CN117187206A

CN117187206A - Fucosyltransferase from intestinal microorganisms and application thereof

Info

Publication number: CN117187206A
Application number: CN202310567618.5A
Authority: CN
Inventors: 李由然; 石贵阳; 吴志勇; 陈权
Original assignee: Wuxi Special Food And Nutrition Health Research Institute Co ltd
Current assignee: Wuxi Special Food And Nutrition Health Research Institute Co ltd
Priority date: 2023-05-19
Filing date: 2023-05-19
Publication date: 2023-12-08

Abstract

The invention discloses fucosyltransferase from intestinal microorganisms and application thereof, belonging to the technical fields of synthetic biology and genetic engineering. The invention provides a fucosyltransferase with alpha-1, 2 specificity screened from intestinal microorganisms and a coding gene thereof, the coding genes of the fucosyltransferase are heterologously expressed in bacillus licheniformis by utilizing a gene recombination technology, and the obtained recombinant strain can be used for fermentation production of 2' -fucosyllactose. The fucosyltransferase has high catalytic activity, and has great potential in applications such as fermentation production of breast milk oligosaccharide when the optimal catalytic temperature is matched with high-temperature fermentation of bacteria.

Description

Fucosyltransferase from intestinal microorganisms and application thereof

Technical Field

The invention relates to fucosyltransferase from intestinal microorganisms and application thereof, belonging to the technical fields of synthetic biology and genetic engineering.

Background

The breast milk oligosaccharide is an important oligosaccharide only existing in breast milk, and plays an irreplaceable role in promoting the growth of intestinal flora of infants, improving the function of an immune system, promoting the development of brains and the like. Wherein 2 '-fucosyllactose (2' -FL) accounts for 30% of total amount of breast milk oligosaccharides, and is one of the highest ratio. It can be synthesized from L-fucose and lactose by the catalysis of fucosyltransferase.

In recent years, researchers have begun to widely use microbial fermentation methods to produce 2' -FL, with the host bacteria used primarily including E.coli, B.subtilis, B.licheniformis, and the like. The wild-type genome of these microorganisms already contains various enzymes of the 2' -FL synthesis pathway, such as mannomutase, mannose-1-phosphate-guanylate transferase, etc., but none of them has fucosyltransferase. The most widely used fucosyltransferase genes in the construction of 2' -FL recombinant strains at present are FutC derived from helicobacter pylori and wbgL derived from Escherichia coli O126. Although recombinant bacteria capable of producing 2' -FL have been obtained by the construction of fucosyltransferases derived from the above two microorganisms, the fucosyltransferases encoded by futC and wbgL have extremely low catalytic efficiency at temperatures above 40 ℃. The high-temperature fermentation of industrial microorganisms is very beneficial to reducing the risk of bacteria contamination, improving the fermentation efficiency and reducing the fermentation cost, so that the fucosyltransferase which has high catalytic efficiency and optimal catalytic temperature matched with the high-temperature fermentation conditions of bacteria has great potential in the applications of 2' -FL fermentation production and the like.

Intestinal microorganisms are a class of microorganisms that have important interactions with breast milk oligosaccharides, and the hydrolysis or transglycosylation reactions mediated by enzymes produced by them can alter the structure of breast milk oligosaccharides. Thus, intestinal microorganisms are likely to be a source of novel glycosyltransferases. However, intestinal microorganisms are mostly difficult to perform pure culture and isolation under in vitro standard conditions, which hinders the screening of high catalytic efficiency and high temperature resistant fucosyltransferases from intestinal microorganism sources.

Disclosure of Invention

Aiming at the technical problems of lack of high catalytic efficiency and high temperature resistance of fucosyltransferase and difficulty in screening, the invention aims to provide fucosyltransferase with high catalytic activity of intestinal microorganisms and optimal catalytic temperature matched with high-temperature fermentation of bacteria and encoding gene thereof by utilizing a metagenomic technology. The novel enzyme screened by the detection of the fermentation and the metabolite detection of the recombinant microorganism carrying the gene can be efficiently synthesized into 2' -FL at 42 ℃, thereby having great potential in the applications such as fermentation production of breast milk oligosaccharide.

