CN114921434B - Recombinant glycosyltransferases catalyzing Reb a to produce Reb M - Google Patents

Recombinant glycosyltransferases catalyzing Reb a to produce Reb M Download PDF

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CN114921434B
CN114921434B CN202210589049.XA CN202210589049A CN114921434B CN 114921434 B CN114921434 B CN 114921434B CN 202210589049 A CN202210589049 A CN 202210589049A CN 114921434 B CN114921434 B CN 114921434B
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reb
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catalyzing
glycosyltransferase
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CN114921434A (en
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马媛媛
王保淇
汪振洋
魏晓珍
宋浩
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Tianjin University
Sinochem Health Co Ltd
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Sinochem Health Co Ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin

Abstract

The invention discloses a recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, and the nucleotide sequence of a recombinant gene for encoding the recombinant glycosyltransferase is shown as SEQ ID NO.1-SEQ ID NO. 8; the recombinant glycosyltransferase for catalyzing Reb A to produce Reb M can directly catalyze and produce Reb M by taking Reb A as a substrate with high efficiency, and Reb M production by Reb A two-step catalysis or Reb D catalysis is avoided. The manner of producing Reb M with Reb D having low solubility and more high cost is avoided, and the production cost of the substrate is greatly saved. Compared with the traditional two-step in-vitro cascade catalysis method, the method realizes one-step catalysis, simplifies the whole operation flow, effectively reduces consumable loss and operation time, and reduces production cost; the method has high Reb M generation rate.

Description

Recombinant glycosyltransferases catalyzing Reb a to produce Reb M
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, recombinant genes for encoding the recombinant glycosyltransferase, recombinant strain for expressing the recombinant glycosyltransferase for catalyzing Reb A to produce Reb M and application of the recombinant glycosyltransferase.
Background
Steviol glycosides (Stevia), also known as steviol (Stevia sugar), are novel sweeteners extracted from Stevia leaves. Steviol glycosides have been used as non-nutritive sweeteners in some countries for hundreds of years for sweet foods because they do not produce calories, nor increase blood glucose levels, and have a sweetness 300 times higher than sucrose. Rebaudioside a (Reb a) is the predominant steviol glycoside therein, 300 times the sweetness of sucrose. However, the bitter taste of Reb a affects the mouthfeel, and research shows that Rebaudioside M (Reb M) tastes good and has little bitter taste, and the sweetness can be 400 times that of sucrose, and is considered to be a new generation of stevia-derived sweetener. In 2014, stevia-derived sweeteners containing Reb M were certified by the U.S. food and drug administration (USFDA) for safety (Generally Recognized as Safe, GRAS).
However, the Reb A content is 3.8% of the dry cell weight, the Reb M content is 0.4% of the dry cell weight, and only trace amounts exist in stevia leaves, so that the conventional extraction method is difficult to expand the production scale. And with the identification of the gene encoding the synthetic stevioside Reb M, the enzymatic synthesis of Reb M is possible. Studies have reported that Reb A can be catalyzed by uridine diphosphate glycosyltransferase (uridine diphosphate glycosyltransferase, UGTs) in natural stevia leaves to produce Rebaudioside D (Reb D), and then Reb D can be catalyzed to produce Reb M.
However, reb D is less water soluble (0.04%) than Reb a (0.8%), and is more expensive compared to Reb a. Therefore, in the current method of producing Reb M by using Reb D as a substrate, the substrate Reb D has the disadvantages of low water solubility, high price and the like. There are studies on the production of Reb M using Reb a as a substrate using a mixture of two enzymes for dual enzyme in vitro cascade catalysis. However, the low water solubility of the intermediate product Reb D limits the forward progress of the reaction, and the production rate of the product Reb M is only 37.9%, which limits the industrial application thereof.
Therefore, the existing method for producing Reb M has the problems of high production cost, poor substrate water solubility, low yield and the like. The method for producing Reb M by using Reb a catalysis can reduce the substrate production cost, but cannot avoid accumulation of byproducts and lower catalysis efficiency, and effective measures are needed to reduce the accumulation of byproducts and improve the catalysis efficiency.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide recombinant glycosyltransferase for catalyzing Reb A to produce Reb M.
A second object of the present invention is to provide a recombinant gene encoding the above recombinant glycosyltransferase.
It is a third object of the present invention to provide a recombinant strain expressing a recombinant glycosyltransferase catalyzing the production of Reb M from Reb a.
It is a fourth object of the present invention to provide an induced expression of the recombinant strain described above.
It is a fifth object of the present invention to provide a method for catalyzing Reb a production of Reb M by a bacterial cell containing a recombinant glycosyltransferase that catalyzes Reb a production of Reb M.
It is a sixth object of the present invention to provide a method for catalyzing Reb a production of Reb M by an enzyme solution of a recombinant glycosyltransferase that catalyzes Reb a production of Reb M.
The technical scheme of the invention is summarized as follows:
recombinant glycosyltransferases catalyzing Reb a to produce Reb M.
The nucleotide sequence of the recombinant gene is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
Recombinant strains expressing recombinant glycosyltransferases catalyzing the production of Reb M from Reb a were constructed as follows: constructing the recombinant gene into an expression vector to obtain a recombinant vector; the recombinant vector is transformed into a host strain to obtain a recombinant strain.
The expression vector is preferably pPIC9K, pPIC9, pPink. Alpha. -HC, pET-28a, pMAL or pBAD30.
The host strain is preferably E.coli or Pichia pastoris.
The induction expression of the recombinant strain comprises the following steps: culturing the recombinant strain, adding an inducer, and carrying out induced expression for 6-27h at 15-37 ℃ to obtain a bacterial cell containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M; and (3) breaking the bacterial cells to obtain an enzyme solution of the recombinant glycosyltransferase for catalyzing the production of the Reb M by the Reb A.
A method for catalyzing Reb a production of Reb M by a bacterial cell containing a recombinant glycosyltransferase that catalyzes Reb a production of Reb M, comprising the steps of:
get final OD 600 25-250 of the recombinant glycosyltransferase for catalyzing production of Reb M from Reb A or the cell containing the recombinant glycosyltransferase for catalyzing production of Reb M from Reb A after the permeabilization treatment, adding 1-10g/L of Reb A, 4-10mM uridine diphosphate glucose and 0.4-1mM Mn 2+ Adding PBS with final concentration of 50mM and pH of 7.2-8.0, reacting at 26-42deg.C and rotation speed of 50-250rpm for 10-24 hr, and catalyzing to obtain Reb M;
the permeabilized bacterial cells containing the recombinant glycosyltransferase that catalyzes the production of Reb M from Reb a are prepared in the following manner:
mode one: freezing the bacterial cells containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M at-20 degrees to-80 ℃ for 0.5-24 hours;
mode two: re-suspending the bacterial cells containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M with cell penetrating agent with final concentration of 0.15-0.45g/L, and standing at 20-30deg.C for 5min-1h; the cell penetrating agent is cetyl trimethyl ammonium bromide, triton X-100, polysorbate-80 or sodium dodecyl sulfate.
A method for catalyzing Reb a to produce Reb M using an enzyme solution of a recombinant glycosyltransferase that catalyzes Reb a to produce Reb M, comprising the steps of:
taking the enzyme solution of the recombinant glycosyltransferase for catalyzing the production of the Reb M by the Reb A, wherein the final concentration of the enzyme solution is 1-5g/L, adding 1-10g/LReb A, 1-8mM uridine diphosphate glucose and 0.1-6mM metal ion, adding 50mM buffer solution with the pH of 5.5-10.5, reacting for 12-96 hours at the temperature of 26-42 ℃ and catalyzing to generate the Reb M;
the metal ion is Mg 2+ 、Mn 2+ 、Ba 2+ 、Ca 2+ Or Pb 2+ 。
THE ADVANTAGES OF THE PRESENT INVENTION
(1) The recombinant glycosyltransferase for catalyzing Reb A to produce Reb M can directly catalyze Reb A as a substrate to produce Reb M with high efficiency, and Reb M production by Reb A two-step catalysis or Reb D catalysis is avoided. The manner of producing Reb M with Reb D having low solubility and more high cost is avoided, and the production cost of the substrate is greatly saved.
(2) Compared with the traditional two-step in-vitro cascade catalysis method, the method realizes one-step catalysis, simplifies the whole operation flow, effectively reduces consumable loss and operation time, and reduces production cost;
(3) The method has higher substrate accessibility, can effectively avoid the limitation of low solubility of the cascade catalysis intermediate product Reb D on catalytic activity, remarkably improves the yield of Reb M, and lays a foundation for industrial application.
Drawings
FIG. 1 is an SDS-PAGE gel of the crude enzyme solution of the recombinant bacterium obtained in example 2.
Detailed Description
The substances referred to herein are abbreviated as follows:
rebaudioside a is abbreviated Reb a; rebaudioside D is abbreviated Reb D; uridine diphosphate glucose is abbreviated as UDPG; isopropyl-beta-D-thiogalactoside is abbreviated as IPTG.
EXAMPLE 1 construction of recombinant Strain expressing recombinant glycosyltransferase catalyzing production of Reb M by Reb A
The inventors of the present invention designed and synthesized 12 recombinant genes catalyzing Reb A to produce Reb M recombinant glycosyltransferases from Huada gene, SL1E (SEQ ID NO. 1), SL2E (SEQ ID NO. 2), SL3E (SEQ ID NO. 3), SL4E (SEQ ID NO. 4), SL5E (SEQ ID NO. 5), SL6E (SEQ ID NO. 6), S1SL2E (SEQ ID NO. 7), EL5S (SEQ ID NO. 8), EL1S (SEQ ID NO. 9), EL3S (SEQ ID NO. 10), EL4S (SEQ ID NO. 11) and EL6S (SEQ ID NO. 12), respectively, and constructed on pET28a linearization vectors after double cleavage of NcoI and XhoI by seamless cloning technique to obtain corresponding recombinant plasmids pET28a-SL1E, pET a-SL2E, pET28a-SL3E, pET a-SL4E, pET a-SL4E, pET a-5 SL 28 a-3728 a-6 EL 28 a-6 EL 28 a-9728 a-6 EL3S (SEQ ID NO. 12), respectively.
The recombinant plasmids are respectively transformed into E.coli BL21 (DE 3) competent cells, LB (1% peptone, 0.5% yeast powder, 1% NaCl,1.6% agar powder) solid plates containing 50 mug/ml kanamycin are adopted for screening, colony PCR identification is carried out on the screened monoclonal transformants, and corresponding recombinant strains BL21 (pET 28a-SL 1E), BL21 (pET 28a-SL 2E), BL21 (pET 28a-SL 3E), BL21 (pET 28a-SL 4E), BL21 (pET 28a-SL 5E), BL21 (pET 28a-SL 6E) are obtained,
BL21 (pET 28a-S1SL 2E), BL21 (pET 28a-EL 5S), BL21 (pET 28a-EL 1S), BL21 (pET 28a-EL 3S), BL21 (pET 28a-EL 4S) and BL21 (pET 28a-EL 6S).
Experiments prove that the commercial expression vectors pPIC9K, pPIC, pPink alpha-HC, pMAL or pBAD30 are used for replacing pET-28a, and corresponding recombinant plasmids can be obtained.
The recombinant plasmid can also be respectively transformed into competent cells of escherichia coli such as Tuner (DE 3), BL21star (DE 3), BL21-AI and the like or competent cells of pichia pastoris such as X33, KM71, GS115 and the like to obtain corresponding recombinant strains.
