CN115960856A - Glycosyltransferase fusion enzyme variant and application thereof in AA-2G preparation - Google Patents
Glycosyltransferase fusion enzyme variant and application thereof in AA-2G preparation Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a glycosyltransferase fusion enzyme variant and application thereof in AA-2G preparation, belonging to the technical field of enzyme engineering. The CGTase-TB fusion enzyme is obtained by fusing the front 1-331 sections of amino acids of the CGTase-Tx and the rear 327-681 sections of amino acids of the CGTase-Bs, and the catalytic efficiency of the enzyme is improved. The invention also makes the CGTase-TB fusion enzyme mutated, so that the catalytic activity of the mutant is further improved compared with that of the fusion enzyme, the specific activity reaches 9.21-19.53U/mg, the AA-2G yield is improved to more than 70%, the thermal stability of the mutant is obviously improved compared with that of a parent, and the activity of the enzyme still remains 90% after the mutant is treated for 4 hours at 70 ℃.
Description
Technical Field
The invention relates to a glycosyltransferase fusion enzyme variant and application thereof in AA-2G preparation, belonging to the technical field of enzyme engineering.
Background
At present, almost all L-ascorbic acid-2-glucoside (AA-2G) production adopts enzyme catalysis, the used enzymes mainly comprise cyclodextrin glucosyltransferase (CGTase) and sucrose phosphorylase, the glycosyl donor used in the former mainly comprises liquefied starch, maltodextrin, cyclodextrin and the like, the glycosyl donor used in the latter is sucrose, and the glycosyl acceptor is vitamin C. Different from sucrose phosphorylase, the production of AA-2G by cyclodextrin glycosyltransferase catalysis usually takes beta-cyclodextrin, liquefied starch or maltodextrin as glycosyl donor and vitamin C as acceptor, and the reaction is carried out under certain temperature and pH condition, at this time, a large amount of AA-2Gn and AA-2G exist in the reaction process, saccharifying enzyme is needed to be added for further saccharifying treatment after the catalysis is finished, and redundant glycosyl on AA-2Gn is cut off to obtain AA-2G, so that the catalytic reaction is finished.
However, CGTase in the prior art has low catalytic efficiency, the AA-2G enzyme catalysis preparation period is generally long (24-72 hours are needed), in addition, the reaction has serious balance problem, and the maximum AA-2G yield can only reach about 40-60%.
Disclosure of Invention
The invention provides a cyclodextrin glucosyltransferase fusion enzyme, which contains an amino acid sequence shown in SEQ ID NO. 1.
The invention also provides a cyclodextrin glucosyltransferase fusion enzyme mutant, which is obtained by mutating one or more sites of leucine at position 46, tyrosine at position 152, glycine at position 190, asparagine at position 269 and isoleucine at position 325 on the basis of the fusion enzyme shown in SEQ ID NO. 1.
In one embodiment, the mutant is a mutant L46F obtained by mutating leucine at position 46 to phenylalanine.
In one embodiment, the mutant is a mutant Y152G obtained by mutating tyrosine 152 to glycine.
In one embodiment, the mutant is a mutant G190W obtained by mutating glycine 190 to tryptophan.
In one embodiment, the mutant is a mutant N269A obtained by mutating asparagine to alanine at position 269.
In one embodiment, the mutant is a mutant I325K obtained by mutating isoleucine to lysine at position 325.
In one embodiment, the mutant is a mutant L46F/Y152G/N269A/I325K obtained by mutating leucine at position 46 to phenylalanine, tyrosine at position 152 to glycine, asparagine at position 269 to alanine, and isoleucine at position 325 to lysine.
In one embodiment, the mutant is L46F/G190W/I325K obtained by mutating leucine at position 46 to phenylalanine, glycine at position 190 to tryptophan, and isoleucine at position 325 to lysine.
In one embodiment, the mutant is L46F/Y152G/I325K obtained by mutating leucine at position 46 to phenylalanine, tyrosine at position 152 to glycine, and isoleucine at position 325 to lysine.
The present invention also provides a gene encoding the fusion enzyme or the fusion enzyme mutant.
In one embodiment, the gene encoding the fusion enzyme comprises the nucleotide sequence set forth in SEQ ID No. 2.
The invention also provides a recombinant plasmid carrying the gene.
In one embodiment, the plasmid includes, but is not limited to, a pET series plasmid.
The invention also provides a recombinant microbial cell expressing the fusion enzyme or the fusion enzyme mutant.