The invention provides a fucosyltransferase with alpha-1, 2 specificity from intestinal microorganisms, wherein the amino acid sequence of the fucosyltransferase is shown as SEQ ID NO.1 and named as HM3.

The invention provides a coding gene of fucosyltransferase with alpha-1, 2 specificity from intestinal microorganisms, wherein the coding gene of fucosyltransferase is obtained by screening from intestinal metagenome; the nucleotide sequence for encoding HM3 is shown as SEQ ID NO. 2;

the recombinant bacterium for expressing the fucosyltransferase provided by the invention takes bacteria or yeast as a host to express the fucosyltransferase.

In one embodiment of the invention, the host is bacillus licheniformis.

The construction method of the recombinant bacterium BLM2 for expressing the fucosyltransferase HM3, provided by the invention, comprises the following steps:

by homologous weightThe group has nucleotide sequence shown as P shown in SEQ ID NO.3 _Lan The promoter and terminator ter of xylose isomerase gene with nucleotide sequence shown as SEQ ID NO.4 are respectively fused with mannose mutase gene manB shown as SEQ ID NO.5, mannose-1-phosphate-guanylate transferase gene manC shown as SEQ ID NO.6, GDP-mannose-4, 6-dehydratase gene gmd shown as SEQ ID NO.7, GDP-L-fucose synthase gene wcaG shown as SEQ ID NO.8 and fucosyltransferase gene HM3 to obtain corresponding gene expression fragments which are respectively named as H1, H2, H3, H4 and M5;

the primer pairs are used for amplifying gene expression fragments H1, H2, H3, H4 and M5 respectively, so that enzyme cutting sites are introduced at two ends of the gene fragments. And sequentially connecting the gene expression fragments H1, H2 and H3 into pHY300PLK plasmid by using a homologous recombination method to obtain recombinant plasmid pHY-H123. Then sequentially connecting gene expression fragments H4 and M5 into pHT43 plasmid by using a homologous recombination method to obtain recombinant plasmid pHT-M45, and further verifying the construction condition of the plasmid by using 1% agarose gel electrophoresis;

converting the successfully constructed recombinant plasmid pHYH123 into bacillus licheniformis ATCC 9945A to obtain bacillus licheniformis engineering bacteria BLH1, and converting the recombinant plasmid pHT-M45 into bacillus licheniformis engineering bacteria BLH1 by the same method to obtain recombinant bacillus licheniformis bacteria BLM2;

the method for producing 2' -fucosyllactose by recombinant bacteria expressing fucosyltransferase comprises streak-activating the recombinant bacteria on an LB plate, culturing at 37 ℃ for 16h, selecting single colony, inoculating in 15mL LB culture medium at 37 ℃ for 250 r.min ^-1 Culturing for 16-18 h as seed solution, transferring 1mL of seed solution into 30mL of shake flask fermentation medium, shake flask fermenting, controlling initial OD to 0.5-1, and culturing at 37deg.C or 42deg.C for 250r min ^-1 Culturing;

or subjecting the recombinant strain to fed-batch fermentation in a fermenter at a fermentation temperature of 42 ℃ and an initial pH of 7.5. When the pH in the fermentation process is reduced to 7.0, the pH in the fermentation process is maintained at about 7.0 by adding 50% ammonia water, the aeration rate in the fermentation process is controlled at 0.5vvm, the DO is controlled at about 30% by coupling stirring and DO, and the upper limit of the rotating speed is set at 800rpm. Sucrose was continuously fed after 8h fermentation, maintaining it unconsumed.

In one embodiment of the invention, the use of intestinal metagenome-derived fucosyltransferases and recombinant bacteria expressing fucosyltransferases in the production of 2' -FL fermentation production is presented.

The invention has the beneficial effects that:

(1) The invention provides a novel fucosyltransferase HM3 from intestinal microorganisms, which has high catalytic efficiency and good catalytic function under high-temperature fermentation (42 ℃), and is beneficial to reducing the risk of bacteria contamination, improving the fermentation efficiency and reducing the fermentation cost in industrial application.