Example 2 inducible expression of a recombinant strain comprising the steps of:
(1) Each recombinant strain obtained in example 1 was cultured to OD at 37℃under conditions of 220rpm in LB liquid medium (1% peptone, 0.5% yeast powder, 0.5% NaCl) containing 50. Mu.g/ml kanamycin 600 0.5 to 0.9;
(2) Adding inducer IPTG to make the final concentration 0.1mM, and inducing expression at 18 deg.C for 18h;
(3) Centrifugation (12000 rpm, 4 ℃,10 min), removal of supernatant, and collection of bacterial cells. Washing the collected cells with 10mM PBS (pH 7.2) for 1 time to remove the residual culture medium on the cells; the cells were resuspended in 10mM PBS (pH 7.2) to obtain cells containing recombinant glycosyltransferases catalyzing the production of Reb M by Reb A and designated C1-C12, respectively;
ultrasonic cell disruption of each cell in an ice bath under the conditions: 150W, working for 5s, intermittently for 8s and fully for 15min, centrifuging the cytoblast liquid (12000 g, 4 ℃ for 10 min), collecting supernatant, namely crude enzyme liquid of recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, respectively named as E1-E12, and purifying the obtained crude enzyme liquid.
mu.L of each of the crude enzyme solutions E1 to E12 was added to 5. Mu.L of 5 Xprotein loading buffer, and after mixing, denaturation and inactivation treatment was performed at 100℃for 10min, followed by centrifugation (12000 g, 4℃for 2 min), and the supernatant was subjected to 10% SDS-PAGE gel protein electrophoresis, and the results were shown in FIG. 1.SDS-PAGE analysis shows that all recombinant strains have a distinct band at about 108kDa, which is substantially consistent with the predicted size of the target protein, indicating successful expression of the recombinant fusion protein. Wherein the E1-E8 target protein bands are brighter, and the expression level is higher; E9-E12 expression levels were lower.
Example 3 inducible expression of a recombinant strain comprising the steps of:
(1) Recombinant strain BL21 (pET 28a-SL 2E) was cultured to OD at 37℃and 220rpm in LB liquid medium (1% peptone, 0.5% yeast powder, 0.5% NaCl) containing 50. Mu.g/ml kanamycin 600 0.5 to 0.9;
(2) Adding inducer IPTG to make the final concentration 0.1mM, and inducing expression at 15 deg.C for 27h;
(3) As in step (3) of example 2, a bacterial cell containing a recombinant glycosyltransferase catalyzing the production of Reb M by Reb A was obtained and designated as 2C2.
Cell 2C2 cells were sonicated in an ice bath under the following conditions: 150W, working for 5s, intermittently for 8s and fully for 15min, centrifuging the cytoblast liquid (12000 g, 4 ℃ for 10 min), collecting supernatant, namely a crude enzyme liquid of recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, named as 2E2, and purifying the obtained crude enzyme liquid.
SDS-PAGE analysis showed that 2E2 had a distinct band at about 108kDa, which was substantially consistent with the predicted size of the protein of interest, indicating successful expression of the recombinant fusion protein.
Example 4 inducible expression of a recombinant strain comprising the steps of:
(1) Recombinant strain BL21 (pET 28a-SL 2E) was cultured to OD at 37℃and 220rpm in LB liquid medium (1% peptone, 0.5% yeast powder, 0.5% NaCl) containing 50. Mu.g/ml kanamycin 600 0.5 to 0.9;
(2) Adding inducer IPTG to make the final concentration 0.1mM, and inducing expression at 37 deg.C for 6h;
(3) As in step (3) of example 2, a bacterial cell containing a recombinant glycosyltransferase catalyzing the production of Reb M by Reb A was obtained and designated as 3C2.
Cell 3C2 was sonicated in an ice bath under the following conditions: 150W, working for 5s, intermittently for 8s and fully for 15min, centrifuging the cytoblast liquid (12000 g, 4 ℃ for 10 min), collecting supernatant, namely a crude enzyme liquid of recombinant glycosyltransferase for catalyzing RebA to produce RebM, named 3E2, and purifying the obtained crude enzyme liquid.
SDS-PAGE analysis showed that 3E2 had a distinct band at about 108kDa, which was substantially consistent with the predicted size of the protein of interest, indicating successful expression of the recombinant fusion protein.
Example 5
A method for catalyzing Reb a to produce Reb M by bacterial cells C1-C12 (obtained in example 2), comprising the steps of:
will end OD 600 160C 1-C12 were added with 1g/L Reb A, 6mM UDPG, and 1mM MnCl, respectively 2 And PBS with a final concentration of 50mM and a pH of 7.5, and the reaction was catalyzed to form Reb M at 34℃and a rotation speed of 150rpm for 10 hours.
After completion of the reaction, 200. Mu.L of chromatographic acetonitrile was added, and after shaking and mixing, the mixture was allowed to stand for 10 minutes, centrifuged at 12000rpm at room temperature for 10 minutes, and the supernatant was subjected to an organic film of 0.22. Mu.m, and then analyzed by HPLC. HPLC adopts a Luna C18 reverse phase bonded silica gel separation column (4.6 mm multiplied by 250mm,5 μm), mobile phase adopts 25% acetonitrile, flow rate is 1mL/min, column temperature is 40 ℃, ultraviolet detector VWD is adopted, wavelength of the VWD detector is 210nm, and sample injection amount is 5 mu L. The concentration changes of the substrate Reb A and the product Reb M were found by liquid phase analysis, and the Reb M production rates of C1-C12 are shown in Table 1.
Example 6
A method for producing Reb M by catalyzing Reb a with bacterial cell 2C2 (obtained in example 3), comprising the steps of:
will end OD 600 25 of 2C2 was added with 5g/L Reb A, 10mM UDPG, 0.4mM MnCl 2 And PBS with a final concentration of 50mM and a pH of 7.2, and was reacted at 26℃and a rotation speed of 50rpm for 24 hours to catalyze the formation of Reb M, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 7
A method for producing Reb M by catalyzing Reb a with bacterial cell 3C2 (obtained in example 4), comprising the steps of:
will end OD 600 250 of 3C2 was added with 10g/L Reb A final concentration, 4mM UDPG final concentration, 0.7mM MnCl final concentration 2 And PBS with a final concentration of 50mM and a pH of 8.0, and was reacted at 42℃and a rotation speed of 250rpm for 14 hours to catalyze the formation of Reb M, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 8 preparation of somatic cells FC1-FC12 and method for catalyzing Reb a to produce Reb M comprising the steps of:
freezing C1-C12 at-20deg.C for 24 hr, and re-suspending with 10mM PBS (pH 7.5) to obtain cell containing recombinant glycosyltransferase for catalyzing rebA to produce rebM, named FC1-FC12;
get final OD 600 FC1-FC12 at 160 was added with 1g/L RebA, 6mM UDPG, 1mM MnCl, respectively 2 And PBS with a final concentration of 50mM and a pH of 7.5, and was reacted at 34℃and a rotation speed of 150rpm for 10 hours to catalyze the formation of RebM, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 9 preparation of somatic cell F2C2 and method for catalyzing Reb a to produce Reb M comprising the steps of:
freezing 2C2 at-50deg.C for 12 hr for permeability treatment, and re-suspending with 10mM PBS (pH 7.5) to obtain cell containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, named F2C2;
get final OD 600 25F 2C2 was added with 5g/L RebA, 10mM UDPG, 0.4mM MnCl 2 And PBS with a final concentration of 50mM and a pH of 7.2, and was reacted at 26℃and a rotation speed of 50rpm for 24 hours to catalyze the formation of RebM, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 10 preparation of somatic cell F3C2 and method for catalyzing Reb a to produce Reb M comprising the steps of:
freezing 3C2 at-80deg.C for 0.5 hr, and re-suspending with 10mM PBS (pH 7.5) to obtain cell containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, named F3C2;
will end OD 600 F3C2 at 250 was added with 10g/L RebA at a final concentration, 4mM UDPG at a final concentration, and MnCl at a final concentration of 0.7mM 2 And PBS with a final concentration of 50mM and a pH of 8.0, and was reacted at 42℃and a rotation speed of 250rpm for 14 hours to catalyze the formation of RebM, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 11 preparation of bacterial cells PC1-PC12 and method for catalyzing production of Reb M by Reb A comprising the steps of:
C1-C12 were resuspended in cetyltrimethylammonium bromide as a cell permeabilizer at a final concentration of 0.3g/L, and after standing at 20℃for 5min, the cells were resuspended in 10mM PBS (pH 7.5), designated as PC1-PC12, respectively;
will end OD 600 160 PC1-PC12 was added with 1g/L RebA, 6mM UDPG and 1mM MnCl 2 And PBS with a final concentration of 50mM and a pH of 7.5, and was reacted at 34℃and a rotation speed of 150rpm for 10 hours to catalyze the formation of Reb M, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 12 preparation of somatic cell P2C2 and method for catalyzing production of Reb M by Reb A comprising the steps of:
2C2 was resuspended with polysorbate-80, a cell-permeabilizing agent, at a final concentration of 0.15g/L, and allowed to stand at 30℃for 30min, and the cells were resuspended with 10mM PBS (pH 7.5), designated P2C2;
will end OD 600 25P 2C2 was added with 5g/L RebA, 10mM UDPG and 1mM MnCl 2 And PBS with a final concentration of 50mM and a pH of 7.2, and was reacted at 26℃and a rotation speed of 50rpm for 24 hours to catalyze the formation of Reb M, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Example 13 preparation of somatic cell P3C2 and method for catalyzing production of Reb M by Reb A comprising the steps of:
3C2 was resuspended in triton X-100 (experiments demonstrated that sodium dodecyl sulfate was also available) using a final concentration of 0.45g/L, and after standing at 25℃for 1h, the cells were resuspended in 10mM PBS (pH 7.5), designated P3C2;
will end OD 600 The final concentration of P3C2 was 250 to 10g/L Reb A, 4mM UDPG and 0.7mM MnCl 2 And PBS with a final concentration of 50mM and a pH of 8.0, and was reacted at 42℃and a rotation speed of 250rpm for 14 hours to catalyze the formation of Reb M, and the formation rate of Reb M was analyzed by HPLC and shown in Table 1.
Table 1 shows the results of the activity of the bacterial cells of examples 5-13 containing recombinant glycosyltransferases catalyzing the production of Reb M from Reb A.
Reaction numbering Cell of fungus Reaction system Reb M generation rate Reaction numbering Cell of fungus Reaction system Reb M generation rate
1-1 C1 Example 5 65％ 1-22 FC8 Example 8 63％
1-2 C2 Example 5 75％ 1-23 FC9 Example 8 43％
1-3 C3 Example 5 69％ 1-24 FC10 Example 8 55％
1-4 C4 Example 5 78％ 1-25 FC11 Example 8 51％
1-5 C5 Example 5 70％ 1-26 FC12 Example 8 47％
1-6 C6 Example 5 66％ 1-27 F2C2 Example 9 63％
1-7 C7 Example 5 63％ 1-28 F3C2 Example 10 65％
1-8 C8 Example 5 61％ 1-29 PC1 Example 11 87％
1-9 C9 Example 5 40％ 1-30 PC2 Example 11 95％
1-10 C10 Example 5 53％ 1-31 PC3 Example 11 89％
1-11 C11 Example 5 51％ 1-32 PC4 Example 11 100％
1-12 C12 Example 5 44％ 1-33 PC5 Example 11 93％
1-13 2C2 Example 6 61％ 1-34 PC6 Example 11 81％
1-14 3C2 Example 7 65％ 1-35 PC7 Example 11 86％
1-15 FC1 Example 8 64％ 1-36 PC8 Example 11 84％
1-16 FC2 Example 8 79％ 1-37 PC9 Example 11 54％
1-17 FC3 Example 8 71％ 1-38 PC10 Example 11 63％
1-18 FC4 Example 8 81％ 1-39 PC11 Example 11 66％
1-19 FC5 Example 8 76％ 1-40 PC12 Example 11 58％
1-20 FC6 Example 8 66％ 1-41 P2C2 Example 12 83％
1-21 FC7 Example 8 69％ 1-42 P3C2 Example 13 88％
Example 14 enzyme solution of recombinant glycosyltransferase catalyzing production of Reb M by Reb a (E1-E12 obtained in example 2) a method of catalyzing production of Reb M by Reb a comprising the steps of:
adding Reb A with final concentration of 2.5g/L E1-E12, 6mM UDPG and 1mM MnCl 2 And PBS with final concentration of 50mM and pH of 7.5, and reacting at 37 ℃ for 48 hours to catalyze and generate Reb M; analysis of Reb M formation using HPLC is shown in table 2.
