg 1020

gctgctaaca aagcccgaaa 1040

<210> 6

<211> 906

<212> DNA

<213>helicobacter pylori ()

<400> 6

atggctttta aagtggtgca aatttgcgga gggcttggga atcaaatgtt tcaatacgct 60

ttcgctaaaa gtttgcaaaa acactctaat acgcctgtgc tgttagatat tacttctttt 120

gattggagca ataggaaaat gcaattagag cttttcccta ttgatttacc ctatgcgaat 180

gcaaaagaaa tcgctatagc taaaatgcaa cacctcccca agctagtaag agatacgctc 240

aaatacatgg gatttgatag ggtgagtcaa gaaatcgtgt ttgaatacga gcctaaattg 300

ttaaagccaa gccgcttgac ttatttttat ggctattttc aagatccacg atattttgat 360

gctatatccc ctttaatcaa gcaaactttc accctacccc accccccccc ccccgaaaat 420

ggaaataata aaaaaaaaga ggaagaatac caccgcaaac ttgctttgat tttagccgct 480

caaaacagcg tgtttgtgca tataagaaga ggggattatg tggggattgg ctgtcagctt 540

ggcattgact atcaaaaaaa ggcgcttgag tatatggcaa aacgcgtgcc aaacatggaa 600

cttttcgtgt tttgcgaaga cttagaattc acgcaaaatc ttgatcttgg ctaccctttt 660

atggacatga ccactaggga tagagaagaa gaggcgtatt gggatatgct gctcatgcaa 720

tcctgtcagc atggcattat cgctaatagc acttatagct ggtgggcggc ttatttgata 780

gaaaatccag aaaaaatcat tattggcccc aaacactggc tttttgggca tgagaatatc 840

ctttgtgagg aatgggtgaa aatagaatcc cattttgagg taaaatccca aaagtataac 900

gcttaa 906

<210> 7

<211> 32

<212> DNA

<213>artificial sequence ()

<400> 7

ggggtaccat gacaaagtat gcattagtcg gt 32

<210> 8

<211> 32

<212> DNA

<213>artificial sequence ()

<400> 8

gctctagatt acagaatgtg acctaaggtc tg 32

<210> 9

<211> 29

<212> DNA

<213>artificial sequence ()

<400> 9

gctctagaat gaaaaaatta acctgcttt 29

<210> 10

<211> 27

<212> DNA

<213>artificial sequence ()

<400> 10

cgggatcctt actcgttcag caacgtc 27

<210> 11

<211> 32

<212> DNA

<213>artificial sequence ()

<400> 11

cgggatccat gacaaagtat gcattagtcg gt 32

<210> 12

<211> 32

<212> DNA

<213>artificial sequence ()

<400> 12

ggggtacctt acagaatgtg acctaaggtc tg 32

<210> 13

<211> 29

<212> DNA

<213>artificial sequence ()

<400> 13

catgccatgg atgtcaaaag tctctcatc 29

<210> 14

<211> 27

<212> DNA

<213>artificial sequence ()

<400> 14

cccaagcttt atgactccag cgcgatc 27

<210> 15

<211> 35

<212> DNA

<213>artificial sequence ()

<400> 15

catgccatgg atgagtaaac aacgagtttt tattg 35

<210> 16

<211> 25

<212> DNA

<213>artificial sequence ()

<400> 16

cccaagcttt acccccgaaa gcggt 25

<210> 17

<211> 29

<212> DNA

<213>artificial sequence ()

<400> 17

gctctagaat ggcttttaaa agtgtgcaa 29

<210> 18

<211> 32

<212> DNA

<213>artificial sequence ()

<400> 18

ggggtacctt aagcgttata cttttgggat tt 32

Claims (8)

1. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose, which is characterized in that with efficient table Lactococcus lactis recombinant bacterium up to hexokinase, mannose-phosphate mutase, -1 guanosine 5-monophosphate transferase of mannose is production bacterium Strain synthesizes GDP- mannose using mannose as substrate, recycles GDP- mannose 4,6- dehydratase, GDP-4- ketone -6- deoxidation are sweet Reveal 3,5- of sugar mutarotase/4- reductase, α 1,2- fucosyltransferase is in vitro catalyzed GDP- mannose, to close At 2'-Fucosyl lactose.

2. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as described in claim 1, It is characterized in that, after Lactococcus lactis synthesizes GDP- mannose as substrate using mannose, will be used for after clasmatosis centrifuging and taking supernatant The synthesis of 2'-Fucosyl lactose.

3. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as described in claim 1, It is characterized in that, the hexokinase, mannose-phosphate mutase, -1 guanosine 5-monophosphate transferase of mannose are all from Escherichia coli E.coli K12, nucleotide sequence is as shown in sequence table SEQ ID NO.1-3.

4. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as described in claim 1, It is characterized in that, the host cell of the Lactococcus lactis recombinant bacterium is Lactococcus lactis NZ3900；Expression vector is plasmid pNZ8149。

5. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as described in claim 1, It is characterized in that, the GDP- mannose 4,6- dehydratase, GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reductase, α The encoding gene of 1,2- fucosyltransferase is respectively as shown in SEQ ID NO.4-6.

6. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as described in claim 1, It is characterized in that, specific as follows:

(1) cell fermentation: with Lactococcus lactis recombinant bacterium be production bacterial strain, to containing mannose be substrate fermentation medium into Row fermentation, synthesizes GDP- mannose；

(2) cell and mixed enzyme fermentation synthesize human milk fucosyllactose: utilizing GDP- mannose 4,6- dehydratase, GDP-4- ketone- The production that fermentation obtains in 6-de-oxy-L-mannose 3,5- mutarotase/4- reductase, α 1,2- fucosyltransferase catalytic step (1) The lactose of object GDP- mannose and external addition, synthesizes 2'-Fucosyl lactose；

GDP- mannose 4,6- dehydratase in every mL reaction system: GDP-4- ketone -6-de-oxy-L-mannose 3,5- mutarotase/4- reduction Enzyme: α 1,2- fucosyltransferase: GDP- mannose: lactose=2U:2U:3U:1mg:2mg.

7. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as claimed in claim 6, It is characterized in that, the fermentation condition of step (1) are as follows: 30-37 DEG C of fermentation temperature, shaking speed 160-220r/min, fermentation time 36- 48h。

8. a kind of method using recombinant bacterial strain and mixed enzyme fermentation synthesis 2'-Fucosyl lactose as claimed in claim 6, It is characterized in that, step (2) carries out the syncatalytic condition of enzyme are as follows: 30-40 DEG C of catalytic temperature, reaction time 6-10h.