(2) The invention constructs the bacillus licheniformis recombinant strain BLM2 for expressing the fucosyltransferase HM3, and 2' -FL of 2.91g/L can be obtained after the recombinant strain BLM2 is fermented for 36 hours in a shaking way under the culture condition of 42 ℃; 2' -FL output reaches 56g/L after the recombinant bacteria BLM2 is fed-batch fermented for 46h in a 30L fermentation tank, and the maximum OD ₆₀₀ 72.1.

Drawings

Fig. 1: recombinant plasmid pHY-H123 plasmid map.

Fig. 2: recombinant plasmid pHT-M45 plasmid map.

Fig. 3: an electropherogram of recombinant plasmid verification; m, standard molecular weight Marker; lane 1, pHY-H123 was double digested with SmaI and EcoRI; lane 2, pHT-M45 was cut with EcoRV; lanes 3-10, transformant colonies PCR validated samples.

Fig. 4: protein electrophoresis detection of recombinant fucosyltransferase HM3 pure enzyme; m, standard protein molecular weight Marker; lane 1, recombinant fucosyltransferase HM3 pure enzyme.

Fig. 5: enzyme activity of recombinant fucosyltransferase HM3 pure enzyme under different temperature conditions.

Fig. 6: HPLC quantitative analysis of 2' -FL in fermentation supernatant.

Detailed Description

The strains and primers referred to in the examples below.

TABLE 1 strains involved in the invention

TABLE 2 primer sequences for introduction of cleavage sites

TABLE 3 primer sequences

The gene sequences used in the invention are all synthesized artificially.

The detection method and the calculation method related by the invention are as follows:

the enzyme activity detection method comprises the following steps: the reaction system was 20. Mu.L containing 0.5. Mu. Mol/L substrate GDP-fucose, 20mmol/L MnCI ₂ 1% Triton X-100, 50mmol/L of arsinic acid salt-hydrochloric acid buffer (pH 5.8), 0.5nmol/L of benzene-. Beta. -D-galactose, and recombinant bacterium cell disruption supernatant (containing 0.5mg of total protein) were added in a total volume of 100. Mu.L. Protein concentration was determined by the Bradeford method using the Bio-Rad protein assay kit with BSA as the standard protein. After incubating the mixture at 37℃for 1 hour, the reaction was terminated with 300. Mu.L of chloroform/methanol (2:1, v/v), and after mixing, the mixture was centrifuged at 12000rpm for 3 minutes, and the supernatant was subjected to HPLC detection, whereby the enzyme activity of fucosyltransferase HM3 in the disrupted supernatant of the recombinant cell was calculated by detecting the amount of the product produced in the reaction system by HPLC. The enzyme activity was defined as the amount of enzyme required to produce 1pmol of product under the set reaction conditions for 1h was 1U.

HPLC detection 2' -FL:

the sample treatment method comprises the following steps:

1. fermenting liquor 12000r/min, centrifuging for 20min,

2. 500ul of supernatant is taken, absolute ethanol with the final concentration of 70 percent is added, the temperature is minus 20 ℃ for 2 hours,

3. and then 12000r/min, centrifuging for 20min, and taking 200ul of supernatant, and placing the supernatant into a liquid phase sample injection bottle.

Chromatography column Dikma CarboPac Ca2 +300X8.0 mm,6 μm (Cat. No: 99304) mobile phase: water flow rate: 1.0mL/min detector: RI column temperature: 80℃sample injection amount: 10. Mu.L.

Protein concentration detection:

protein content determination reference coomassie brilliant blue dye solution method: adding 40 μl of sample to be tested into 200 μl of coomassie brilliant blue solution, slowly mixing, standing for 5min, detecting the absorbance at 595nm, and measuring the absorbance at 595nm according to protein concentration standard curve y= 0.2215x-0.0006 (x is absorbance at 595nm, y is protein concentration mg·mL) ^-1 ，R ² =0.9975) calculates the protein concentration in the sample.