In one embodiment, the microorganism includes, but is not limited to, E.coli.
In one embodiment, the microbial cell is an escherichia coli host, and the mutant is expressed by taking pET-28a (+) as an expression vector.
The invention also provides a cell catalyst containing the microbial cell.
In one embodiment, the cell catalyst is a cell culture fluid having cyclodextrin glycosyltransferase activity that is collected by culturing the recombinant microbial cell in a culture medium for a period of time.
In one embodiment, the cell catalyst is prepared as follows: culturing the recombinant microorganism in a culture medium until the OD of a bacterial liquid reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) to induce the expression of enzyme, and collecting thalli after inducing for a period of time to obtain the cell catalyst.
In one embodiment, the medium is LB medium or TB medium.
In one embodiment, the inducer is added in an amount of 0.05 to 0.15mmol/L.
In one embodiment, the induction is at 20-25 ℃ for 20-28 h.
The invention also provides application of the mutant or the cell catalyst in catalytic production of L-ascorbic acid-2-glucoside.
In one embodiment, the mutant or the cell catalyst is used for catalyzing and synthesizing the L-ascorbic acid-2-glucoside in a reaction system containing beta-cyclodextrin and L-ascorbic acid.
In one embodiment, the application takes beta-cyclodextrin as glycosyl donor, ascorbic acid as acceptor, the cyclodextrin glucosyltransferase fusion enzyme or the cyclodextrin glucosyltransferase fusion enzyme mutant or the cell catalyst for catalytic reaction for 3-10 h, and then saccharifying enzyme is added for reaction for at least 4h.
In one embodiment, the application takes beta-cyclodextrin as glycosyl donor, takes ascorbic acid as acceptor, adopts cell catalyst to react for 3 to 10 hours at 25 to 30 ℃, then raises the temperature to 50 ℃ and uses saccharifying enzyme to react for at least 4 hours.
In one embodiment, the beta-cyclodextrin and ascorbic acid are present in a mass ratio of 1:0.8 to 1.2.
Has the advantages that:
1. the invention obtains the CGTase-TB variant by fusing the first 1-331 amino acids of the CGTase-Tx and the last 327-681 amino acids of the CGTase-Bs, the catalytic efficiency is further improved compared with the CGTase-Tx, the AA-2G yield is higher, and no by-product is generated.
2. The fusion mutant CGTase-TB is further mutated, so that the catalytic activity of the mutant is further improved compared with that of the parent, the yield of AA-2G is further improved to more than 70%, and the specific activity reaches 9.21-19.53U/mg; and the heat stability of the mutant is obviously improved compared with that of the parent, and 90 percent of enzyme activity is still remained after the mutant is treated for 4 hours at 70 ℃.
Drawings
FIG. 1 is a schematic diagram of the catalytic production of AA-2G by CGTase.
Detailed Description
The invention will be further elucidated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The examples relate to experimental materials, reagents and equipment as shown in tables 1 to 2.
TABLE 1 reagents
| PrimeStar Max enzyme | Takara treasureNissan Tech Co Ltd |
| Seamless Cloning Kit | Biotechnology engineering Co., ltd |
| Taq DNA polymerase | Takara Baoriri doctor Tech Co Ltd |
| Ex Taq Buffer | Takara Baoriri doctor Tech Co Ltd |
| dNTP Mix(2.5mM) | Takara Baoriri doctor Tech Co Ltd |
| Restriction enzymes Nco I, ecoRI and Dpn I | Takara Baoriri doctor Tech Co Ltd |
| DL 10000marker/DL 10000marker | Takara Baoriri doctor Tech technology Co Ltd |
| T4 DNA Ligase | Biotechnology engineering Co Ltd |
| Yeast powder | Aladdin Biotechnology Ltd |
| Tryptone | Alantin Biochemical technology Ltd |
| Agar powder | Biochemical family of AladdinTechnology and resources Co Ltd |
| NaCl | Aladdin Biotechnology Ltd |
| Beta-cyclodextrin | Alantin Biochemical technology Ltd |
| L-ascorbic acid | Aladdin Biotechnology Ltd |
TABLE 2 instruments and apparatus
Example 1: construction of CGTase fusion enzymes