Example 15 enzyme solution of recombinant glycosyltransferase catalyzing production of Reb M by Reb a (2E 2 obtained in example 3) a method for producing Reb M by Reb a comprises the steps of:
to 1 g/L2E 2, 5g/L Reb A, 8mM UDPG and 6mM MgCl were added 2 And a final concentration of 50mM of citric acid-sodium citrate buffer at pH 5.5, at 26℃for 96 hours, catalyzing the formation of Reb M; analysis of Reb M formation using HPLC is shown in table 2.
BaCl is added to 2 Or CaCl 2 Instead of MgCl of this embodiment 2 Otherwise, the rate of Reb M generation is similar to that of the present embodiment.
Example 16 enzymatic solution of recombinant glycosyltransferase catalyzing production of Reb M by Reb a (3E 2 obtained in example 4) a method of catalyzing production of Reb M by Reb a comprising the steps of:
to 5 g/L3E 2, 10g/L Reb A was added at a final concentration of 1mM UDPG and 0.1mM (CH) 3 COO) 2 Pb and final concentration of 50mM glycine-sodium hydroxide buffer solution with pH of 10.5, at 42 ℃ for 12 hours, catalyzing to generate Reb M; analysis of Reb M formation using HPLC is shown in table 2.
TABLE 2 Activity results of enzyme solutions catalyzing recombinant glycosyltransferases for Reb A production of Reb M
Reaction numbering Enzyme solution Reaction system Reb M generation rate Reaction numbering Enzyme solution Reaction system Reb M generation rate
2-1 E1 Example 14 62％ 2-8 E8 Example 14 68％
2-2 E2 Example 14 73％ 2-9 E9 Example 14 42％
2-3 E3 Example 14 66％ 2-10 E10 Example 14 51％
2-4 E4 Example 14 74％ 2-11 E11 Example 14 49％
2-5 E5 Example 14 70％ 2-12 E12 Example 14 44％
2-6 E6 Example 14 63％ 2-13 2E2 Example 15 61％
2-7 E7 Example 14 65％ 2-14 3E2 Example 16 63％
Sequence listing
<110> health industry development Co., ltd
Tianjin University
<120> recombinant glycosyltransferase catalyzing production of Reb M by Reb a
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2814
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggagaaca agaccgaaac caccgtgcgt cgtcgtcgtc gtatcattct gtttccggtt 60
ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta cagcaaaggt 120
tttagcatca ccatttttca caccaacttc aacaagccga aaaccagcaa ctatccgcac 180
ttcacctttc gtttcatcct ggacaacgat ccgcaggacg agcgtattag caacctgccg 240
acccacggcc cgctggcggg tatgcgtatc ccgatcatta acgagcacgg cgcggatgaa 300
ctgcgtcgtg agctggaact gctgatgctg gcgagcgagg aagacgagga agttagctgc 360
ctgattaccg atgcgctgtg gtacttcgcg caaagcgtgg cggacagcct gaacctgcgt 420
cgtctggttc tgatgaccag cagcctgttt aacttccacg cgcacgtgag cctgccgcag 480
tttgacgagc tgggctacct ggacccggac gataagaccc gtctggagga acaagcgagc 540
ggtttcccga tgctgaaggt taaagatatc aaaagcgcgt atagcaactg gcagatcctg 600
aaggaaattc tgggcaagat gatcaaacaa accaaggcga gcagcggtgt gatttggaac 660
agctttaagg agctggagga aagcgagctg gaaaccgtta tccgtgaaat tccggcgccg 720
agcttcctga tcccgctgcc gaaacacctg accgcgagca gcagcagcct gctggaccac 780
gatcgtaccg tgttccagtg gctggaccag caaccgccga gcagcgtgct gtacgttagc 840
tttggcagca ccagcgaggt ggacgaaaaa gatttcctgg agattgcgcg tggtctggtt 900
gacagcaagc agagcttcct gtgggtggtt cgtccgggct tcgtgaaagg tagcacctgg 960
gttgagccgc tgccggatgg ttttctgggc gaacgtggtc gtatcgtgaa atgggttccg 1020
caacaagaag tgctggcgca cggcgcgatt ggtgcgttct ggacccacag cggttggaac 1080
agcaccctgg agagcgtgtg cgaaggcgtt ccgatgatct ttagcgactt cggtctggat 1140
cagccgctga acgcgcgtta catgagcgat gttctgaaag tgggcgttta tctggagaac 1200
ggctgggagc gtggtgaaat cgcgaacgcg attcgtcgtg tgatggttga cgaggaaggt 1260
gagtacatcc gtcagaacgc gcgtgtgctg aagcaaaaag cggatgttag cctgatgaaa 1320
ggtggcagca gctacgagag cctggaaagc ctggttagct atattagcag cctgggtggc 1380
ggtggctcgg gcggtggtgg gtcgatggac agcggttaca gcagcagcta cgcggcggcg 1440
gcgggtatgc acgtggttat ctgcccgtgg ctggcgtttg gtcacctgct gccgtgcctg 1500
gatctggcgc agcgtctggc gagccgtggc caccgtgtta gcttcgtgag caccccgcgt 1560
aacattagcc gtctgccgcc ggttcgtccg gcgctggcgc cgctggttgc gttcgtggcg 1620
ctgccgctgc cgcgtgtgga gggtctgccg gatggtgcgg aaagcaccaa cgatgttccg 1680
cacgaccgtc cggatatggt ggagctgcat cgtcgtgcgt ttgatggtct ggcggcgccg 1740
ttcagcgaat ttctgggtac cgcgtgcgcg gactgggtga tcgttgatgt gtttcatcac 1800
tgggctgcgg cggcggcgct ggagcacaag gttccgtgcg cgatgatgct gctgggtagc 1860
gcgcacatga tcgcgagcat tgcggatcgt cgtctggaac gtgcggaaac cgagagcccg 1920
gcggcggcgg gtcaaggtcg tccggctgcg gcgccgacct ttgaggtggc gcgtatgaag 1980
ctgatccgta ccaaaggtag cagcggcatg agcctggcgg aacgtttcag cctgaccctg 2040
agccgtagca gcctggtggt tggtcgtagc tgcgttgaat ttgagccgga aaccgtgccg 2100
ctgctgagca ccctgcgtgg caagccgatt accttcctgg gtctgatgcc gccgctgcat 2160
gagggtcgtc gtgaggacgg cgaagatgcg accgttcgtt ggctggatgc gcagccggcg 2220
aagagcgtgg tttatgttgc gctgggtagc gaggtgccgc tgggcgttga gaaagtgcac 2280
gaactggcgc tgggtctgga actggcgggt acccgttttc tgtgggcgct gcgtaaaccg 2340
accggtgtga gcgatgcgga tctgctgccg gcgggtttcg aggaacgtac ccgtggtcgt 2400
ggcgtggttg cgacccgttg ggttccgcaa atgagcattc tggcgcatgc ggcggtgggt 2460
gcgtttctga cccactgcgg ctggaacagc accattgaag gtctgatgtt cggccacccg 2520
ctgatcatgc tgccgatttt tggtgaccag ggcccgaacg cgcgtctgat tgaggcgaag 2580
aacgcgggtc tgcaagttgc gcgtaacgac ggtgatggca gctttgatcg tgaaggcgtg 2640
gctgcggcga tccgtgcggt tgcggtggag gaagagagca gcaaggtttt ccaggcgaaa 2700
gcgaagaaac tgcaagagat tgtggcggac atggcgtgcc acgaacgtta catcgatggt 2760
ttcattcagc aactgcgtag ctataaagat ctcgagcacc accaccacca ccac 2814
<210> 2
<211> 2859
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggagaaca agaccgaaac caccgtgcgt cgtcgtcgtc gtatcattct gtttccggtt 60
ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta cagcaaaggt 120
tttagcatca ccatttttca caccaacttc aacaagccga aaaccagcaa ctatccgcac 180
ttcacctttc gtttcatcct ggacaacgat ccgcaggacg agcgtattag caacctgccg 240
acccacggcc cgctggcggg tatgcgtatc ccgatcatta acgagcacgg cgcggatgaa 300
ctgcgtcgtg agctggaact gctgatgctg gcgagcgagg aagacgagga agttagctgc 360
ctgattaccg atgcgctgtg gtacttcgcg caaagcgtgg cggacagcct gaacctgcgt 420
cgtctggttc tgatgaccag cagcctgttt aacttccacg cgcacgtgag cctgccgcag 480
tttgacgagc tgggctacct ggacccggac gataagaccc gtctggagga acaagcgagc 540
ggtttcccga tgctgaaggt taaagatatc aaaagcgcgt atagcaactg gcagatcctg 600
aaggaaattc tgggcaagat gatcaaacaa accaaggcga gcagcggtgt gatttggaac 660
agctttaagg agctggagga aagcgagctg gaaaccgtta tccgtgaaat tccggcgccg 720
agcttcctga tcccgctgcc gaaacacctg accgcgagca gcagcagcct gctggaccac 780
gatcgtaccg tgttccagtg gctggaccag caaccgccga gcagcgtgct gtacgttagc 840
tttggcagca ccagcgaggt ggacgaaaaa gatttcctgg agattgcgcg tggtctggtt 900
gacagcaagc agagcttcct gtgggtggtt cgtccgggct tcgtgaaagg tagcacctgg 960
gttgagccgc tgccggatgg ttttctgggc gaacgtggtc gtatcgtgaa atgggttccg 1020
caacaagaag tgctggcgca cggcgcgatt ggtgcgttct ggacccacag cggttggaac 1080
agcaccctgg agagcgtgtg cgaaggcgtt ccgatgatct ttagcgactt cggtctggat 1140
cagccgctga acgcgcgtta catgagcgat gttctgaaag tgggcgttta tctggagaac 1200
ggctgggagc gtggtgaaat cgcgaacgcg attcgtcgtg tgatggttga cgaggaaggt 1260
gagtacatcc gtcagaacgc gcgtgtgctg aagcaaaaag cggatgttag cctgatgaaa 1320
ggtggcagca gctacgagag cctggaaagc ctggttagct atattagcag cctgggtggc 1380
ggtggctcgg gtggcggtgg ctcgggtggc ggtggatccg aagccgcggc gaaagaagcc 1440
gcggcgaaaa tggacagcgg ttacagcagc agctacgcgg cggcggcggg tatgcacgtg 1500
gttatctgcc cgtggctggc gtttggtcac ctgctgccgt gcctggatct ggcgcagcgt 1560
ctggcgagcc gtggccaccg tgttagcttc gtgagcaccc cgcgtaacat tagccgtctg 1620
ccgccggttc gtccggcgct ggcgccgctg gttgcgttcg tggcgctgcc gctgccgcgt 1680
gtggagggtc tgccggatgg tgcggaaagc accaacgatg ttccgcacga ccgtccggat 1740
atggtggagc tgcatcgtcg tgcgtttgat ggtctggcgg cgccgttcag cgaatttctg 1800
ggtaccgcgt gcgcggactg ggtgatcgtt gatgtgtttc atcactgggc tgcggcggcg 1860
gcgctggagc acaaggttcc gtgcgcgatg atgctgctgg gtagcgcgca catgatcgcg 1920
agcattgcgg atcgtcgtct ggaacgtgcg gaaaccgaga gcccggcggc ggcgggtcaa 1980
ggtcgtccgg ctgcggcgcc gacctttgag gtggcgcgta tgaagctgat ccgtaccaaa 2040