Calculation of specific enzyme Activity (U/mg):

the enzyme activity (U) per ml of sample solution was divided by its protein concentration (mg).

EXAMPLE 1 determination of the fucosyltransferase Gene sequence

Comparing infant intestinal metagenome collected in tin-free city with CAZyme database by using dbCAN2 comparison tool, screening and comparing to obtain the gene sequence of family 1 glycotransferase (GT 1), screening out fucosyltransferase gene with strong structural stability by using protein primary structure simulation prediction (ExpASY-ProtParam), secondary structure simulation prediction (SOPMA) and three-dimensional structure template prediction (SWISS-MODEL), artificially synthesizing the gene fragment, named HM3, and carrying out heterologous expression and activity test.

In particular, P is obtained by homologous recombination _Lan The promoter (shown as a nucleotide sequence SEQ ID NO. 3) and a terminator ter (shown as a nucleotide sequence SEQ ID NO. 4) of the xylose isomerase gene are cloned to a vector pHY300-PLK respectively together with a nucleotide sequence (shown as a nucleotide sequence SEQ ID NO. 2) for encoding fucosyltransferase HM3, so as to obtain a recombinant plasmid pHY-HM3.

The specific method for constructing the recombinant plasmid pHY-HM3 comprises the following steps: first, using Bacillus licheniformis genome as a template, amplifying P by using a primer pair PF11-F/R _Lan Promoter sequence, terminator sequence was amplified using primer pair PF13-F/RA column; the nucleotide sequence shown in SEQ ID NO.2 synthesized chemically is used as a template, and a primer pair PF12-F/R is used for amplifying the HM3 coding gene sequence. Then, the three fragments were once ligated to pHY300-PLK, which had been tangential using HindIII and BamHI enzymes, by homologous recombination to transform E.coli, and positive transformants were picked up and plasmids were extracted to obtain recombinant plasmid pHY-HM3.

The recombinant plasmid pHY-HM3 was transformed into Bacillus licheniformis ATCC 9945A to obtain recombinant bacterial BHM3. Culturing recombinant strain on LB plate, culturing at 37deg.C for 16 hr, picking single colony, inoculating into 15mL LB culture medium at 37deg.C for 250 r.min ^-1 Culturing for 16-18 h as seed solution, transferring 1mL of seed solution into 30mL of shake flask fermentation medium, controlling initial OD to 0.5-1, and culturing at 37deg.C or 42deg.C and 250r.min ^-1 Culturing. Fermenting for 48h, collecting cells, ultrasonic crushing to obtain crude enzyme solution, and detecting the enzyme activity of fucosyltransferase in the supernatant of the crushed solution. The result shows that the enzyme activity in the supernatant of the recombinant BHM3 cell disruption solution is 11.2U/mL.

EXAMPLE 2 cloning of fucosyltransferase HM3 and construction of 2' -FL-producing recombinant bacterium

The nucleotide sequence of SEQ ID NO.2 was first chemically synthesized, and the recombinant plasmid pHY-HM3 was obtained in the same manner as in example 1.

Then P is subjected to homologous recombination _Lan The promoter (nucleotide sequence SEQ ID NO. 3) and terminator ter (nucleotide sequence SEQ ID NO. 4) of xylose isomerase gene were ligated to vector pHY300-PLK, respectively, with mannose mutase gene manB (nucleotide sequence SEQ ID NO. 5), mannose-1-phosphate-guanylate transferase gene manC (nucleotide sequence SEQ ID NO. 6), GDP-mannose-4, 6-dehydratase gene gmd (nucleotide sequence SEQ ID NO. 7), GDP-L-fucose synthase gene wcaG (nucleotide sequence SEQ ID NO. 8) to construct plasmid pHY-manB, pHY-manC, pHY-gmd, pHY-wcaG containing 4 expression cassettes.