The sequence conservation from the V323 to M331 site of CGTase-Pm was found to be high by sequence analysis of CGTase-Pm (Genbank accession No.: WP _ 036618292.1), CGTase-Nc (UniProtKB accession No.: P43379.1), CGTase-Bs (shown in SEQ ID NO.5, uniProtKB accession No.: P31797.1 of the original sequence) and CGTase-Tx (shown in SEQ ID NO.3, genbank accession No.: WP _013787419.1 of the original sequence). Respectively defining sequences before and after M331 in the CGTase-Pm amino acid sequence as a domain 1 and a domain 2, namely, the domain 1 of the CGTase-Pm is M1-M331, and the domain 2 is D332-N688; . The sequences before and after M332 in the CGTase-Nc amino acid sequence are respectively defined as a domain 1 and a domain 2, namely the domain 1 of the CGTase-Nc is M1-M332, and the domain 2 is E333-P689; respectively defining sequences before and after M326 in the amino acid sequence of the CGTase-Bs as a domain 1 and a domain 2, wherein the domain 1 of the CGTase-Bs is M1-M326, and the domain 2 is D327-N681; the sequences before and after M331 in the CGTase-Tx amino acid sequence are defined as domain 1 and domain 2, respectively, i.e., domain 1 of CGTase-Tx is M1 to M331, and domain 2 is D332 to Q684. The domains 1 and 2 of CGTase-Pm, CGTase-Nc, CGTase-Bs, and CGTase-Tx were subjected to staggered recombinant fusion (as shown in table 3), and there were theoretically 12 fusion variants, CGTase-PN (Pm 1-Nc 2), CGTase-PB, (Pm 1-Bs 2), CGTase-PT (Pm 1-Tx 2), CGTase-TP (Tx 1-Pm 2), CGTase-TN, (Tx 1-Nc 2), CGTase-TB (Tx 1-Bs 2), CGTase-BT (Bs 1-Tx 2), CGTase-BT, (Bs 1-Pm 2), CGTase-Bs, (Bs 1-Pm 2), CGTase-BN (Bs 1-Nc 2), CGTase-NP (Nc 1-Pm 2), CGTase-NT (Nc 1-Tx 2), CGTase-NB (Nc 1-Bs 2), and CGTase-NB (ncbp 2), respectively, and the catalytic yield of these fusion variants on AA2G was verified.
TABLE 3 list of fusion variants
After the genes of CGTase-Tx, CGTase-Bs, CGTase-Pm and CGTase-Nc are subjected to codon optimization (nucleotide sequences are respectively shown in SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 and SEQ ID No. 10), the genes encoding V323 site-M331 site segments in 4 CGTases are adjusted to be the same sequence after optimization, the genes are synthesized by a general biological company, and are connected to pET-28a (+) vectors to construct gene expression vectors after being subjected to enzyme digestion by NcoI and EcoRI. The recombinant plasmid was transformed into E.coli TOP10 competent cells by chemical transformation for storage and cloning of the expression vector. Designing primers for obtaining DNA fragments of a domain 1 and a domain 2 of CGTase-Pm, CGTase-Nc, CGTase-Bs and CGTase-Tx, wherein the specific primers are as follows:
domains 1-F: GCGGATAACAATTCCCCTCTAGAAATAATT;
domains 1-R: ATCATGATTATCGATGATGAAGGGTCACCATACCG;
domain 2-F: GACCTTCATCGATAATCATGGATCGTT;
domain 2-R: TCTAGAGGGAATTGTTATCCGCTCACAATT;
the domain 1 and domain 2 fragments of CGTase-Pm, CGTase-Nc, CGTase-Bs and CGTase-Tx were amplified by PCR using each CGTase expression vector DNA as a template. And (3) PCR system: a50. Mu.L system with PrimeStar Max enzyme 25. Mu.L, upstream and downstream primers 2. Mu.L each, template DNA 1. Mu.L, made up to 50. Mu.L with water. And (3) PCR reaction conditions: after denaturation at 98 ℃ for 5min, circulation is started, after denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 1min, 30 cycles are carried out, and extension is carried out at 72 ℃ for 10min. The size of the domain 1 fragment of each CGTase obtained by amplification is about 1000bp, while the size of the domain 2 fragment is about 6300bp because the fragment contains a plasmid skeleton. After the PCR reaction was completed, the PCR fragment was recovered using a DNA fragment recovery kit (Aisika), and the specific method was according to the manufacturer's instructions. After completion of the recovery, the concentration of the DNA product was measured, and the concentration of the domain 1 and domain 2 fragments of each gene was adjusted to 50 ng/. Mu.L, at which the recombinant fusion reaction of the domain 1 and domain 2 fragments was carried out by using the Seamless Cloning kit (Biyun).