ggtagcagcg gcatgagcct ggcggaacgt ttcagcctga ccctgagccg tagcagcctg 2100
gtggttggtc gtagctgcgt tgaatttgag ccggaaaccg tgccgctgct gagcaccctg 2160
cgtggcaagc cgattacctt cctgggtctg atgccgccgc tgcatgaggg tcgtcgtgag 2220
gacggcgaag atgcgaccgt tcgttggctg gatgcgcagc cggcgaagag cgtggtttat 2280
gttgcgctgg gtagcgaggt gccgctgggc gttgagaaag tgcacgaact ggcgctgggt 2340
ctggaactgg cgggtacccg ttttctgtgg gcgctgcgta aaccgaccgg tgtgagcgat 2400
gcggatctgc tgccggcggg tttcgaggaa cgtacccgtg gtcgtggcgt ggttgcgacc 2460
cgttgggttc cgcaaatgag cattctggcg catgcggcgg tgggtgcgtt tctgacccac 2520
tgcggctgga acagcaccat tgaaggtctg atgttcggcc acccgctgat catgctgccg 2580
atttttggtg accagggccc gaacgcgcgt ctgattgagg cgaagaacgc gggtctgcaa 2640
gttgcgcgta acgacggtga tggcagcttt gatcgtgaag gcgtggctgc ggcgatccgt 2700
gcggttgcgg tggaggaaga gagcagcaag gttttccagg cgaaagcgaa gaaactgcaa 2760
gagattgtgg cggacatggc gtgccacgaa cgttacatcg atggtttcat tcagcaactg 2820
cgtagctata aagatctcga gcaccaccac caccaccac 2859
<210> 3
<211> 2859
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atggagaaca agaccgaaac caccgtgcgt cgtcgtcgtc gtatcattct gtttccggtt 60
ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta cagcaaaggt 120
tttagcatca ccatttttca caccaacttc aacaagccga aaaccagcaa ctatccgcac 180
ttcacctttc gtttcatcct ggacaacgat ccgcaggacg agcgtattag caacctgccg 240
acccacggcc cgctggcggg tatgcgtatc ccgatcatta acgagcacgg cgcggatgaa 300
ctgcgtcgtg agctggaact gctgatgctg gcgagcgagg aagacgagga agttagctgc 360
ctgattaccg atgcgctgtg gtacttcgcg caaagcgtgg cggacagcct gaacctgcgt 420
cgtctggttc tgatgaccag cagcctgttt aacttccacg cgcacgtgag cctgccgcag 480
tttgacgagc tgggctacct ggacccggac gataagaccc gtctggagga acaagcgagc 540
ggtttcccga tgctgaaggt taaagatatc aaaagcgcgt atagcaactg gcagatcctg 600
aaggaaattc tgggcaagat gatcaaacaa accaaggcga gcagcggtgt gatttggaac 660
agctttaagg agctggagga aagcgagctg gaaaccgtta tccgtgaaat tccggcgccg 720
agcttcctga tcccgctgcc gaaacacctg accgcgagca gcagcagcct gctggaccac 780
gatcgtaccg tgttccagtg gctggaccag caaccgccga gcagcgtgct gtacgttagc 840
tttggcagca ccagcgaggt ggacgaaaaa gatttcctgg agattgcgcg tggtctggtt 900
gacagcaagc agagcttcct gtgggtggtt cgtccgggct tcgtgaaagg tagcacctgg 960
gttgagccgc tgccggatgg ttttctgggc gaacgtggtc gtatcgtgaa atgggttccg 1020
caacaagaag tgctggcgca cggcgcgatt ggtgcgttct ggacccacag cggttggaac 1080
agcaccctgg agagcgtgtg cgaaggcgtt ccgatgatct ttagcgactt cggtctggat 1140
cagccgctga acgcgcgtta catgagcgat gttctgaaag tgggcgttta tctggagaac 1200
ggctgggagc gtggtgaaat cgcgaacgcg attcgtcgtg tgatggttga cgaggaaggt 1260
gagtacatcc gtcagaacgc gcgtgtgctg aagcaaaaag cggatgttag cctgatgaaa 1320
ggtggcagca gctacgagag cctggaaagc ctggttagct atattagcag cctgggtggc 1380
ggtggctcgg aagccgcggc gaaagaagcg gccgcgaaag aagccgcggc gaaaggtggc 1440
ggtggctcga tggacagcgg ttacagcagc agctacgcgg cggcggcggg tatgcacgtg 1500
gttatctgcc cgtggctggc gtttggtcac ctgctgccgt gcctggatct ggcgcagcgt 1560
ctggcgagcc gtggccaccg tgttagcttc gtgagcaccc cgcgtaacat tagccgtctg 1620
ccgccggttc gtccggcgct ggcgccgctg gttgcgttcg tggcgctgcc gctgccgcgt 1680
gtggagggtc tgccggatgg tgcggaaagc accaacgatg ttccgcacga ccgtccggat 1740
atggtggagc tgcatcgtcg tgcgtttgat ggtctggcgg cgccgttcag cgaatttctg 1800
ggtaccgcgt gcgcggactg ggtgatcgtt gatgtgtttc atcactgggc tgcggcggcg 1860
gcgctggagc acaaggttcc gtgcgcgatg atgctgctgg gtagcgcgca catgatcgcg 1920
agcattgcgg atcgtcgtct ggaacgtgcg gaaaccgaga gcccggcggc ggcgggtcaa 1980
ggtcgtccgg ctgcggcgcc gacctttgag gtggcgcgta tgaagctgat ccgtaccaaa 2040
ggtagcagcg gcatgagcct ggcggaacgt ttcagcctga ccctgagccg tagcagcctg 2100
gtggttggtc gtagctgcgt tgaatttgag ccggaaaccg tgccgctgct gagcaccctg 2160
cgtggcaagc cgattacctt cctgggtctg atgccgccgc tgcatgaggg tcgtcgtgag 2220
gacggcgaag atgcgaccgt tcgttggctg gatgcgcagc cggcgaagag cgtggtttat 2280
gttgcgctgg gtagcgaggt gccgctgggc gttgagaaag tgcacgaact ggcgctgggt 2340
ctggaactgg cgggtacccg ttttctgtgg gcgctgcgta aaccgaccgg tgtgagcgat 2400
gcggatctgc tgccggcggg tttcgaggaa cgtacccgtg gtcgtggcgt ggttgcgacc 2460
cgttgggttc cgcaaatgag cattctggcg catgcggcgg tgggtgcgtt tctgacccac 2520
tgcggctgga acagcaccat tgaaggtctg atgttcggcc acccgctgat catgctgccg 2580
atttttggtg accagggccc gaacgcgcgt ctgattgagg cgaagaacgc gggtctgcaa 2640
gttgcgcgta acgacggtga tggcagcttt gatcgtgaag gcgtggctgc ggcgatccgt 2700
gcggttgcgg tggaggaaga gagcagcaag gttttccagg cgaaagcgaa gaaactgcaa 2760
gagattgtgg cggacatggc gtgccacgaa cgttacatcg atggtttcat tcagcaactg 2820
cgtagctata aagatctcga gcaccaccac caccaccac 2859
<210> 4
<211> 2859
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atggagaaca agaccgaaac caccgtgcgt cgtcgtcgtc gtatcattct gtttccggtt 60
ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta cagcaaaggt 120
tttagcatca ccatttttca caccaacttc aacaagccga aaaccagcaa ctatccgcac 180
ttcacctttc gtttcatcct ggacaacgat ccgcaggacg agcgtattag caacctgccg 240
acccacggcc cgctggcggg tatgcgtatc ccgatcatta acgagcacgg cgcggatgaa 300
ctgcgtcgtg agctggaact gctgatgctg gcgagcgagg aagacgagga agttagctgc 360
ctgattaccg atgcgctgtg gtacttcgcg caaagcgtgg cggacagcct gaacctgcgt 420
cgtctggttc tgatgaccag cagcctgttt aacttccacg cgcacgtgag cctgccgcag 480
tttgacgagc tgggctacct ggacccggac gataagaccc gtctggagga acaagcgagc 540
ggtttcccga tgctgaaggt taaagatatc aaaagcgcgt atagcaactg gcagatcctg 600
aaggaaattc tgggcaagat gatcaaacaa accaaggcga gcagcggtgt gatttggaac 660
agctttaagg agctggagga aagcgagctg gaaaccgtta tccgtgaaat tccggcgccg 720
agcttcctga tcccgctgcc gaaacacctg accgcgagca gcagcagcct gctggaccac 780
gatcgtaccg tgttccagtg gctggaccag caaccgccga gcagcgtgct gtacgttagc 840
tttggcagca ccagcgaggt ggacgaaaaa gatttcctgg agattgcgcg tggtctggtt 900
gacagcaagc agagcttcct gtgggtggtt cgtccgggct tcgtgaaagg tagcacctgg 960
gttgagccgc tgccggatgg ttttctgggc gaacgtggtc gtatcgtgaa atgggttccg 1020
caacaagaag tgctggcgca cggcgcgatt ggtgcgttct ggacccacag cggttggaac 1080
agcaccctgg agagcgtgtg cgaaggcgtt ccgatgatct ttagcgactt cggtctggat 1140
cagccgctga acgcgcgtta catgagcgat gttctgaaag tgggcgttta tctggagaac 1200
ggctgggagc gtggtgaaat cgcgaacgcg attcgtcgtg tgatggttga cgaggaaggt 1260
gagtacatcc gtcagaacgc gcgtgtgctg aagcaaaaag cggatgttag cctgatgaaa 1320
ggtggcagca gctacgagag cctggaaagc ctggttagct atattagcag cctgggtggc 1380
ggtggctcgg aagccgcggc gaaaggtggc ggtggatccg aagccgcggc gaaaggtggc 1440
ggtggctcga tggacagcgg ttacagcagc agctacgcgg cggcggcggg tatgcacgtg 1500
gttatctgcc cgtggctggc gtttggtcac ctgctgccgt gcctggatct ggcgcagcgt 1560
ctggcgagcc gtggccaccg tgttagcttc gtgagcaccc cgcgtaacat tagccgtctg 1620
ccgccggttc gtccggcgct ggcgccgctg gttgcgttcg tggcgctgcc gctgccgcgt 1680
gtggagggtc tgccggatgg tgcggaaagc accaacgatg ttccgcacga ccgtccggat 1740
atggtggagc tgcatcgtcg tgcgtttgat ggtctggcgg cgccgttcag cgaatttctg 1800
ggtaccgcgt gcgcggactg ggtgatcgtt gatgtgtttc atcactgggc tgcggcggcg 1860
gcgctggagc acaaggttcc gtgcgcgatg atgctgctgg gtagcgcgca catgatcgcg 1920
agcattgcgg atcgtcgtct ggaacgtgcg gaaaccgaga gcccggcggc ggcgggtcaa 1980
ggtcgtccgg ctgcggcgcc gacctttgag gtggcgcgta tgaagctgat ccgtaccaaa 2040
ggtagcagcg gcatgagcct ggcggaacgt ttcagcctga ccctgagccg tagcagcctg 2100
gtggttggtc gtagctgcgt tgaatttgag ccggaaaccg tgccgctgct gagcaccctg 2160
cgtggcaagc cgattacctt cctgggtctg atgccgccgc tgcatgaggg tcgtcgtgag 2220
gacggcgaag atgcgaccgt tcgttggctg gatgcgcagc cggcgaagag cgtggtttat 2280
gttgcgctgg gtagcgaggt gccgctgggc gttgagaaag tgcacgaact ggcgctgggt 2340
ctggaactgg cgggtacccg ttttctgtgg gcgctgcgta aaccgaccgg tgtgagcgat 2400
gcggatctgc tgccggcggg tttcgaggaa cgtacccgtg gtcgtggcgt ggttgcgacc 2460
cgttgggttc cgcaaatgag cattctggcg catgcggcgg tgggtgcgtt tctgacccac 2520
tgcggctgga acagcaccat tgaaggtctg atgttcggcc acccgctgat catgctgccg 2580
atttttggtg accagggccc gaacgcgcgt ctgattgagg cgaagaacgc gggtctgcaa 2640
gttgcgcgta acgacggtga tggcagcttt gatcgtgaag gcgtggctgc ggcgatccgt 2700
gcggttgcgg tggaggaaga gagcagcaag gttttccagg cgaaagcgaa gaaactgcaa 2760
gagattgtgg cggacatggc gtgccacgaa cgttacatcg atggtttcat tcagcaactg 2820