The construction method of the recombinant plasmid is as follows:

taking the construction method of the recombinant plasmid pHY-manB as an example: p was amplified separately by the same method as in example 1 _Lan PromotersSequence and ter terminator sequence; amplifying a manB coding gene sequence by using a nucleotide sequence shown in a chemically synthesized SEQ ID NO.5 as a template and using a primer pair PF42-F/R, then connecting the three fragments to pHY300-PLK which is tangentially treated by using HindIII and BamHI double enzymes at one time through homologous recombination, transforming escherichia coli, picking up positive transformants, extracting plasmids, and obtaining a recombinant plasmid pHY-manB.

The nucleotide sequence shown in SEQ ID No.6 synthesized chemically is used as a template, a primer pair PF52-F/R is used for amplifying a manC coding gene sequence, and the gene sequence is connected with a plasmid subjected to corresponding double enzyme digestion by the same method, so that a recombinant plasmid pHY-manC is obtained. The nucleotide sequence shown in SEQ ID No.7 synthesized chemically is used as a template, a primer pair PF62-F/R is used for amplifying the gmd coding gene sequence, and the gene sequence is connected with the plasmid subjected to corresponding double enzyme digestion by the same method to obtain the recombinant plasmid pHY-gmd. The nucleotide sequence shown in SEQ ID No.8 synthesized chemically is used as a template, a primer pair PF72-F/R is used for amplifying the wcaG coding gene sequence, and the gene sequence is connected with the plasmid after the corresponding double enzyme digestion by the same method, so as to obtain the recombinant plasmid pHY-wcaG.

Amplifying the P1 by using the recombinant plasmid pHY-manB as a template and using a primer pair to obtain the recombinant plasmid pHY-manB containing the enzyme cutting sites _Lan The expression cassette fragments of the promoter, the manB gene and the ter terminator are designated H1. The same method is used, pHY-manC, pHY-gmd, pHY-wcaG and pHY-HM3 are respectively used as templates, and the primer pair P2-P5 is used for respectively amplifying to obtain corresponding expression cassette fragments which are respectively named as H2, H3, H4 and M5. The reaction conditions are as follows: after pre-denaturation at 95 ℃ for 5min, the mixture enters the next cycle of denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃. The gene expression fragments H1, H2 and H3 are connected into pHY300PLK plasmid by homologous recombination to obtain recombinant plasmid pHY-H123 (figure 1). And then the gene expression fragments H4 and M5 are connected into pHT43 plasmid by utilizing a homologous recombination method in sequence to obtain recombinant plasmid pHT-M45 (figure 2). The construction of the plasmid was further verified by 1% agarose gel electrophoresis (FIG. 3).

The successfully constructed recombinant plasmid pHYH123 was transformed according to Li, Y; jin, k; zhang, LA third party; ding, z.; gu, z.; bacillus licheniformis ATCC 9945A was transformed by the method of Shi, G.development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose OPERon. Journal ofAgricultural and Food Chemistry 2018,66,9456-9464 to obtain Bacillus licheniformis engineering strain BLH1. The specific transformation method comprises the following steps: fresh single colonies of Bacillus licheniformis were first picked and inoculated into 50mL LBSP medium (10 g.L ^-1 Peptone, 10 g.L ^-1 Sodium chloride, 5 g.L ^-1 Yeast paste, 0.50 mol.L ^-1 Sorbitol), at 37℃at 200rpm for 12h. Next, 0.4mL of the culture medium was transferred to 50mL of LBS medium, and cultured at 37℃and 200rpm until the cell concentration reached an absorbance (OD) of 600nm ₆₀₀ ) 0.6 to 0.8, and collecting the bacterial cells by freeze centrifugation (3000 g,8 min). With 20mL SHMP medium (0.5 mol.L) ^-1 Sorbitol, 0.5 mol.L ^-1 Mannitol, 10% glycerol) and then freeze-centrifuging to discard the supernatant to wash the cells, for a total of 4 washes. After completion, the somatic cells were resuspended in 1mL SHMP medium, 90. Mu.L was removed and placed in a 1.5mL centrifuge tube, about 200ng of plasmid was added thereto, and the mixture was gently mixed and then ice-cooled for 5min. Transferring the mixture into a precooled electrorotating cup, and placing the electrorotating cup into an electrorotating instrument to carry out electroporation, wherein the conditions are as follows: voltage, 1000V; time, 5ms. 800. Mu.L LBSPG medium (10 g.L) ^-1 Peptone, 10 g.L ^-1 Sodium chloride, 5 g.L ^-1 Yeast paste, 0.50 mol.L ^-1 Sorbitol, 0.38 mol.L ^-1 Mannitol) and gently mixed, incubated at 37℃for 2h at 100rpm, plated on a resistance plate and incubated at 37 ℃.

