A fructosidase mutant, preparation method and application

Abstract

The application discloses a fructosidase mutant, which is obtained by mutating any one or more of positions 347, 365, 405 and 477 of a fructosidase PsFFase with an amino acid sequence shown in SEQ ID NO: 1. The application also discloses a preparation method of the fructosidase mutant and application of the fructosidase mutant in catalyzing synthesis of fructosyl rebaudioside A and fructosyl rubusoside. The application realizes glycosylation preparation of f-RebA and f-Rub by using a PsFFase mutant, and the PsFFase mutant has high catalytic activity and good thermal stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a fructosidase mutant, a preparation method and application. BACKGROUND

[0002] In order to cope with the global health challenges such as obesity, diabetes and other diseases, the demand for low-calorie sweeteners to replace traditional sugars is increasing. Although traditional artificial sweeteners reduce calorie intake, long-term consumption still has some adverse effects on human health. As a class of natural sweeteners extracted from stevia rebaudiana, steviol glycosides (SGs) not only have the advantages of high sweetness and low heat, but also can bring some benefits to human health (anti-cancer, anti-inflammatory, anti-caries, etc.). Therefore, high-purity SGs as food additives have been recognized by the US Food and Drug Administration (FDA) and the Joint Expert Committee on Food Additives of the Food and Agriculture Organization / World Health Organization (JECFA). However, the existing SGs are restricted in their commercial application due to their own bad aftertaste.

[0003] SGs belong to the diterpenoid group of plant secondary metabolites, and their chemical structures are all centered on a glycoside aglycone called steviol, which is connected to different numbers and types of sugar groups at the C19 and C13 positions. Related studies on the relationship between these different types and numbers of sugar groups and the sensory and physicochemical properties of SGs have found that sugar chain length, pyranose substitution, and C16 double bond are important structural features that distinguish the taste characteristics of SGs. In order to improve the taste of SGs, enzymatic modification of their carbohydrate moieties is an efficient method.

[0004] By screening fructosidases in the database NCBI, it was found that a fructosidase from Pseudoalteromonas (PsFFase) could catalyze the transfructosylation of some steviol glycosides to produce fructosyl steviol glycosides, including Rebaudioside A (RebA) and Rubusoside (Rub). However, PsFFase has poor thermal stability, and its enzyme activity is greatly lost after 1h of catalysis in a 40℃ environment, which severely limits the efficient application of this fructosidase. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a fructosidase mutant, a preparation method and application in view of the deficiencies in the prior art. The present application uses a PsFFase mutant to prepare f-RebA and f-Rub by glycosylation of RebA and Rub, and the PsFFase mutant has high catalytic activity and good thermal stability.

[0006] To solve the above technical problems, the technical scheme of the present application is as follows:

[0007] A fructosidase mutant, which is obtained by mutating any one or more of positions 87, 128, 190, 275, 297, 347, 361, 365, 388, 405, 446, 477 of a fructosidase PsFFase having an amino acid sequence as shown in SEQ ID NO: 1.

[0008] Preferably, the mutation site of the fructosidase mutant is selected from any one or more of positions 347, 365, 405, 477. More preferably, the mutation site is selected from at least one of positions 405, 477.

[0009] Preferably, the amino acid sequence of the fructosidase mutant comprises a mutation of at least one amino acid residue in A87G, S128P, Q190A, A275G, T297K, Q347V, T361H, T365P, M388L, W405P, N446R, Y477N in the amino acid sequence of SEQ ID NO: 1.

[0010] Preferably, the T365P mutant is a mutation of the amino acid at position 365 from threonine (T) to proline (P) in the fructosidase PsFFase sequence having an amino acid sequence as shown in SEQ ID NO: 1.

[0011] Preferably, the W405P mutant is a mutation of the amino acid at position 405 from tryptophan (W) to proline (P) in the fructosidase PsFFase sequence having an amino acid sequence as shown in SEQ ID NO: 1, and the amino acid sequence of the mutant is as shown in SEQ ID NO: 2.