Seamless Cloning reaction system: seamless Cloning enzyme 5. Mu.L, domain 1PCR fragment 1. Mu.L, domain 2PCR fragment 2. Mu.L, supplemented with water to 10. Mu.L, and the reaction was continued at 50 ℃ for 25min. After the fusion reaction is finished, connecting products pET-28a (+) -CGTase-PT, pET-28a (+) -CGTase-PN, pET-28a (+) -CGTase-PB, pET-28a (+) -CGTase-TP, pET-28a (+) -CGTase-TN, pET-28a (+) -CGTase-TB, pET-28a (+) -CGTase-BT, pET-28a (+) -CGTase-BP, pET-28a (+) -CGTase-BN, pET-28a (+) -CGTase-NP, pET-28a (+) -CGTase-NT and pET-28a (+) -CGTase-NB are used for transforming escherichia coli BL21 (DE 3) competent cells, the incubated cells are coated and cultured overnight at 37 ℃, and then the grown fusion enzyme transformant is sent to a Zhou cloning biotechnology limited company for gene sequencing, and preserving and successfully constructing fusion enzyme variants, and 12 fusion enzyme variants are obtained in total.
Example 2: validation of fusion enzyme variant Activity
1mL of LB liquid medium containing 50mg/L kanamycin was added to a 24-deep-well plate, 10. Mu.L of the strain was inoculated, and cultured overnight at 37 ℃ and 250 rpm. Then 40. Mu.L of the bacterial solution was inoculated into 1.5mL of TB liquid medium containing 50mg/L kanamycin and grown to OD at 37 ℃ 600 =0.6-0.8, inducing with IPTG with final concentration of 0.1mmol/L, and cooling to 25 deg.CAnd (5) culturing overnight. After the enzyme production is finished, centrifuging to remove supernatant fermentation liquor, and using 20mmol/LNaH 2 PO 4 -Na 2 HPO 4 The cells were resuspended and washed 2 times in buffer (pH 7.0), the supernatant was discarded by centrifugation, and the remaining cells were washed with 300. Mu.L of 50mmol/L KH 2 PO 4 The solution was resuspended for catalytic reaction. The catalytic reaction method comprises the following steps: to the 300. Mu.L of the above-mentioned bacterial solution (OD) 600 = 16) 1.2mL of the reaction solution was added to prepare a reaction mixture containing 20G/L β -cyclodextrin and 20G/L-ascorbic acid at a final concentration and pH =5.0, and then the mixture was reacted at 30 ℃ for 24 hours, and after the reaction was completed, the content of AA-2G was measured in a liquid phase.
AA-2G liquid phase detection method (HPLC method): after the catalytic reaction is finished, centrifuging the reaction solution (10000 rpm), taking 20 mu L of supernatant, adding pure water to dilute the supernatant to 2000 mu L, filtering the supernatant by using a 0.22 mu m filter membrane, injecting a sample, and substituting an absorption peak area into an external standard calculation formula to calculate the yield of AA-2G. The AA-2G yield is calculated as follows:
wherein A is the AA-2G peak area measured by an AA-2G catalytic reaction liquid sample, N is the dilution multiple, K is the peak area of AA-2G unit concentration, and W is the AA-2G concentration theoretically obtained by converting all ascorbic acid as a substrate into AA-2G.
The activity of the fusion enzyme variant is verified as follows, and the yield of AA-2G of the CGTase-TB variant can reach 34.7%.
TABLE 4 verification of the Activity of the fusion enzyme variants
Example 3: construction of CGTase-TB saturation mutation library
Analyzing the composition of a catalytic pocket of CGTase-TB, selecting L46, Y152, G190, N269 and I325 sites to establish a saturation mutation library, and designing a corresponding saturation mutation primer according to the plasmid information of the CGTase-TB, wherein the method is as follows.