cgtagctata aagatctcga gcaccaccac caccaccac 2859
<210> 5
<211> 2859
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atggagaaca agaccgaaac caccgtgcgt cgtcgtcgtc gtatcattct gtttccggtt 60
ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta cagcaaaggt 120
tttagcatca ccatttttca caccaacttc aacaagccga aaaccagcaa ctatccgcac 180
ttcacctttc gtttcatcct ggacaacgat ccgcaggacg agcgtattag caacctgccg 240
acccacggcc cgctggcggg tatgcgtatc ccgatcatta acgagcacgg cgcggatgaa 300
ctgcgtcgtg agctggaact gctgatgctg gcgagcgagg aagacgagga agttagctgc 360
ctgattaccg atgcgctgtg gtacttcgcg caaagcgtgg cggacagcct gaacctgcgt 420
cgtctggttc tgatgaccag cagcctgttt aacttccacg cgcacgtgag cctgccgcag 480
tttgacgagc tgggctacct ggacccggac gataagaccc gtctggagga acaagcgagc 540
ggtttcccga tgctgaaggt taaagatatc aaaagcgcgt atagcaactg gcagatcctg 600
aaggaaattc tgggcaagat gatcaaacaa accaaggcga gcagcggtgt gatttggaac 660
agctttaagg agctggagga aagcgagctg gaaaccgtta tccgtgaaat tccggcgccg 720
agcttcctga tcccgctgcc gaaacacctg accgcgagca gcagcagcct gctggaccac 780
gatcgtaccg tgttccagtg gctggaccag caaccgccga gcagcgtgct gtacgttagc 840
tttggcagca ccagcgaggt ggacgaaaaa gatttcctgg agattgcgcg tggtctggtt 900
gacagcaagc agagcttcct gtgggtggtt cgtccgggct tcgtgaaagg tagcacctgg 960
gttgagccgc tgccggatgg ttttctgggc gaacgtggtc gtatcgtgaa atgggttccg 1020
caacaagaag tgctggcgca cggcgcgatt ggtgcgttct ggacccacag cggttggaac 1080
agcaccctgg agagcgtgtg cgaaggcgtt ccgatgatct ttagcgactt cggtctggat 1140
cagccgctga acgcgcgtta catgagcgat gttctgaaag tgggcgttta tctggagaac 1200
ggctgggagc gtggtgaaat cgcgaacgcg attcgtcgtg tgatggttga cgaggaaggt 1260
gagtacatcc gtcagaacgc gcgtgtgctg aagcaaaaag cggatgttag cctgatgaaa 1320
ggtggcagca gctacgagag cctggaaagc ctggttagct atattagcag cctgggtggc 1380
ggtggctcgg gtggcggtgg ctcgggtggc ggtggatccg gtggcggtgg ctcgggtggc 1440
ggtggctcga tggacagcgg ttacagcagc agctacgcgg cggcggcggg tatgcacgtg 1500
gttatctgcc cgtggctggc gtttggtcac ctgctgccgt gcctggatct ggcgcagcgt 1560
ctggcgagcc gtggccaccg tgttagcttc gtgagcaccc cgcgtaacat tagccgtctg 1620
ccgccggttc gtccggcgct ggcgccgctg gttgcgttcg tggcgctgcc gctgccgcgt 1680
gtggagggtc tgccggatgg tgcggaaagc accaacgatg ttccgcacga ccgtccggat 1740
atggtggagc tgcatcgtcg tgcgtttgat ggtctggcgg cgccgttcag cgaatttctg 1800
ggtaccgcgt gcgcggactg ggtgatcgtt gatgtgtttc atcactgggc tgcggcggcg 1860
gcgctggagc acaaggttcc gtgcgcgatg atgctgctgg gtagcgcgca catgatcgcg 1920
agcattgcgg atcgtcgtct ggaacgtgcg gaaaccgaga gcccggcggc ggcgggtcaa 1980
ggtcgtccgg ctgcggcgcc gacctttgag gtggcgcgta tgaagctgat ccgtaccaaa 2040
ggtagcagcg gcatgagcct ggcggaacgt ttcagcctga ccctgagccg tagcagcctg 2100
gtggttggtc gtagctgcgt tgaatttgag ccggaaaccg tgccgctgct gagcaccctg 2160
cgtggcaagc cgattacctt cctgggtctg atgccgccgc tgcatgaggg tcgtcgtgag 2220
gacggcgaag atgcgaccgt tcgttggctg gatgcgcagc cggcgaagag cgtggtttat 2280
gttgcgctgg gtagcgaggt gccgctgggc gttgagaaag tgcacgaact ggcgctgggt 2340
ctggaactgg cgggtacccg ttttctgtgg gcgctgcgta aaccgaccgg tgtgagcgat 2400
gcggatctgc tgccggcggg tttcgaggaa cgtacccgtg gtcgtggcgt ggttgcgacc 2460
cgttgggttc cgcaaatgag cattctggcg catgcggcgg tgggtgcgtt tctgacccac 2520
tgcggctgga acagcaccat tgaaggtctg atgttcggcc acccgctgat catgctgccg 2580
atttttggtg accagggccc gaacgcgcgt ctgattgagg cgaagaacgc gggtctgcaa 2640
gttgcgcgta acgacggtga tggcagcttt gatcgtgaag gcgtggctgc ggcgatccgt 2700
gcggttgcgg tggaggaaga gagcagcaag gttttccagg cgaaagcgaa gaaactgcaa 2760
gagattgtgg cggacatggc gtgccacgaa cgttacatcg atggtttcat tcagcaactg 2820
cgtagctata aagatctcga gcaccaccac caccaccac 2859
<210> 6
<211> 2859
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atggagaaca agaccgaaac caccgtgcgt cgtcgtcgtc gtatcattct gtttccggtt 60
ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta cagcaaaggt 120
tttagcatca ccatttttca caccaacttc aacaagccga aaaccagcaa ctatccgcac 180
ttcacctttc gtttcatcct ggacaacgat ccgcaggacg agcgtattag caacctgccg 240
acccacggcc cgctggcggg tatgcgtatc ccgatcatta acgagcacgg cgcggatgaa 300
ctgcgtcgtg agctggaact gctgatgctg gcgagcgagg aagacgagga agttagctgc 360
ctgattaccg atgcgctgtg gtacttcgcg caaagcgtgg cggacagcct gaacctgcgt 420
cgtctggttc tgatgaccag cagcctgttt aacttccacg cgcacgtgag cctgccgcag 480
tttgacgagc tgggctacct ggacccggac gataagaccc gtctggagga acaagcgagc 540
ggtttcccga tgctgaaggt taaagatatc aaaagcgcgt atagcaactg gcagatcctg 600
aaggaaattc tgggcaagat gatcaaacaa accaaggcga gcagcggtgt gatttggaac 660
agctttaagg agctggagga aagcgagctg gaaaccgtta tccgtgaaat tccggcgccg 720
agcttcctga tcccgctgcc gaaacacctg accgcgagca gcagcagcct gctggaccac 780
gatcgtaccg tgttccagtg gctggaccag caaccgccga gcagcgtgct gtacgttagc 840
tttggcagca ccagcgaggt ggacgaaaaa gatttcctgg agattgcgcg tggtctggtt 900
gacagcaagc agagcttcct gtgggtggtt cgtccgggct tcgtgaaagg tagcacctgg 960
gttgagccgc tgccggatgg ttttctgggc gaacgtggtc gtatcgtgaa atgggttccg 1020
caacaagaag tgctggcgca cggcgcgatt ggtgcgttct ggacccacag cggttggaac 1080
agcaccctgg agagcgtgtg cgaaggcgtt ccgatgatct ttagcgactt cggtctggat 1140
cagccgctga acgcgcgtta catgagcgat gttctgaaag tgggcgttta tctggagaac 1200
ggctgggagc gtggtgaaat cgcgaacgcg attcgtcgtg tgatggttga cgaggaaggt 1260
gagtacatcc gtcagaacgc gcgtgtgctg aagcaaaaag cggatgttag cctgatgaaa 1320
ggtggcagca gctacgagag cctggaaagc ctggttagct atattagcag cctggaagcc 1380
gcggcgaaag aagccgcggc gaaagaagcg gccgcgaaag aagccgcggc gaaagaagcc 1440
gcggcgaaaa tggacagcgg ttacagcagc agctacgcgg cggcggcggg tatgcacgtg 1500
gttatctgcc cgtggctggc gtttggtcac ctgctgccgt gcctggatct ggcgcagcgt 1560
ctggcgagcc gtggccaccg tgttagcttc gtgagcaccc cgcgtaacat tagccgtctg 1620
ccgccggttc gtccggcgct ggcgccgctg gttgcgttcg tggcgctgcc gctgccgcgt 1680
gtggagggtc tgccggatgg tgcggaaagc accaacgatg ttccgcacga ccgtccggat 1740
atggtggagc tgcatcgtcg tgcgtttgat ggtctggcgg cgccgttcag cgaatttctg 1800
ggtaccgcgt gcgcggactg ggtgatcgtt gatgtgtttc atcactgggc tgcggcggcg 1860
gcgctggagc acaaggttcc gtgcgcgatg atgctgctgg gtagcgcgca catgatcgcg 1920
agcattgcgg atcgtcgtct ggaacgtgcg gaaaccgaga gcccggcggc ggcgggtcaa 1980
ggtcgtccgg ctgcggcgcc gacctttgag gtggcgcgta tgaagctgat ccgtaccaaa 2040
ggtagcagcg gcatgagcct ggcggaacgt ttcagcctga ccctgagccg tagcagcctg 2100
gtggttggtc gtagctgcgt tgaatttgag ccggaaaccg tgccgctgct gagcaccctg 2160
cgtggcaagc cgattacctt cctgggtctg atgccgccgc tgcatgaggg tcgtcgtgag 2220
gacggcgaag atgcgaccgt tcgttggctg gatgcgcagc cggcgaagag cgtggtttat 2280
gttgcgctgg gtagcgaggt gccgctgggc gttgagaaag tgcacgaact ggcgctgggt 2340
ctggaactgg cgggtacccg ttttctgtgg gcgctgcgta aaccgaccgg tgtgagcgat 2400
gcggatctgc tgccggcggg tttcgaggaa cgtacccgtg gtcgtggcgt ggttgcgacc 2460
cgttgggttc cgcaaatgag cattctggcg catgcggcgg tgggtgcgtt tctgacccac 2520
tgcggctgga acagcaccat tgaaggtctg atgttcggcc acccgctgat catgctgccg 2580
atttttggtg accagggccc gaacgcgcgt ctgattgagg cgaagaacgc gggtctgcaa 2640
gttgcgcgta acgacggtga tggcagcttt gatcgtgaag gcgtggctgc ggcgatccgt 2700
gcggttgcgg tggaggaaga gagcagcaag gttttccagg cgaaagcgaa gaaactgcaa 2760
gagattgtgg cggacatggc gtgccacgaa cgttacatcg atggtttcat tcagcaactg 2820
cgtagctata aagatctcga gcaccaccac caccaccac 2859
<210> 7
<211> 2961
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atggcggagg cggaagcgaa agcgaaagcg gaagcggagg cgaaagcgaa accgaccccg 60
ccgaccaccc cgaccccgcc gactaccccg accccgaccc cgatggagaa caagaccgaa 120
accaccgtgc gtcgtcgtcg tcgtatcatt ctgtttccgg ttccgttcca gggccacatc 180
aacccgattc tgcaactggc gaacgtgctg tacagcaaag gttttagcat caccattttt 240
cacaccaact tcaacaagcc gaaaaccagc aactatccgc acttcacctt tcgtttcatc 300
ctggacaacg atccgcagga cgagcgtatt agcaacctgc cgacccacgg cccgctggcg 360
ggtatgcgta tcccgatcat taacgagcac ggcgcggatg aactgcgtcg tgagctggaa 420
ctgctgatgc tggcgagcga ggaagacgag gaagttagct gcctgattac cgatgcgctg 480
tggtacttcg cgcaaagcgt ggcggacagc ctgaacctgc gtcgtctggt tctgatgacc 540
agcagcctgt ttaacttcca cgcgcacgtg agcctgccgc agtttgacga gctgggctac 600
ctggacccgg acgataagac ccgtctggag gaacaagcga gcggtttccc gatgctgaag 660