[0012] Preferably, the W405P / Y477N mutant is a mutation of the amino acid at position 405 from tryptophan (W) to proline (P) and the amino acid at position 477 from tyrosine (Y) to asparagine (N) in the fructosidase PsFFase sequence having an amino acid sequence as shown in SEQ ID NO: 1, and the amino acid sequence of the mutant is as shown in SEQ ID NO: 3.

[0013] An expression gene encoding any of the above fructosidase mutants.

[0014] A recombinant plasmid linked with the above expression gene.

[0015] A recombinant cell characterized in that the recombinant cell comprises the above expression gene and / or the above recombinant plasmid, and is an empty vector.

[0016] Further, the empty carrier bacteria are preferably Escherichia Coli BL21 (DE3) strains.

[0017] An application of a fructosidase mutant for catalyzing synthesis of fructosyl steviol glycoside.

[0018] A method for catalyzing synthesis of fructosyl steviol glycoside by a fructosidase mutant, comprising the following steps:

[0019] (1) Constructing a recombinant strain of enzyme expression:

[0020] The gene of the fructosidase mutant is constructed into a plasmid to obtain a recombinant plasmid; the recombinant plasmid is transformed into Escherichia Coli BL21 (DE3) competent cells to obtain a recombinant strain containing an enzyme expression system;

[0021] (2) Inducing enzyme production of the recombinant strain: activate the recombinant strain PsFFase, transfer it into LB induction medium at a volume concentration of 1-2%, and culture at 25-40 DEG C until OD600 reaches 0.6-0.8; then add an inducer IPTG to a final concentration of 0.02-1 mM, and induce for 24 h at 20-37 DEG C and 150-300 r / min; after the culture is completed, centrifuge at 8000 rpm and collect the bacterial bodies, resuspend the bacterial bodies in a buffer, ultrasonically break, centrifuge to collect the supernatant, and obtain crude enzyme liquid PsFFase;

[0022] (3) Dissolve steviol glycoside in water, add the above-mentioned crude enzyme liquid PsFFase and sucrose, and react at pH 6.0 and 20-60 DEG C for 1-10 h; after the reaction is completed, terminate the reaction at high temperature, and centrifuge to obtain the supernatant, which is a fructosylated derivative.

[0023] Preferably, in step (3), the concentration of steviol glycoside in the reaction system is 1-500 mM; the concentration of sucrose is 1-3000 mM; and the addition amount of the crude enzyme is 0.1-10 U / ml.

[0024] Thanks to the above technical solutions, the application has the following beneficial effects:

[0025] The application discloses a fructosidase mutant, which is based on fructosidase PsFFase, and single-point and combined mutations are performed on predicted residues; through comparison of relative activity and thermal stability of the mutant strain, catalytic ability of the fructosidase mutant is investigated, and a fructosidase mutant strain with good catalytic effect and good thermal stability is obtained.

[0026] The application utilizes fructosidase PsFFase to catalyze substrates RebA or Rub, adds appropriate amount of sucrose, and catalytically synthesizes fructosyl steviol glycoside, and the catalytic effect of the mutant system is better than that of the non-mutant system, the conversion rate of RebA reaches 70.56% in 4h, and the concentration of f-RebA is 79.66g / L in 4h of reaction; the conversion rate of Rub reaches 53.49% in 4h, and the concentration of f-Rub is 43.06g / L in 4h of reaction. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained according to the provided drawings without creative labor for those skilled in the art.

[0028] Figure 1 Liquid phase diagram of RebA product catalyzed by fructosidase PsFFase;

[0029] Figure 2 Liquid phase diagram of Rub product catalyzed by fructosidase PsFFase. DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained according to the provided drawings without creative labor for those skilled in the art.

[0031] In order to further understand the present application, the preferred embodiments of the present application will be described below in combination with the embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations on the claims of the present application.

[0032] A fructosidase mutant, which is obtained by mutating any one or more of positions 87, 128, 190, 275, 297, 347, 361, 365, 388, 405, 446, and 477 of fructosidase PsFFase with an amino acid sequence as shown in SEQ ID NO: 1.