And then the recombinant plasmid pHT-M45 is transformed into Bacillus licheniformis engineering bacteria BLH1 by the same method to obtain recombinant Bacillus licheniformis bacteria BLM2.

Example 32' -FL shaking flask fermentation

Streaking and activating the bacillus licheniformis engineering bacteria BLM2 constructed in the example 2 on an LB plate, culturing for 16 hours at 37 ℃, picking single colony, inoculating the single colony into 15mL LB culture medium at 37 ℃ and 250 r.min ^-1 Culturing for 16-18 h as seed solution, transferring 1mL of seed solution into 30mL of shake flask fermentation medium, and shake flask fermentingThe initial OD is controlled to be 0.5-1, and the temperature is 37 ℃ or 42 ℃ and the temperature is 250 r.min ^-1 Culturing. Sampling every 12h, fermenting at 4deg.C for 12000 r.min ^-1 Centrifugation was carried out for 10min under conditions and the supernatant was used to detect the product (FIG. 4). The results show that 2.91 g/L2 '-FL can be obtained after fermentation of engineering bacterium BLM2 for 36h under the culture condition of 42 ℃, and the yield of 2' -FL in the fermentation broth for 36h at 37 ℃ is 2.83g/L.

EXAMPLE 4 characterization of enzymatic Properties

(1) Cell disruption of recombinant bacteria culture medium

The recombinant strain BLM2 was fermented using a shake flask fermentation medium at 37℃and 250rpm for 48 hours. After the fermentation broth was centrifuged at 12000rpm for 10min, the supernatant was discarded to collect the cells. Disrupted cell solution A (10 mM sodium phosphate buffer solution (pH=7.0) +0.5 mol.L) was used ^-1 NaCl+3g·L ^-1 Lysozyme) was washed, repeated 3 times, and the cells were still diluted to OD with solution a ₆₀₀ Incubation was carried out at 37℃for 1-1.5h until flocculent precipitate formation was observed, and cell disruption was carried out by placing the cells on ice under conditions such that each 1 second of operation was stopped for 2 seconds and the total disruption time was 8min. And after the liquid is clarified, the crushing is successful. The resulting cell disruption solution was centrifuged at 12000rpm for 10 min.