gttaaagata tcaaaagcgc gtatagcaac tggcagatcc tgaaggaaat tctgggcaag 720
atgatcaaac aaaccaaggc gagcagcggt gtgatttgga acagctttaa ggagctggag 780
gaaagcgagc tggaaaccgt tatccgtgaa attccggcgc cgagcttcct gatcccgctg 840
ccgaaacacc tgaccgcgag cagcagcagc ctgctggacc acgatcgtac cgtgttccag 900
tggctggacc agcaaccgcc gagcagcgtg ctgtacgtta gctttggcag caccagcgag 960
gtggacgaaa aagatttcct ggagattgcg cgtggtctgg ttgacagcaa gcagagcttc 1020
ctgtgggtgg ttcgtccggg cttcgtgaaa ggtagcacct gggttgagcc gctgccggat 1080
ggttttctgg gcgaacgtgg tcgtatcgtg aaatgggttc cgcaacaaga agtgctggcg 1140
cacggcgcga ttggtgcgtt ctggacccac agcggttgga acagcaccct ggagagcgtg 1200
tgcgaaggcg ttccgatgat ctttagcgac ttcggtctgg atcagccgct gaacgcgcgt 1260
tacatgagcg atgttctgaa agtgggcgtt tatctggaga acggctggga gcgtggtgaa 1320
atcgcgaacg cgattcgtcg tgtgatggtt gacgaggaag gtgagtacat ccgtcagaac 1380
gcgcgtgtgc tgaagcaaaa agcggatgtt agcctgatga aaggtggcag cagctacgag 1440
agcctggaaa gcctggttag ctatattagc agcctgggtg gcggtggctc gggtggcggt 1500
ggctcgggtg gcggtggatc cgaagccgcg gcgaaagaag ccgcggcgaa aatggacagc 1560
ggttacagca gcagctacgc ggcggcggcg ggtatgcacg tggttatctg cccgtggctg 1620
gcgtttggtc acctgctgcc gtgcctggat ctggcgcagc gtctggcgag ccgtggccac 1680
cgtgttagct tcgtgagcac cccgcgtaac attagccgtc tgccgccggt tcgtccggcg 1740
ctggcgccgc tggttgcgtt cgtggcgctg ccgctgccgc gtgtggaggg tctgccggat 1800
ggtgcggaaa gcaccaacga tgttccgcac gaccgtccgg atatggtgga gctgcatcgt 1860
cgtgcgtttg atggtctggc ggcgccgttc agcgaatttc tgggtaccgc gtgcgcggac 1920
tgggtgatcg ttgatgtgtt tcatcactgg gctgcggcgg cggcgctgga gcacaaggtt 1980
ccgtgcgcga tgatgctgct gggtagcgcg cacatgatcg cgagcattgc ggatcgtcgt 2040
ctggaacgtg cggaaaccga gagcccggcg gcggcgggtc aaggtcgtcc ggctgcggcg 2100
ccgacctttg aggtggcgcg tatgaagctg atccgtacca aaggtagcag cggcatgagc 2160
ctggcggaac gtttcagcct gaccctgagc cgtagcagcc tggtggttgg tcgtagctgc 2220
gttgaatttg agccggaaac cgtgccgctg ctgagcaccc tgcgtggcaa gccgattacc 2280
ttcctgggtc tgatgccgcc gctgcatgag ggtcgtcgtg aggacggcga agatgcgacc 2340
gttcgttggc tggatgcgca gccggcgaag agcgtggttt atgttgcgct gggtagcgag 2400
gtgccgctgg gcgttgagaa agtgcacgaa ctggcgctgg gtctggaact ggcgggtacc 2460
cgttttctgt gggcgctgcg taaaccgacc ggtgtgagcg atgcggatct gctgccggcg 2520
ggtttcgagg aacgtacccg tggtcgtggc gtggttgcga cccgttgggt tccgcaaatg 2580
agcattctgg cgcatgcggc ggtgggtgcg tttctgaccc actgcggctg gaacagcacc 2640
attgaaggtc tgatgttcgg ccacccgctg atcatgctgc cgatttttgg tgaccagggc 2700
ccgaacgcgc gtctgattga ggcgaagaac gcgggtctgc aagttgcgcg taacgacggt 2760
gatggcagct ttgatcgtga aggcgtggct gcggcgatcc gtgcggttgc ggtggaggaa 2820
gagagcagca aggttttcca ggcgaaagcg aagaaactgc aagagattgt ggcggacatg 2880
gcgtgccacg aacgttacat cgatggtttc attcagcaac tgcgtagcta taaagatctc 2940
gagcaccacc accaccacca c 2961
<210> 8
<211> 2862
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atggacagcg gttacagcag cagctacgcg gcggcggcgg gtatgcacgt ggttatctgc 60
ccgtggctgg cgtttggtca cctgctgccg tgcctggatc tggcgcagcg tctggcgagc 120
cgtggccacc gtgttagctt cgtgagcacc ccgcgtaaca ttagccgtct gccgccggtt 180
cgtccggcgc tggcgccgct ggttgcgttc gtggcgctgc cgctgccgcg tgtggagggt 240
ctgccggatg gtgcggaaag caccaacgat gttccgcacg accgtccgga tatggtggag 300
ctgcatcgtc gtgcgtttga tggtctggcg gcgccgttca gcgaatttct gggtaccgcg 360
tgcgcggact gggtgatcgt tgatgtgttt catcactggg ctgcggcggc ggcgctggag 420
cacaaggttc cgtgcgcgat gatgctgctg ggtagcgcgc acatgatcgc gagcattgcg 480
gatcgtcgtc tggaacgtgc ggaaaccgag agcccggcgg cggcgggtca aggtcgtccg 540
gctgcggcgc cgacctttga ggtggcgcgt atgaagctga tccgtaccaa aggtagcagc 600
ggcatgagcc tggcggaacg tttcagcctg accctgagcc gtagcagcct ggtggttggt 660
cgtagctgcg ttgaatttga gccggaaacc gtgccgctgc tgagcaccct gcgtggcaag 720
ccgattacct tcctgggtct gatgccgccg ctgcatgagg gtcgtcgtga ggacggcgaa 780
gatgcgaccg ttcgttggct ggatgcgcag ccggcgaaga gcgtggttta tgttgcgctg 840
ggtagcgagg tgccgctggg cgttgagaaa gtgcacgaac tggcgctggg tctggaactg 900
gcgggtaccc gttttctgtg ggcgctgcgt aaaccgaccg gtgtgagcga tgcggatctg 960
ctgccggcgg gtttcgagga acgtacccgt ggtcgtggcg tggttgcgac ccgttgggtt 1020
ccgcaaatga gcattctggc gcatgcggcg gtgggtgcgt ttctgaccca ctgcggctgg 1080
aacagcacca ttgaaggtct gatgttcggc cacccgctga tcatgctgcc gatttttggt 1140
gaccagggcc cgaacgcgcg tctgattgag gcgaagaacg cgggtctgca agttgcgcgt 1200
aacgacggtg atggcagctt tgatcgtgaa ggcgtggctg cggcgatccg tgcggttgcg 1260
gtggaggaag agagcagcaa ggttttccag gcgaaagcga agaaactgca agagattgtg 1320
gcggacatgg cgtgccacga acgttacatc gatggtttca ttcagcaact gcgtagctat 1380
aaagatggtg gcggtggctc gggtggcggt ggctcgggtg gcggtggatc cggtggcggt 1440
ggctcgggtg gcggtggctc gatggagaac aagaccgaaa ccaccgtgcg tcgtcgtcgt 1500
cgtatcattc tgtttccggt tccgttccag ggccacatca acccgattct gcaactggcg 1560
aacgtgctgt acagcaaagg ttttagcatc accatttttc acaccaactt caacaagccg 1620
aaaaccagca actatccgca cttcaccttt cgtttcatcc tggacaacga tccgcaggac 1680
gagcgtatta gcaacctgcc gacccacggc ccgctggcgg gtatgcgtat cccgatcatt 1740
aacgagcacg gcgcggatga actgcgtcgt gagctggaac tgctgatgct ggcgagcgag 1800
gaagacgagg aagttagctg cctgattacc gatgcgctgt ggtacttcgc gcaaagcgtg 1860
gcggacagcc tgaacctgcg tcgtctggtt ctgatgacca gcagcctgtt taacttccac 1920
gcgcacgtga gcctgccgca gtttgacgag ctgggctacc tggacccgga cgataagacc 1980
cgtctggagg aacaagcgag cggtttcccg atgctgaagg ttaaagatat caaaagcgcg 2040
tatagcaact ggcagatcct gaaggaaatt ctgggcaaga tgatcaaaca aaccaaggcg 2100
agcagcggtg tgatttggaa cagctttaag gagctggagg aaagcgagct ggaaaccgtt 2160
atccgtgaaa ttccggcgcc gagcttcctg atcccgctgc cgaaacacct gaccgcgagc 2220
agcagcagcc tgctggacca cgatcgtacc gtgttccagt ggctggacca gcaaccgccg 2280
agcagcgtgc tgtacgttag ctttggcagc accagcgagg tggacgaaaa agatttcctg 2340
gagattgcgc gtggtctggt tgacagcaag cagagcttcc tgtgggtggt tcgtccgggc 2400
ttcgtgaaag gtagcacctg ggttgagccg ctgccggatg gttttctggg cgaacgtggt 2460
cgtatcgtga aatgggttcc gcaacaagaa gtgctggcgc acggcgcgat tggtgcgttc 2520
tggacccaca gcggttggaa cagcaccctg gagagcgtgt gcgaaggcgt tccgatgatc 2580
tttagcgact tcggtctgga tcagccgctg aacgcgcgtt acatgagcga tgttctgaaa 2640
gtgggcgttt atctggagaa cggctgggag cgtggtgaaa tcgcgaacgc gattcgtcgt 2700
gtgatggttg acgaggaagg tgagtacatc cgtcagaacg cgcgtgtgct gaagcaaaaa 2760
gcggatgtta gcctgatgaa aggtggcagc agctacgaga gcctggaaag cctggttagc 2820
tatattagca gcctgctcga gcaccaccac caccaccact ga 2862
<210> 9
<211> 2817
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atggacagcg gttacagcag cagctacgcg gcggcggcgg gtatgcacgt ggttatctgc 60
ccgtggctgg cgtttggtca cctgctgccg tgcctggatc tggcgcagcg tctggcgagc 120
cgtggccacc gtgttagctt cgtgagcacc ccgcgtaaca ttagccgtct gccgccggtt 180
cgtccggcgc tggcgccgct ggttgcgttc gtggcgctgc cgctgccgcg tgtggagggt 240
ctgccggatg gtgcggaaag caccaacgat gttccgcacg accgtccgga tatggtggag 300
ctgcatcgtc gtgcgtttga tggtctggcg gcgccgttca gcgaatttct gggtaccgcg 360
tgcgcggact gggtgatcgt tgatgtgttt catcactggg ctgcggcggc ggcgctggag 420
cacaaggttc cgtgcgcgat gatgctgctg ggtagcgcgc acatgatcgc gagcattgcg 480
gatcgtcgtc tggaacgtgc ggaaaccgag agcccggcgg cggcgggtca aggtcgtccg 540
gctgcggcgc cgacctttga ggtggcgcgt atgaagctga tccgtaccaa aggtagcagc 600
ggcatgagcc tggcggaacg tttcagcctg accctgagcc gtagcagcct ggtggttggt 660
cgtagctgcg ttgaatttga gccggaaacc gtgccgctgc tgagcaccct gcgtggcaag 720
ccgattacct tcctgggtct gatgccgccg ctgcatgagg gtcgtcgtga ggacggcgaa 780
gatgcgaccg ttcgttggct ggatgcgcag ccggcgaaga gcgtggttta tgttgcgctg 840
ggtagcgagg tgccgctggg cgttgagaaa gtgcacgaac tggcgctggg tctggaactg 900
gcgggtaccc gttttctgtg ggcgctgcgt aaaccgaccg gtgtgagcga tgcggatctg 960
ctgccggcgg gtttcgagga acgtacccgt ggtcgtggcg tggttgcgac ccgttgggtt 1020
ccgcaaatga gcattctggc gcatgcggcg gtgggtgcgt ttctgaccca ctgcggctgg 1080
aacagcacca ttgaaggtct gatgttcggc cacccgctga tcatgctgcc gatttttggt 1140
gaccagggcc cgaacgcgcg tctgattgag gcgaagaacg cgggtctgca agttgcgcgt 1200
aacgacggtg atggcagctt tgatcgtgaa ggcgtggctg cggcgatccg tgcggttgcg 1260
gtggaggaag agagcagcaa ggttttccag gcgaaagcga agaaactgca agagattgtg 1320