[0033] The amino acid sequence of the fructosidase PsFFase is as shown in SEQ ID NO: 1, and the nucleotide sequence is as shown in SEQ ID NO: 4.

[0034] Preferably, the mutation site of the fructosidase mutant is selected from any one or more of positions 347, 365, 405, 477. More preferably, the mutation site is selected from at least one of positions 405, 477.

[0035] Preferably, the amino acid sequence of the fructosidase mutant comprises a mutation of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 1 at A87G, S128P, Q190A, A275G, T297K, Q347V, T361H, T365P, M388L, W405P, N446R, Y477N.

[0036] Preferably, the amino acid at position 365 in the fructosidase PsFFase sequence having the amino acid sequence as shown in SEQ ID NO: 1 is mutated from threonine (T) to proline (P).

[0037] Preferably, the amino acid at position 405 in the fructosidase PsFFase sequence having the amino acid sequence as shown in SEQ ID NO: 1 is mutated from tryptophan (W) to proline (P), and the amino acid sequence of the mutant is as shown in SEQ ID NO: 2.

[0038] Preferably, the amino acid at position 405 in the fructosidase PsFFase sequence having the amino acid sequence as shown in SEQ ID NO: 1 is mutated from tryptophan (W) to proline (P), and the amino acid at position 477 is mutated from tyrosine (Y) to asparagine (N), and the amino acid sequence of the mutant is as shown in SEQ ID NO: 3.

[0039] The detection method used in the following examples is as follows:

[0040] HPLC detection method: Agilent 5TC-C18 column (250 mm x 4.6 mm) was used, UV 210 nm, UltiMate 3000 was used to determine the concentration of fructosyl steviol glycosides and substrates. The flow rate was 1.0 mL / min, and the column temperature was 55°C. The mobile phase was acetonitrile + 0.1% formic acid (A) and water + 0.1% formic acid (B), and the gradient program was as follows: from 0.0 to 15.0 min, the content of mobile phase A increased from 25% to 43%; from 15.0 to 25.0 min, the content of mobile phase A increased to 100%; from 25.0 to 30.0 min, the content of mobile phase A decreased to 25%.

[0041] Example 1

[0042] Construction and induced expression of wild enzyme system, including the following steps:

[0043] (1) Constructing recombinant strain for enzyme expression:

[0044] The gene fragment encoding fructosidase was codon-optimized and cloned into the Nco I and Xho I enzyme sites of pET28a (Novagen) to obtain a recombinant plasmid named pET-PsFFase (the full plasmid was synthesized by Jin Sui Biotech Co., Ltd.); the optimized PsFFase nucleotide sequence is shown in SEQ ID NO: 4;

[0045] The above recombinant plasmid pET-PsFFase was added to E. coli BL21 (DE3) competent cells (TRebAnsGen Biotech), and the recombinant plasmid and the competent cells were mixed and shaken on ice for 30 min, then placed in a 42°C water bath for heat shock for 90 s, ice-bathed for 2 min, placed in a 37°C shaker for activation for 60 min, and finally the recombinant bacterial liquid was spread on an LB solid plate containing 50 mg / L kanamycin resistance (10 g / L proteose peptone, 5 g / L yeast powder, 10 g / L NaCl, and 15 g / L agar) and cultured in a 37°C incubator for 12 h to obtain the recombinant strain PsFFase;

[0046] (2) Inducing enzyme production of the recombinant strain:

[0047] A single colony was selected from the above solid plate and inoculated into 5 mL LB medium (10 g / L proteose peptone, 5 g / L yeast powder, and 10 g / L NaCl) containing 50 mg / L kanamycin and cultured in a 37°C incubator for 12 h. A 1% volume was inoculated into 200 mL LB medium (10 g / L proteose peptone, 5 g / L yeast powder, and 10 g / L NaCl) containing 50 mg / L kanamycin resistance and cultured at 37°C and 200 rpm until the absorbance (OD600) reached 0.6. IPTG was added to the medium to a final concentration of 0.1 mM, and finally incubated at 20°C for 24 h.