TB-L46-F：ATACCAGTNNKAAAAAATATTTTGGCGGTG；
TB-L46-R：TATTTTTTMNNACTGGTATGGGTCGGATCA；
TB-Y152-F：ATCCGACCNNKGCCGAAAATGGCCGTCTGT；
TB-Y152-R：TTTTCGGCMNNGGTCGGATCGGTTTCACTT；
TB-G190-F：ATGAAGATNNKATCTATCGCAATCTGTTTG；
TB-G190-R：CGATAGATMNNATCTTCATAACTGCTAAAA；
TB-N269-F：TGGATGCCNNKAATACCTATTTTGCAAATG；
TB-N269-R：TAGGTATTMNNGGCATCCACTTCATTGGTG；
TB-I325-F：TGACCTTCNNKGATAATCATGATATGGATC；
TB-I325-R：TGATTATCMNNGAAGGTCACCATATCGTTA；
PCR amplification was performed using PrimeStar Max enzyme using pET-28a (+) -CGTase-TB plasmid DNA as a template. The PCR reaction was carried out in a 50. Mu.L system using PrimeStar Max enzyme 25. Mu.L, upstream and downstream primers 2. Mu.L each, and template DNA 1. Mu.L, and purified water to make up to 50. Mu.L. The reaction conditions were that denaturation at 98 ℃ was carried out for 5min and then circulation was started, denaturation at 98 ℃ was carried out for 30s, annealing at 58 ℃ was carried out for 15s, and elongation at 72 ℃ was carried out for 10s, and after 30 cycles in total, elongation at 72 ℃ was carried out for 10min. After the amplification is finished, a fragment product of about 7300bp is obtained, the product is digested by Dpn I enzyme and transformed into competent cells of escherichia coli BL21 (DE 3), the competent cells are coated and cultured overnight at 37 ℃, and then grown clones are picked to a 96-pore plate to obtain a saturated mutation library.
Example 4: screening of saturated mutant libraries
Clones in the saturation mutation library were picked up in a 96-well plate, 200. Mu.L of LB liquid medium containing 50mg/L kanamycin per well was added, and cultured overnight at 37 ℃ and 250rpm to obtain a primary seed solution. Transferring 40. Mu.L of the primary seed solution into the same 50mg/L kanamycin-containing TB liquid medium, and culturing at 37 ℃ to OD 600 =0.6-0.8, use final concentration 0.1mmolIPTG induction was performed at/L, the induction temperature was adjusted to 25 ℃ and the cells were cultured overnight, while CGTase-TB parent enzyme strain was used as a control. After the induction, the supernatant broth was discarded by centrifugation, 600. Mu.L of 0.2mol/L sodium sulfite solution was added to resuspend the cells, and the cells were treated on a shaker at 30 ℃ and 250rpm for 2 hours, and then the supernatant was discarded by centrifugation. Adding 20mmol/L NaH into thallus 2 PO 4 -Na 2 HPO 4 The cells were resuspended and washed 2 times in buffer (pH 7.0), the supernatant was discarded by centrifugation, and the cells were washed with 300. Mu.L of 50mmol/L KH 2 PO 4 The solution was resuspended again. 1.2mL of a reaction mixture (containing ascorbic acid at a concentration of 25g/L, beta-cyclodextrin at a concentration of 25g/L, 50mmol/L KH) was added to the resuspended suspension 2 PO 4 Solution, pH = 5.0), making the final concentration of ascorbic acid in the reaction system be 20g/L and the final concentration of beta-cyclodextrin be 20g/L, reacting at 30 ℃ for 24h, centrifuging, taking supernatant, diluting by 100 times, taking 100 mu L of diluent, adding 100 mu L of phosphomolybdic acid solution to carry out color development reaction, making the final concentration of phosphomolybdic acid be 7mg/mL, reacting for 10min, and measuring the light absorption value of the solution at 660 nm. The solution light absorption value is in positive correlation with the L-ascorbic acid content, the residual L-ascorbic acid content in the reaction solution is calculated according to the standard curve by establishing a phosphomolybdic acid chromogenic reaction standard curve of the L-ascorbic acid, and the AA-2G yield is calculated by the following formula. The relative activity of the CGTase-TB wild type was defined as 100%.
Wherein, W0 represents the content of L-ascorbic acid in the initial reaction solution;
w-the content of L-ascorbic acid in the final reaction solution.
The mutants with improved activity are obtained by primary screening and then rescreened according to the method of example 2, the beneficial mutants obtained by rescreening are sent to Hangzhou Ongke Biotechnology Limited for gene sequencing, and the sequencing results and the corresponding activities of the beneficial mutants are shown in Table 5.