gcggacatgg cgtgccacga acgttacatc gatggtttca ttcagcaact gcgtagctat 1380
aaagatggtg gcggtggctc gggcggtggt gggtcgatgg agaacaagac cgaaaccacc 1440
gtgcgtcgtc gtcgtcgtat cattctgttt ccggttccgt tccagggcca catcaacccg 1500
attctgcaac tggcgaacgt gctgtacagc aaaggtttta gcatcaccat ttttcacacc 1560
aacttcaaca agccgaaaac cagcaactat ccgcacttca cctttcgttt catcctggac 1620
aacgatccgc aggacgagcg tattagcaac ctgccgaccc acggcccgct ggcgggtatg 1680
cgtatcccga tcattaacga gcacggcgcg gatgaactgc gtcgtgagct ggaactgctg 1740
atgctggcga gcgaggaaga cgaggaagtt agctgcctga ttaccgatgc gctgtggtac 1800
ttcgcgcaaa gcgtggcgga cagcctgaac ctgcgtcgtc tggttctgat gaccagcagc 1860
ctgtttaact tccacgcgca cgtgagcctg ccgcagtttg acgagctggg ctacctggac 1920
ccggacgata agacccgtct ggaggaacaa gcgagcggtt tcccgatgct gaaggttaaa 1980
gatatcaaaa gcgcgtatag caactggcag atcctgaagg aaattctggg caagatgatc 2040
aaacaaacca aggcgagcag cggtgtgatt tggaacagct ttaaggagct ggaggaaagc 2100
gagctggaaa ccgttatccg tgaaattccg gcgccgagct tcctgatccc gctgccgaaa 2160
cacctgaccg cgagcagcag cagcctgctg gaccacgatc gtaccgtgtt ccagtggctg 2220
gaccagcaac cgccgagcag cgtgctgtac gttagctttg gcagcaccag cgaggtggac 2280
gaaaaagatt tcctggagat tgcgcgtggt ctggttgaca gcaagcagag cttcctgtgg 2340
gtggttcgtc cgggcttcgt gaaaggtagc acctgggttg agccgctgcc ggatggtttt 2400
ctgggcgaac gtggtcgtat cgtgaaatgg gttccgcaac aagaagtgct ggcgcacggc 2460
gcgattggtg cgttctggac ccacagcggt tggaacagca ccctggagag cgtgtgcgaa 2520
ggcgttccga tgatctttag cgacttcggt ctggatcagc cgctgaacgc gcgttacatg 2580
agcgatgttc tgaaagtggg cgtttatctg gagaacggct gggagcgtgg tgaaatcgcg 2640
aacgcgattc gtcgtgtgat ggttgacgag gaaggtgagt acatccgtca gaacgcgcgt 2700
gtgctgaagc aaaaagcgga tgttagcctg atgaaaggtg gcagcagcta cgagagcctg 2760
gaaagcctgg ttagctatat tagcagcctg ctcgagcacc accaccacca ccactga 2817
<210> 10
<211> 2862
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atggacagcg gttacagcag cagctacgcg gcggcggcgg gtatgcacgt ggttatctgc 60
ccgtggctgg cgtttggtca cctgctgccg tgcctggatc tggcgcagcg tctggcgagc 120
cgtggccacc gtgttagctt cgtgagcacc ccgcgtaaca ttagccgtct gccgccggtt 180
cgtccggcgc tggcgccgct ggttgcgttc gtggcgctgc cgctgccgcg tgtggagggt 240
ctgccggatg gtgcggaaag caccaacgat gttccgcacg accgtccgga tatggtggag 300
ctgcatcgtc gtgcgtttga tggtctggcg gcgccgttca gcgaatttct gggtaccgcg 360
tgcgcggact gggtgatcgt tgatgtgttt catcactggg ctgcggcggc ggcgctggag 420
cacaaggttc cgtgcgcgat gatgctgctg ggtagcgcgc acatgatcgc gagcattgcg 480
gatcgtcgtc tggaacgtgc ggaaaccgag agcccggcgg cggcgggtca aggtcgtccg 540
gctgcggcgc cgacctttga ggtggcgcgt atgaagctga tccgtaccaa aggtagcagc 600
ggcatgagcc tggcggaacg tttcagcctg accctgagcc gtagcagcct ggtggttggt 660
cgtagctgcg ttgaatttga gccggaaacc gtgccgctgc tgagcaccct gcgtggcaag 720
ccgattacct tcctgggtct gatgccgccg ctgcatgagg gtcgtcgtga ggacggcgaa 780
gatgcgaccg ttcgttggct ggatgcgcag ccggcgaaga gcgtggttta tgttgcgctg 840
ggtagcgagg tgccgctggg cgttgagaaa gtgcacgaac tggcgctggg tctggaactg 900
gcgggtaccc gttttctgtg ggcgctgcgt aaaccgaccg gtgtgagcga tgcggatctg 960
ctgccggcgg gtttcgagga acgtacccgt ggtcgtggcg tggttgcgac ccgttgggtt 1020
ccgcaaatga gcattctggc gcatgcggcg gtgggtgcgt ttctgaccca ctgcggctgg 1080
aacagcacca ttgaaggtct gatgttcggc cacccgctga tcatgctgcc gatttttggt 1140
gaccagggcc cgaacgcgcg tctgattgag gcgaagaacg cgggtctgca agttgcgcgt 1200
aacgacggtg atggcagctt tgatcgtgaa ggcgtggctg cggcgatccg tgcggttgcg 1260
gtggaggaag agagcagcaa ggttttccag gcgaaagcga agaaactgca agagattgtg 1320
gcggacatgg cgtgccacga acgttacatc gatggtttca ttcagcaact gcgtagctat 1380
aaagatggtg gcggtggctc ggaagccgcg gcgaaagaag cggccgcgaa agaagccgcg 1440
gcgaaaggtg gcggtggctc gatggagaac aagaccgaaa ccaccgtgcg tcgtcgtcgt 1500
cgtatcattc tgtttccggt tccgttccag ggccacatca acccgattct gcaactggcg 1560
aacgtgctgt acagcaaagg ttttagcatc accatttttc acaccaactt caacaagccg 1620
aaaaccagca actatccgca cttcaccttt cgtttcatcc tggacaacga tccgcaggac 1680
gagcgtatta gcaacctgcc gacccacggc ccgctggcgg gtatgcgtat cccgatcatt 1740
aacgagcacg gcgcggatga actgcgtcgt gagctggaac tgctgatgct ggcgagcgag 1800
gaagacgagg aagttagctg cctgattacc gatgcgctgt ggtacttcgc gcaaagcgtg 1860
gcggacagcc tgaacctgcg tcgtctggtt ctgatgacca gcagcctgtt taacttccac 1920
gcgcacgtga gcctgccgca gtttgacgag ctgggctacc tggacccgga cgataagacc 1980
cgtctggagg aacaagcgag cggtttcccg atgctgaagg ttaaagatat caaaagcgcg 2040
tatagcaact ggcagatcct gaaggaaatt ctgggcaaga tgatcaaaca aaccaaggcg 2100
agcagcggtg tgatttggaa cagctttaag gagctggagg aaagcgagct ggaaaccgtt 2160
atccgtgaaa ttccggcgcc gagcttcctg atcccgctgc cgaaacacct gaccgcgagc 2220
agcagcagcc tgctggacca cgatcgtacc gtgttccagt ggctggacca gcaaccgccg 2280
agcagcgtgc tgtacgttag ctttggcagc accagcgagg tggacgaaaa agatttcctg 2340
gagattgcgc gtggtctggt tgacagcaag cagagcttcc tgtgggtggt tcgtccgggc 2400
ttcgtgaaag gtagcacctg ggttgagccg ctgccggatg gttttctggg cgaacgtggt 2460
cgtatcgtga aatgggttcc gcaacaagaa gtgctggcgc acggcgcgat tggtgcgttc 2520
tggacccaca gcggttggaa cagcaccctg gagagcgtgt gcgaaggcgt tccgatgatc 2580
tttagcgact tcggtctgga tcagccgctg aacgcgcgtt acatgagcga tgttctgaaa 2640
gtgggcgttt atctggagaa cggctgggag cgtggtgaaa tcgcgaacgc gattcgtcgt 2700
gtgatggttg acgaggaagg tgagtacatc cgtcagaacg cgcgtgtgct gaagcaaaaa 2760
gcggatgtta gcctgatgaa aggtggcagc agctacgaga gcctggaaag cctggttagc 2820
tatattagca gcctgctcga gcaccaccac caccaccact ga 2862
<210> 11
<211> 2862
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atggacagcg gttacagcag cagctacgcg gcggcggcgg gtatgcacgt ggttatctgc 60
ccgtggctgg cgtttggtca cctgctgccg tgcctggatc tggcgcagcg tctggcgagc 120
cgtggccacc gtgttagctt cgtgagcacc ccgcgtaaca ttagccgtct gccgccggtt 180
cgtccggcgc tggcgccgct ggttgcgttc gtggcgctgc cgctgccgcg tgtggagggt 240
ctgccggatg gtgcggaaag caccaacgat gttccgcacg accgtccgga tatggtggag 300
ctgcatcgtc gtgcgtttga tggtctggcg gcgccgttca gcgaatttct gggtaccgcg 360
tgcgcggact gggtgatcgt tgatgtgttt catcactggg ctgcggcggc ggcgctggag 420
cacaaggttc cgtgcgcgat gatgctgctg ggtagcgcgc acatgatcgc gagcattgcg 480
gatcgtcgtc tggaacgtgc ggaaaccgag agcccggcgg cggcgggtca aggtcgtccg 540
gctgcggcgc cgacctttga ggtggcgcgt atgaagctga tccgtaccaa aggtagcagc 600
ggcatgagcc tggcggaacg tttcagcctg accctgagcc gtagcagcct ggtggttggt 660
cgtagctgcg ttgaatttga gccggaaacc gtgccgctgc tgagcaccct gcgtggcaag 720
ccgattacct tcctgggtct gatgccgccg ctgcatgagg gtcgtcgtga ggacggcgaa 780
gatgcgaccg ttcgttggct ggatgcgcag ccggcgaaga gcgtggttta tgttgcgctg 840
ggtagcgagg tgccgctggg cgttgagaaa gtgcacgaac tggcgctggg tctggaactg 900
gcgggtaccc gttttctgtg ggcgctgcgt aaaccgaccg gtgtgagcga tgcggatctg 960
ctgccggcgg gtttcgagga acgtacccgt ggtcgtggcg tggttgcgac ccgttgggtt 1020
ccgcaaatga gcattctggc gcatgcggcg gtgggtgcgt ttctgaccca ctgcggctgg 1080
aacagcacca ttgaaggtct gatgttcggc cacccgctga tcatgctgcc gatttttggt 1140
gaccagggcc cgaacgcgcg tctgattgag gcgaagaacg cgggtctgca agttgcgcgt 1200
aacgacggtg atggcagctt tgatcgtgaa ggcgtggctg cggcgatccg tgcggttgcg 1260
gtggaggaag agagcagcaa ggttttccag gcgaaagcga agaaactgca agagattgtg 1320
gcggacatgg cgtgccacga acgttacatc gatggtttca ttcagcaact gcgtagctat 1380
aaagatggtg gcggtggctc ggaagccgcg gcgaaaggtg gcggtggatc cgaagccgcg 1440
gcgaaaggtg gcggtggctc gatggagaac aagaccgaaa ccaccgtgcg tcgtcgtcgt 1500
cgtatcattc tgtttccggt tccgttccag ggccacatca acccgattct gcaactggcg 1560
aacgtgctgt acagcaaagg ttttagcatc accatttttc acaccaactt caacaagccg 1620
aaaaccagca actatccgca cttcaccttt cgtttcatcc tggacaacga tccgcaggac 1680
gagcgtatta gcaacctgcc gacccacggc ccgctggcgg gtatgcgtat cccgatcatt 1740
aacgagcacg gcgcggatga actgcgtcgt gagctggaac tgctgatgct ggcgagcgag 1800
gaagacgagg aagttagctg cctgattacc gatgcgctgt ggtacttcgc gcaaagcgtg 1860
gcggacagcc tgaacctgcg tcgtctggtt ctgatgacca gcagcctgtt taacttccac 1920
gcgcacgtga gcctgccgca gtttgacgag ctgggctacc tggacccgga cgataagacc 1980
cgtctggagg aacaagcgag cggtttcccg atgctgaagg ttaaagatat caaaagcgcg 2040
tatagcaact ggcagatcct gaaggaaatt ctgggcaaga tgatcaaaca aaccaaggcg 2100
agcagcggtg tgatttggaa cagctttaag gagctggagg aaagcgagct ggaaaccgtt 2160