[0048] The bacterial liquid PsFFase was collected and centrifuged (4°C, 8000 rpm, 5 min), and the supernatant was discarded to obtain bacterial slurry, which was washed twice with PBS buffer. 5 mL of PBS buffer was added per 1 g of bacterial slurry for resuspension, placed in an ice-water mixture, and the bacterial bodies were broken by ultrasonic disruption with parameters set as Ф6, 300 W for 15 min. The bacterial bodies were centrifuged at 4°C and 8000 rpm for 20 min, and the supernatant was collected as the crude enzyme liquid PsFFase, which was stored in a 4°C refrigerator for use.

[0049] Example 2

[0050] Construction and induced expression of single mutant enzyme system:

[0051] (1) Selection of mutation sites:

[0052] Using the method of homology modeling, the spatial structure of fructosidase PsFFase was simulated, and the potential thermal stability related residues were predicted by combining the consensus sequence, the key sites were 87th, 128th, 190th, 275th, 297th, 347th, 361st, 365th, 388th, 405th, 446th, 477th, the amino acid at the 365th of the amino acid sequence was mutated from threonine (T) to proline (P), the amino acid at the 405th of the amino acid sequence was mutated from tryptophan (W) to proline (P), and the amino acid at the 477th of the amino acid sequence was mutated from tyrosine (Y) to asparagine (N); the simulation mutation was carried out on these amino acid residues, and the following multiple mutants which were beneficial to improve the enzyme activity and stability of PsFFase were obtained to continue the experimental verification.

[0053] The following is the single mutation primer of PsFFase:

[0054] The primer for A87G site-directed mutation is:

[0055] Forward primer: 5'-GAGAATGgcAACCAGTATTCCGTGAACGGC-3',

[0056] Reverse primer: 5'-CTGGTTgcCATTCTCGTCGGTCAGCGG-3';

[0057] The primer for S128P site-directed mutation is:

[0058] Forward primer: 5'-GCGGGTATCccgGCTGACCAACGTCCAGAG-3',

[0059] Reverse primer: 5'-GTCAGCcggGATACCCGCTGGACGGTAGAA-3';

[0060] The primer for Q190A site-directed mutation is:

[0061] Forward primer: 5'-TATCGCGATgcAAACGGCGGTCCGGATATT3',

[0062] Reverse primer: 5'-ACCGCCGTTTgcATCGCGATAAAACGCAAC-3';

[0063] The primer for A275G site-directed mutation is:

[0064] Forward primer: 5'-ATGGATCGTGgcACGGCTCAGTGTGATGCG-3', reverse primer: 5'- CTGAGCCGTgcCACGATCCATCGCGGTGTT-3'; the primer for site-directed mutation of T297K is:

[0065] Forward primer: 5'-GAGACAGTGAaaCAAGTTAACGCCAGCGGC-3', reverse primer: 5'- GTTAACTTGttTCACTGTCTCCGCGTACGG-3'; the primer for site-directed mutation of Q347V is:

[0066] Forward primer: 5'-CAGATTTATgtGCAGGATGGTAAATATTACCTGT-3', reverse primer: 5'- ACCATCCTGCacATAAATCTGCGGACG-3';

[0067] The primer for site-directed mutation of T361H is:

[0068] Forward primer: 5'-AGCCACAGCcatACCTACGCGACGGGCATC-3', reverse primer: 5'- CGCGTAGGTatgGCTGTGGCTAATCGTGAA-3'; the primer for site-directed mutation of T365P is:

[0069] Forward primer: 5'-ACCTACGCGcCGGGCATCACCGGTCCGGAA-3', reverse primer: 5'- GGTGATGCCCGgCGCGTAGGTGGTGCTGTG-3'; the primer for site-directed mutation of M388L is:

[0070] Forward primer: 5'-TACCAACCGcTGAATCAAGGTAGTGGCTTA-3', reverse primer: 5'- ACCTTGATTCAgCGGTTGGTAATCGGAACG-3'; the primer for site-directed mutation of W405P is:

[0071] Forward primer: 5'-TTGAATTATccGCCGGGTTCGCCGTTCGCC-3', reverse primer: 5'- CGAACCCGGCggATAATTCAAATTCGTCGG-3'; the primer for site-directed mutation of N446R is:

[0072] Forward primer: 5'-ACCAAAGAAcgcTTCCGCCGTGGTGGTACT-3', reverse primer: 5'- ACGGCGGAAgcgTTCTTTGGTGCCAATGGT-3'; the primer for site-directed mutation of Y477N is:

[0073] Forward primer: 5'-GTCAACGGTaatGGGTTGGGAGGTTGGGCA-3', Reverse primer: 5'-TCCCAACCCattACCGTTGACGCCGTAGGA-3';

[0074] (2) Construction of recombinant strains expressing single mutant enzymes:

[0075] PCR amplification was performed using the recombinant plasmid pET-PsFFase as the template and the above single mutation primers and PrimeSTAR DNA polymerase (TaKaRA) (50 μL amplification reaction system: 1 μL template plasmid pET-PsFFase, 1 μL single point mutation forward primer, 1 μL single point mutation reverse primer, 22 μL ddH2O, 25 μL PrimeSTAR DNA polymerase); after agarose gel electrophoresis verification, 1 μL of Dpn I restriction enzyme (TaKaRA) was added to the PCR reaction product and incubated at 37°C for 30 min to digest the template, and then the digested PCR reaction product was transformed into E. coli BL21 (DE3) according to the method in Example 1, and finally a single colony on the plate was picked and inoculated into LB medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin, and cultured at 37°C, 200 rpm for 12 h, then the plasmid was extracted and sent for identification, and the recombinant plasmids pET-PsFFase-A87G, pET-PsFFase-S128P, pET-PsFFase-Q190A, pET-PsFFase-A275G, pET-PsFFase-T297K, pET-PsFFase-Q347V, pET-PsFFase-T361H, pET-PsFFase-T365P, pET-PsFFase-M388L, pET-PsFFase-W405P, pET-PsFFase-N446R, pET-PsFFase-Y477N were obtained.

[0076] (3) Induced expression of single mutant enzymes:

[0077] The above identified recombinant plasmids were transformed into E. coli BL21 (DE3) according to the method in Example 1. The next day, single colonies were picked from the solid plate into 5 mL LB medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin and incubated in a 37 °C incubator for 12 h, then inoculated into 200 mL LB medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin resistance at 1% volume, and incubated at 37 °C, 200 rpm until the absorbance (OD600) reached 0.6, then IPTG was added to the medium to a final concentration of 0.1 mM, and finally incubated at 20 °C for 24 h;

[0078] The fermentation broth was collected and centrifuged at 4 °C, 8000 rpm for 5 min, and the supernatant was discarded to obtain the bacterial slurry, which was washed with PBS buffer. 5 mL of PBS buffer was added per 1 g of bacterial slurry, resuspended in an ice-water mixture, and the bacterial cells were broken by ultrasonic disruption with a parameter setting of Ф6, 300 W for 15 min. Then the bacterial cells were centrifuged at 4 °C, 8000 rpm for 20 min, and the supernatant was collected as the crude enzyme solution of the mutant, which was stored in a 4 °C refrigerator for later use.

[0079] Example 3

[0080] Construction and induced expression of multi-mutant enzyme system

[0081] (1) Selection of mutation sites

[0082] Using the method of homology modeling, the spatial structure of fructosidase PsFFase was simulated, and the potential thermal stability related residues were predicted by combining the consensus sequence, the key sites were the 365th, 405th and 477th amino acids, the 365th amino acid in the amino acid sequence was mutated from threonine (T) to proline (P), the 405th amino acid in the amino acid sequence was mutated from tryptophan (W) to proline (P), and the 477th amino acid in the amino acid sequence was mutated from tyrosine (Y) to asparagine (N); these amino acid residues were subjected to simulated combinatorial mutation to obtain the following multiple mutants that are beneficial to improve the enzyme activity and stability of PsFFase for further experimental verification.