TABLE 5 determination of the Activity of the high Activity CGTase-TB mutant
| Mutation site | 46 | 152 | 190 | 269 | 325 | Relative Activity% | AA-2G yield% |
| CGTase-TB wild type | L | Y | G | N | I | 100 | 34.7 |
| CGTase-TB-L46F | F | - | - | - | - | 126 | 43.7 |
| CGTase-TB-Y152G | - | G | - | - | - | 103 | 35.9 |
| CGTase-TB-G190W | - | - | W | - | - | 111 | 38.4 |
| CGTase-TB-N269A | - | - | - | A | - | 130 | 45.2 |
| CGTase-TB-I325K | - | - | - | - | K | 152 | 52.9 |
Example 5: construction of combinatorial mutation library by staggered extension PCR and screening
The beneficial mutations obtained from the saturation mutation of example 4 were subjected to random combinatorial mutagenesis. According to the plasmid information of CGTase-TB, primers for establishing an L46, Y152, G190, N269 and I325 staggered extension PCR system are designed.
TB-StEP-F：GAATTGTGAGCGGATAACAATTCCCCTC
TB-StEP-R：GCAAGCTTGTCGACGGAGCTCGAATTCT
Plasmids carrying CGTase-TB mutants L46F, Y152G, G190W, N269A or I325K were extracted using a plasmid extraction kit (Aisijin), the above plasmid DNAs were mixed at the same DNA concentration, and staggered extension PCR was performed using Taq DNA polymerase (Takara) using the mixed plasmid DNA as a template. The PCR reaction was carried out in a 50. Mu.L system: taq DNA polymerase 1. Mu.L, each 2. Mu.L after the upstream and downstream primers, template DNA 1. Mu.L, 10 XEx Taq Buffer 5. Mu.L, dNTP Mix (2.5 mM) 4. Mu.L, and purified water to make up to 50. Mu.L. The reaction conditions were 30s for denaturation at 98 ℃ and 30s for renaturation at 55 ℃ and this was repeated 80 times. After the amplification, a fragment product of about 2000bp was obtained, and the PCR fragment was recovered using a DNA fragment recovery kit (Aisijin). PCR amplification was then performed using PrimeStar Max enzyme using the PCR fragment as a template. The PCR reaction was carried out in a 50. Mu.L system using 25. Mu.L of PrimeStar Max enzyme, 2. Mu.L of each of the upstream and downstream primers, and 1. Mu.L of template DNA, and purified water was added to make up to 50. Mu.L. The reaction conditions are that the cycle is started after the denaturation at 98 ℃ is carried out for 5min, then the denaturation at 98 ℃ is carried out for 30s, the annealing at 58 ℃ is carried out for 15s, the extension at 72 ℃ is carried out for 30s, and after 30 cycles, the extension at 72 ℃ is carried out for 10min. After the amplification, a fragment product of about 2000bp was obtained, and the PCR fragment was recovered using a DNA fragment recovery kit (Aisijin). The recovered PCR product and pET-28a (+) -CGTase-TB plasmid are subjected to enzyme digestion by Nco I and EcoR I, after the T4 ligase is enzymatically connected, the ligation product is transformed into an escherichia coli BL21 (DE 3) competent cell, the competent cell is cultured overnight at 37 ℃ after being coated, and then the grown clone is picked up to a 96-well plate to obtain a saturated mutation library. The sequencing results and the corresponding activities of the beneficial mutants are shown in Table 6 and named CGTase-TB1 (L46F/Y152G/N269A/I325K), CGTase-TB2 (L46F/G190W/I325K) and CGTase-TB3 (L46F/Y152G/I325K), respectively.
TABLE 6 determination of Activity of highly active CGTase-TB Complex mutants
| Mutation site | 46 | 152 | 190 | 269 | 325 | Relative activity% | AA-2G yield% |
| CGTase-TB wild type | L | Y | G | N | I | 100 | 34.7 |
| CGTase-TB1 | F | G | G | A | K | 209 | 72.5 |
| CGTase-TB2 | F | Y | W | N | K | 212 | 73.4 |
| CGTase-TB3 | F | G | G | N | K | 202 | 70.1 |
Example 6: preparation of enzyme preparations
Strains expressing the mutants CGTase-TB-L46F, CGTase-TB-Y152G, CGTase-TB-G190W, CGTase-TB-N269A, CGTase-TB-I325K, CGTase-TB1, CGTase-TB2 or CGTase-TB3 were inoculated from a stock tube at 1% inoculum size (50. Mu.L) into 5mL of LB medium containing kanamycin to a final concentration of 50mg/L, cultured overnight at 37 ℃ and 200rpm for 15h. Then, the first-class strain is inoculated into 50mL TB medium containing 50mg/L kanamycin at the final concentration according to the inoculation amount of 4 percent, the first-class strain is cultured at 37 ℃ and 220rpm until the OD of a bacterial liquid is 0.6-0.8, IPTG at the final concentration of 0.1mmol/L is added, the bacterial liquid is induced and expressed at 25 ℃ and 220rpm, after induction is carried out for 24 hours, the fermentation liquid is removed by centrifugation, and thalli are collected, so that the cell catalyst is obtained.