atccgtgaaa ttccggcgcc gagcttcctg atcccgctgc cgaaacacct gaccgcgagc 2220
agcagcagcc tgctggacca cgatcgtacc gtgttccagt ggctggacca gcaaccgccg 2280
agcagcgtgc tgtacgttag ctttggcagc accagcgagg tggacgaaaa agatttcctg 2340
gagattgcgc gtggtctggt tgacagcaag cagagcttcc tgtgggtggt tcgtccgggc 2400
ttcgtgaaag gtagcacctg ggttgagccg ctgccggatg gttttctggg cgaacgtggt 2460
cgtatcgtga aatgggttcc gcaacaagaa gtgctggcgc acggcgcgat tggtgcgttc 2520
tggacccaca gcggttggaa cagcaccctg gagagcgtgt gcgaaggcgt tccgatgatc 2580
tttagcgact tcggtctgga tcagccgctg aacgcgcgtt acatgagcga tgttctgaaa 2640
gtgggcgttt atctggagaa cggctgggag cgtggtgaaa tcgcgaacgc gattcgtcgt 2700
gtgatggttg acgaggaagg tgagtacatc cgtcagaacg cgcgtgtgct gaagcaaaaa 2760
gcggatgtta gcctgatgaa aggtggcagc agctacgaga gcctggaaag cctggttagc 2820
tatattagca gcctgctcga gcaccaccac caccaccact ga 2862
<210> 12
<211> 2862
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
atggacagcg gttacagcag cagctacgcg gcggcggcgg gtatgcacgt ggttatctgc 60
ccgtggctgg cgtttggtca cctgctgccg tgcctggatc tggcgcagcg tctggcgagc 120
cgtggccacc gtgttagctt cgtgagcacc ccgcgtaaca ttagccgtct gccgccggtt 180
cgtccggcgc tggcgccgct ggttgcgttc gtggcgctgc cgctgccgcg tgtggagggt 240
ctgccggatg gtgcggaaag caccaacgat gttccgcacg accgtccgga tatggtggag 300
ctgcatcgtc gtgcgtttga tggtctggcg gcgccgttca gcgaatttct gggtaccgcg 360
tgcgcggact gggtgatcgt tgatgtgttt catcactggg ctgcggcggc ggcgctggag 420
cacaaggttc cgtgcgcgat gatgctgctg ggtagcgcgc acatgatcgc gagcattgcg 480
gatcgtcgtc tggaacgtgc ggaaaccgag agcccggcgg cggcgggtca aggtcgtccg 540
gctgcggcgc cgacctttga ggtggcgcgt atgaagctga tccgtaccaa aggtagcagc 600
ggcatgagcc tggcggaacg tttcagcctg accctgagcc gtagcagcct ggtggttggt 660
cgtagctgcg ttgaatttga gccggaaacc gtgccgctgc tgagcaccct gcgtggcaag 720
ccgattacct tcctgggtct gatgccgccg ctgcatgagg gtcgtcgtga ggacggcgaa 780
gatgcgaccg ttcgttggct ggatgcgcag ccggcgaaga gcgtggttta tgttgcgctg 840
ggtagcgagg tgccgctggg cgttgagaaa gtgcacgaac tggcgctggg tctggaactg 900
gcgggtaccc gttttctgtg ggcgctgcgt aaaccgaccg gtgtgagcga tgcggatctg 960
ctgccggcgg gtttcgagga acgtacccgt ggtcgtggcg tggttgcgac ccgttgggtt 1020
ccgcaaatga gcattctggc gcatgcggcg gtgggtgcgt ttctgaccca ctgcggctgg 1080
aacagcacca ttgaaggtct gatgttcggc cacccgctga tcatgctgcc gatttttggt 1140
gaccagggcc cgaacgcgcg tctgattgag gcgaagaacg cgggtctgca agttgcgcgt 1200
aacgacggtg atggcagctt tgatcgtgaa ggcgtggctg cggcgatccg tgcggttgcg 1260
gtggaggaag agagcagcaa ggttttccag gcgaaagcga agaaactgca agagattgtg 1320
gcggacatgg cgtgccacga acgttacatc gatggtttca ttcagcaact gcgtagctat 1380
aaagatgaag ccgcggcgaa agaagccgcg gcgaaagaag cggccgcgaa agaagccgcg 1440
gcgaaagaag ccgcggcgaa aatggagaac aagaccgaaa ccaccgtgcg tcgtcgtcgt 1500
cgtatcattc tgtttccggt tccgttccag ggccacatca acccgattct gcaactggcg 1560
aacgtgctgt acagcaaagg ttttagcatc accatttttc acaccaactt caacaagccg 1620
aaaaccagca actatccgca cttcaccttt cgtttcatcc tggacaacga tccgcaggac 1680
gagcgtatta gcaacctgcc gacccacggc ccgctggcgg gtatgcgtat cccgatcatt 1740
aacgagcacg gcgcggatga actgcgtcgt gagctggaac tgctgatgct ggcgagcgag 1800
gaagacgagg aagttagctg cctgattacc gatgcgctgt ggtacttcgc gcaaagcgtg 1860
gcggacagcc tgaacctgcg tcgtctggtt ctgatgacca gcagcctgtt taacttccac 1920
gcgcacgtga gcctgccgca gtttgacgag ctgggctacc tggacccgga cgataagacc 1980
cgtctggagg aacaagcgag cggtttcccg atgctgaagg ttaaagatat caaaagcgcg 2040
tatagcaact ggcagatcct gaaggaaatt ctgggcaaga tgatcaaaca aaccaaggcg 2100
agcagcggtg tgatttggaa cagctttaag gagctggagg aaagcgagct ggaaaccgtt 2160
atccgtgaaa ttccggcgcc gagcttcctg atcccgctgc cgaaacacct gaccgcgagc 2220
agcagcagcc tgctggacca cgatcgtacc gtgttccagt ggctggacca gcaaccgccg 2280
agcagcgtgc tgtacgttag ctttggcagc accagcgagg tggacgaaaa agatttcctg 2340
gagattgcgc gtggtctggt tgacagcaag cagagcttcc tgtgggtggt tcgtccgggc 2400
ttcgtgaaag gtagcacctg ggttgagccg ctgccggatg gttttctggg cgaacgtggt 2460
cgtatcgtga aatgggttcc gcaacaagaa gtgctggcgc acggcgcgat tggtgcgttc 2520
tggacccaca gcggttggaa cagcaccctg gagagcgtgt gcgaaggcgt tccgatgatc 2580
tttagcgact tcggtctgga tcagccgctg aacgcgcgtt acatgagcga tgttctgaaa 2640
gtgggcgttt atctggagaa cggctgggag cgtggtgaaa tcgcgaacgc gattcgtcgt 2700
gtgatggttg acgaggaagg tgagtacatc cgtcagaacg cgcgtgtgct gaagcaaaaa 2760
gcggatgtta gcctgatgaa aggtggcagc agctacgaga gcctggaaag cctggttagc 2820
tatattagca gcctgctcga gcaccaccac caccaccact ga 2862

Claims (7)

1. Recombinant glycosyltransferases that catalyze Reb a to produce Reb M; the recombinant glycosyltransferase is characterized in that the nucleotide sequence of a recombinant gene for encoding the recombinant glycosyltransferase is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. A recombinant strain expressing a recombinant glycosyltransferase that catalyzes Reb a production of Reb M, constructed by: constructing the recombinant gene of claim 1 to an expression vector to obtain a recombinant vector; the recombinant vector is transformed into a host strain to obtain a recombinant strain.
3. Recombinant strain according to claim 2, characterized in that the expression vector is pPIC9K, pPIC9, pPink α -HC, pET-28a, pMAL or pBAD30.
4. Recombinant strain according to claim 2, characterized in that the host strain is escherichia coli or pichia pastoris.
5. The method for inducible expression of a recombinant strain according to claim 2, characterized by comprising the steps of: culturing the recombinant strain of claim 2, adding an inducer, and inducing expression for 6-27h at 15-37 ℃ to obtain a somatic cell containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M; and (3) breaking the bacterial cells to obtain an enzyme solution of the recombinant glycosyltransferase for catalyzing the production of the Reb M by the Reb A.
6. A method for producing Reb M by catalyzing Reb a with a bacterial cell containing a recombinant glycosyltransferase that catalyzes Reb a production Reb M, comprising the steps of:
get final OD 600 To 25-250 of the recombinant glycosyltransferase of claim 5 containing the enzyme catalyzing production of Reb M by Reb A or the permeabilized recombinant glycosyltransferase of claim 5 containing the enzyme catalyzing production of Reb M by Reb A, adding 1-10g/L of Reb A, 4-10mM uridine diphosphate glucose and 0.4-1mM Mn 2+ Adding PBS with final concentration of 50mM and pH of 7.2-8.0, reacting at 26-42deg.C and rotation speed of 50-250rpm for 10-24 hr, and catalyzing to obtain Reb M;
the permeabilized bacterial cells containing the recombinant glycosyltransferase that catalyzes the production of Reb M from Reb a are prepared in the following manner:
mode one: freezing the bacterial cells containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M at-20 degrees to-80 ℃ for 0.5-24 hours;
mode two: re-suspending the bacterial cells containing recombinant glycosyltransferase for catalyzing Reb A to produce Reb M with cell penetrating agent with final concentration of 0.15-0.45g/L, and standing at 20-30deg.C for 5min-1h; the cell penetrating agent is cetyl trimethyl ammonium bromide, triton X-100, polysorbate-80 or sodium dodecyl sulfate.
7. A method for producing Reb M by catalyzing Reb a with an enzyme solution of a recombinant glycosyltransferase that catalyzes Reb a production Reb M, comprising the steps of:
taking enzyme solution of recombinant glycosyltransferase for catalyzing Reb A to produce Reb M, which is described in claim 5, with a final concentration of 1-5g/L, adding Reb A with a final concentration of 1-10g/L, uridine diphosphate glucose with a final concentration of 1-8mM and metal ions with a final concentration of 0.1-6mM, adding buffer solution with a final concentration of 50mM and pH of 5.5-10.5, reacting for 12-96h at 26-42 ℃ to catalyze and generate Reb M;
the metal ion is Mg 2+ 、Mn 2+ 、Ba 2+ 、Ca 2+ Or Pb 2+ 。
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