(2) Separation and purification of recombinant fucosyltransferase HM3

The purification mode adopts Mag-beams His-Tag protein purification magnetic Beads for purification. Every 15mL centrifuge tube contains 2-3mL of protein purification magnetic beads and is equipped with a magnetic rack. The relevant operation steps are as follows: (1) regenerating magnetic beads: add 5mL ddH ₂ O, mixing uniformly and magnetically separating; adding 5mL of regeneration buffer solution, uniformly mixing, and magnetically separating for 2 times; add 5mL ddH ₂ O, mixing uniformly, rotating for 5min at room temperature, and magnetically separating for 2 times; add 5mLNi ²⁺ Regenerating buffer solution, uniformly mixing for 20min at room temperature, and magnetically separating; add 5mL ddH ₂ O, mixing evenly, and carrying out magnetic separation operation for 4 times. (2) Pretreatment of magnetic beads: adding 5mL of binding buffer solution, uniformly mixing, and magnetically separating for 2 times; (3) binding of the protein of interest to the magnetic beads: adding 2mg of protein sample into the pretreated centrifuge tube, placing the centrifuge tube on a vortex oscillator to oscillate for 15s, and placing the centrifuge tube into a rotary mixer after fully and uniformly mixingAnd (3) placing the sample on a synthesizer at room temperature for 20-30min to ensure the full combination of the target protein and the magnetic beads, and performing magnetic separation after finishing. (4) Washing magnetic beads: after 10mL of wash buffer was added and gently turned over, the mixture was magnetically separated, and the mixture was twice subjected to a double operation, and the decanted mixture was collected. The purpose is as follows: washing off the impurity proteins and unbound proteins. To avoid nonspecific adsorption of proteins on the walls of the original centrifuge tube, the beads can be transferred to a new centrifuge tube. (5) Eluting target protein: 5-8mL of elution buffer was added, the magnetic separation was performed 3 times, and the decanted liquid was collected. (6) Post-treatment of magnetic beads: adding 5mL of water, and magnetically separating for 2 times; adding 5mL of the magnetic bead preservation solution, and preserving in a refrigerator at 4 ℃. The purified enzyme solution is collected, transferred to a dialysis bag with proper size and pretreatment, imidazole and salt are removed by overnight dialysis, the dialysis solution is replaced for 2-3 times, and the protein concentration is measured after the completion. If necessary, the enzyme solution may be subjected to ultrafiltration concentration.

(3) Specific enzyme activity determination of recombinant fucosyltransferase HM3

The purified recombinant fucosyltransferase HM3 purified enzyme was reacted in a reaction system of 20. Mu.L containing 0.5. Mu. Mol/L substrate GDP-fucose and 20mmol/LMnCI ₂ 1% Triton X-100, 50mmol/L of arsinic acid salt-hydrochloric acid buffer (pH 5.8), 0.5nmol/L of benzene-beta-D-galactose and 0.5mg of recombinant fucosyltransferase HM3 pure enzyme, the total volume was 100. Mu.L, after incubating the mixture at 37℃for 1 hour, the reaction was terminated with 300. Mu.L of chloroform/methanol (2:1, v/v), after mixing evenly, centrifugation was carried out at 12000rpm for 3 minutes, the supernatant was taken for HPLC detection, and the enzyme activity of recombinant fucosyltransferase HM3 was calculated indirectly by detecting the production amount of the product in the reaction system by HPLC, and the specific enzyme activity was calculated. The results showed that the specific enzyme activity of the recombinant fucosyltransferase HM3 pure enzyme was 50.9U/mg.

(4) Characterization of temperature characteristics of recombinant fucosyltransferase HM3

Referring to the above method for enzyme activity detection reaction, the purified recombinant fucosyltransferase HM3 purified enzyme was reacted at 30℃at 37℃at 42℃and at 50℃respectively. After the reaction, the enzyme activity of the recombinant fucosyltransferase HM3 was calculated indirectly by detecting the decrease in the substrate and the product formation in the reaction system by HPLC. The results showed that the enzyme activity at 42℃reached 141.8U/mL, which is 1.4 times that at 37℃and that the enzyme activity at 50℃was also higher than 37 ℃.

EXAMPLE 5 engineering bacterium BLM2 fermentation production of 2' -FL in 20L fermenter

Activating the Bacillus licheniformis engineering bacteria BLM2 constructed in the example 2, culturing at 37 ℃ for 16 hours, picking single colony, inoculating in 15mL LB culture medium at 37 ℃ for 250 r.min ^-1 Culturing for 16-18 h as seed solution, transferring 1mL of seed solution into 30mL of shake flask fermentation medium, controlling initial OD to be 0.5-1, and performing fed-batch fermentation in a fermentation tank at a fermentation temperature of 42 ℃ and an initial pH of 7.5. When the pH in the fermentation process is reduced to 7.0, the pH in the fermentation process is maintained at about 7.0 by adding 50% ammonia water, the aeration rate in the fermentation process is controlled at 0.5vvm, the DO is controlled at about 30% by coupling stirring and DO, and the upper limit of the rotating speed is set at 800rpm. After 8h fermentation, a 50% sucrose solution was continuously fed at a rate of 20mL/h, maintaining it unconsumed.