The cell catalyst is subjected to wall breaking, protein purification and determination of the specific enzyme activity of the mutant, and the result shows that the specific enzyme activity of CGTase-TB-L46F is 11.60U/mg, the specific enzyme activity of CGTase-TB-Y152G is 9.49U/mg, the specific enzyme activity of CGTase-TB-G190W is 10.22U/mg, the specific enzyme activity of CGTase-TB-N269A is 11.97U/mg, the specific enzyme activity of CGTase-TB-I325K is 14.00U/mg, the specific enzyme activity of CGTase-TB1 is 19.25U/mg, the specific enzyme activity of CGTase-TB2 is 19.53U/mg and the specific enzyme activity of CGTase-TB3 is 18.6U/mg.
Example 7: application of CGTase-TB1 in catalytic synthesis of AA-2G
Preparing 100mL of an enzyme-catalyzed reaction system containing (by final concentration) 200g/L of beta-cyclodextrin (glycosyl donor) and 200g/L of ascorbic acid (glycosyl acceptor), 50g/L of CGTase-TB1 wet cells prepared by the method of reference example 6; the pH of the solution was adjusted to 5.0 using sodium hydroxide and the reaction temperature was 30 ℃. The reaction progress was monitored by sampling at regular intervals and the reaction time was 5 hours, and the AA2G content did not increase any more. Adding 500000U/L saccharifying enzyme (gold source organism) into the reaction system, adjusting pH to 5.0, maintaining at 50 deg.C for 4 hr, and performing liquid phase detection after reaction to obtain 20G L-ascorbic acid, wherein the yield of AA-2G (27.61G) and AA-2G (71.9%) obtained by enzyme-catalyzed conversion.
AA-2G liquid phase detection method (HPLC method): after the catalytic reaction is finished, centrifuging the reaction solution (10000 rpm), taking 20 mu L of supernatant, adding pure water to dilute the supernatant to 2000 mu L, filtering the supernatant by using a 0.22 mu m filter membrane, injecting a sample, substituting the sample into an external standard calculation formula according to the absorption peak area, and calculating the yield of AA-2G. The AA-2G yield is calculated as follows:
wherein A is the AA-2G peak area measured by an AA-2G catalytic reaction liquid sample, N is the dilution multiple, K is the peak area of AA-2G unit concentration, and W is the AA-2G concentration theoretically obtained by converting all ascorbic acid as a substrate into AA-2G.
Example 8: application of CGTase-TB2 in catalytic synthesis of AA-2G
Preparing 100mL of an enzyme-catalyzed reaction system containing (by final concentration) 200g/L of beta-cyclodextrin (glycosyl donor) and 200 g/LL-ascorbic acid (glycosyl acceptor), 50g/L of CGTase-TB2 wet cells prepared by the method of reference example 6; the pH of the solution was adjusted to 5.0 using sodium hydroxide and the reaction temperature was 30 ℃. The reaction progress was monitored by sampling at regular intervals and the reaction time was 3 hours, and the AA2G content did not increase any more. Adding 500000U/L saccharifying enzyme (gold source organism) into the reaction system, adjusting pH of the solution to 5.0, keeping at 50 ℃ for 4 hours, and performing liquid phase detection after the reaction is finished, wherein the result shows that 27.9G of AA-2G prepared by 20G of L-ascorbic acid through enzyme catalytic conversion and the yield of AA-2G reaches 72.8%.
Example 9: application of CGTase-TB3 in catalytic synthesis of AA-2G
Preparing 100mL of an enzyme-catalyzed reaction system containing (by final concentration) 200g/L of beta-cyclodextrin (glycosyl donor) and 200 g/LL-ascorbic acid (glycosyl acceptor), and 50g/L of CGTase-TB3 wet cells prepared by the method of reference example 6; the pH of the solution was adjusted to 5.0 using sodium hydroxide and the reaction temperature was 30 ℃. The reaction progress was monitored by sampling at regular intervals and the reaction time was 10 hours, and the AA2G content did not increase any more. Adding 500000U/L saccharifying enzyme (gold source organism) into the reaction system, adjusting pH of the solution to 5.0, keeping the temperature at 50 ℃ for 4 hours, and detecting a liquid phase after the reaction is finished, wherein the result shows that 25.8G of AA-2G prepared by 20G of L-ascorbic acid through enzyme catalytic conversion and the yield of AA-2G reaches 67.3%.
Example 10: analysis of thermal stability of each mutant
The fermentation broth of each mutant was centrifuged to remove the supernatant, the cells were retained, and 50mM KH was used 2 PO 4 The solution (pH 5.0) was resuspended, incubated in water at 70 ℃ for 4 hours, and the residual enzyme activity of the cells after incubation was measured in example 2. The thermal stability of CGTase-TB1, CGTase-TB2 and CGTase-TB3 is obviously improved compared with that of CGT-Tx, and 90 percent of enzyme activity remains after treatment for 4 hours at 70 ℃.
TABLE 7 thermostability of the different mutants
| Mutation site | AA2G yield before incubation | AA2G yield after incubation | Enzyme activity residue |
| CGTase-Tx wild type | 20.5 | 4.2 | 20％ |
| CGTase-TB1 | 72.5 | 66.1 | 91％ |
| CGTase-TB2 | 73.4 | 67.8 | 92％ |
| CGTase-TB3 | 70.1 | 63.1 | 90％ |
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 (10)
1. Cyclodextrin glucosyltransferase fusion enzyme comprising an amino acid sequence as set forth in SEQ ID No. 1.
2. A cyclodextrin glucosyltransferase fusion enzyme mutant characterized in that one or more of leucine 46, tyrosine 152, glycine 190, asparagine 269, and isoleucine 325 is/are mutated in the fusion enzyme represented by SEQ ID NO. 1.
3. The cyclodextrin glucosyltransferase fusion enzyme mutant according to claim 2, wherein leucine at position 46 is mutated to phenylalanine, tyrosine at position 152 is mutated to glycine, and isoleucine at position 325 is mutated to lysine based on the fusion enzyme represented by SEQ id No. 1; or
The 46 th leucine is mutated into phenylalanine, the 190 th glycine is mutated into tryptophan, and the 325 th isoleucine is mutated into lysine; or
Leucine 46 was mutated to phenylalanine, tyrosine 152 to glycine, asparagine 269 to alanine, and isoleucine 325 to lysine.
4. A gene encoding the fusion enzyme of claim 1 or the mutant fusion enzyme of any one of claims 2 to 3.
5. A recombinant plasmid carrying the gene of claim 4.
6. A recombinant microbial cell expressing the fusion enzyme of claim 1 or the mutant fusion enzyme of any one of claims 2 to 3.
7. A recombinant Escherichia coli which expresses the fusion enzyme of claim 1 or the fusion enzyme mutant of any one of claims 2 to 3 using pET-28a (+) as an expression vector.
8. A cell catalyst comprising the microbial cell according to claim 6 or comprising the recombinant Escherichia coli according to claim 7.
9. An enzyme preparation comprising the fusion enzyme according to claim 1 or the mutant fusion enzyme according to any one of claims 2 to 3.
10. A method for producing L-ascorbic acid-2-glucoside is characterized in that a biocatalyst is reacted in a reaction system containing beta-cyclodextrin and L-ascorbic acid for at least 4 hours; the biocatalyst comprises the fusion enzyme of claim 1, or the fusion enzyme mutant of any one of claims 2 to 3, or the recombinant microbial cell of claim 6, or the recombinant Escherichia coli of claim 7, or the cell catalyst of claim 8, or the enzyme preparation of claim 9.
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| CN103122341A (en) * | 2013-01-08 | 2013-05-29 | 江南大学 | Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof |
| CN108018268A (en) * | 2018-01-15 | 2018-05-11 | 江南大学 | A kind of yclodextrin glycosyltransferase mutant of raising AA-2G yield |
| CN112301012A (en) * | 2020-10-15 | 2021-02-02 | 江南大学 | Cyclodextrin glucosyltransferase mutant and construction method thereof |
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| CN103122341A (en) * | 2013-01-08 | 2013-05-29 | 江南大学 | Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof |
| CN108018268A (en) * | 2018-01-15 | 2018-05-11 | 江南大学 | A kind of yclodextrin glycosyltransferase mutant of raising AA-2G yield |
| CN112301012A (en) * | 2020-10-15 | 2021-02-02 | 江南大学 | Cyclodextrin glucosyltransferase mutant and construction method thereof |
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