Fermentation medium: 30g/L cottonseed protein, 75g/L sucrose, 80g/L, K lactose ₂ HPO ₄ ·3H ₂ O 9.12g/L、KH ₂ PO ₄ 1.36g/L、FeCl ₃ 0.5 g/L、(NH ₄ ) ₂ HPO ₄ 10g/L (pH 7.5). 30L of fermentation tank liquid amount is 15L. The 2' -FL yield after 46h of fed-batch fermentation on a fermenter scale reached 56g/L, maximum OD ₆₀₀ 72.1.

Comparative example 1:

specific embodiment the same as in example 2 except that the fucosyltransferase gene HM3 was replaced with fucosyltransferase futC (GenBank: ABO 61750.1) derived from helicobacter pylori (Helicobacter pylori), and the recombinant bacteria thus constructed were fermented by the fermentation methods shown in example 3 and example 5 at 42℃and the production amount of 2' -FL by shake flask fermentation was 1.02g/L; the 2' -FL yield of the 20L fermenter fermentation was 23g/L.

Comparative example 2:

specific embodiment the same as in example 2 except that the fucosyltransferase gene HM3 was changed to fucosyltransferase wbgL (GenBank: ADN 43847.1) derived from E.coli (Escherichia coli O126), and the recombinant bacteria thus constructed were fermented by the fermentation methods shown in examples 3 and 5 at 42℃and the production yield of 2' -FL by shake flask fermentation was 0.82g/L; the 2' -FL yield of the 20L fermenter fermentation was 17g/L.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The fucosyltransferase is characterized in that the amino acid sequence of the fucosyltransferase is shown in SEQ ID NO. 1.

2. A gene encoding the fucosyltransferase of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.

3. A recombinant bacterium for expressing a fucosyltransferase according to claim 1, wherein the fucosyltransferase is expressed in a bacterium or yeast.

4. The recombinant bacterium of claim 3, wherein the host is bacillus licheniformis.

5. A method of constructing the recombinant bacterium of claim 3 or 4, comprising the steps of:

(1) Respectively fusing a promoter shown in SEQ ID NO.3 and a terminator shown in SEQ ID NO.4 at two ends of a phosphomannose mutase gene manB, a mannose-1-phosphate-guanylate transferase gene manC, a GDP-mannose-4, 6-dehydratase gene gmd, a GDP-L-fucose synthase gene wcaG and a fucosyltransferase gene to obtain an expression fragment containing the genes;

(2) Connecting the expression fragment constructed in the step (1) to a plasmid pHY300PLK in the sequence of manB, manC and gmd to obtain a recombinant plasmid 1; then the gene wcaG and the gene of claim 2 are connected to plasmid pHT43 to obtain recombinant plasmid 2;

(3) And (3) converting the recombinant plasmid 1 and the recombinant plasmid 2 constructed in the step (2) into bacillus licheniformis to obtain recombinant bacillus licheniformis.

6. A method for producing 2' -fucosyllactose, characterized in that the recombinant bacterium of claim 3 or 4 is used for fermentation at 37 ℃ to 42 ℃ for at least 46 hours.

7. The method according to claim 6, wherein the medium for fermentation comprises: cottonseed protein 5-30g/L, sucrose 40-75g/L, lactose 40-80g/L, K ₂ HPO ₄ ·3H ₂ O7.28-9.12g/L、KH ₂ PO ₄ 1.36-3.15g/L、FeCl ₃ 0.5-1g/L and (NH) ₄ ) ₂ HPO ₄ 5-10g/L; the initial pH is 6.5-7.5.

8. The method of claim 7, wherein the fermentation process is further fed with sucrose after 6-10 hours of fermentation.

9. Use of a fucosyltransferase according to claim 1, or a gene according to claim 2, or a recombinant bacterium according to claim 3 or 4, or a method according to claim 5, or a method according to any one of claims 6-8 for the production of 2 '-fucosyllactose and products containing 2' -fucosyllactose.

10. The use according to claim 9, wherein the product comprises an infant formula.