[0083] The primers for Q347V / W405P mutation are as follows:

[0084] Forward primer: 5'-CAGATTTATgtGCAGGATGGTAAATATTACCTGT-3',

[0085] Reverse primer: 5'-ACCATCCTGCacATAAATCTGCGGACG-3';

[0086] Mutant primers for T365P / W405P are as follows:

[0087] Forward primer: 5'-ACCTACGCGcCGGGCATCACCGGTCCGGAA-3',

[0088] Reverse primer: 5'-GGTGATGCCCGgCGCGTAGGTGGTGCTGTG-3';

[0089] Mutant primers for W405P / Y477N are as follows:

[0090] Forward primer: 5'-GTCAACGGTaatGGGTTGGGAGGTTGGGCA-3'

[0091] Reverse primer: 5'-TCCCAACCCattACCGTTGACGCCGTAGGA-3';

[0092] Mutant primers for T365P / W405P / Y477N are as follows:

[0093] Forward primer: 5'-ACCTACGCGcCGGGCATCACCGGTCCGGAA-3',

[0094] Reverse primer: 5'-GGTGATGCCCGgCGCGTAGGTGGTGCTGTG-3';

[0095] (2) Construction of recombinant strains for expressing multi-mutant enzymes:

[0096] Take the preparation of recombinant plasmid pET-PsFFase-W405P-Y477N as an example.

[0097] The mutation method in step (2) of Example 2 was used to perform combined mutation of Q347V, T365P or Y477N, and the final test identification. The specific steps include the following: using the recombinant plasmid pET-PsFFase-W405P as a template, using the above-mentioned W405P / Y477N mutation primer and PrimeSTAR DNA polymerase (TaKaRA) to perform PCR amplification (50 μL reaction system: 1 μL template plasmid pET-PsFFase-W405P, 1 μL single-point mutation forward primer, 1 μL single-point mutation reverse primer, 22 μL of ddH2O, 25 μL of PrimeSTAR DNA polymerase); after verification by agarose gel electrophoresis, 1 μL of Dpn I restriction enzyme (TaKaRA) was added to the PCR reaction product and incubated at 37°C for 30 min to digest the template, and then the digested PCR reaction product was transformed into E. coli BL21 (DE3) according to the method in Example 1, and finally a single colony on the plate was picked and inoculated into LB medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin, and incubated at 37°C, 200 rpm for 12 h. The plasmid was extracted and sent for identification, and the recombinant plasmid pET-PsFFase-W405P-Y477N was obtained. The PsFFase mutant W405P-Y477N nucleotide sequence is shown in SEQ ID NO: 5;

[0098] (3) Induced expression of multi-mutant enzyme:

[0099] The above-identified recombinant plasmid pET-PsFFase-W405P-Y477N was transformed into E. coli BL21 (DE3) according to the method in Example 1. The next day, a single colony was selected from the solid plate and inoculated into 5 mL LB medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin, and incubated at 37°C for 12 h. Then 1% of the volume was inoculated into 200 mL LB medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin resistance, and incubated at 37°C, 200 rpm until the absorbance (OD600) reached 0.6. IPTG was added to the culture medium to a final concentration of 0.1 mM, and finally incubated at 20°C for 24 h;

[0100] The fermentation broth was collected and centrifuged (4°C, 8000 rpm, 5 min) and the supernatant was discarded to obtain the bacterial slurry, which was washed twice with PBS buffer. 5 mL of PBS buffer was added to 1 g of the bacterial slurry, which was resuspended in an ice-water mixture and the bacterial cells were broken by ultrasonic disruption with a parameter setting of Ф6, 300 W for 15 min. The bacterial cells were then centrifuged at 4°C and 8000 rpm for 20 min, and the supernatant was obtained as the crude enzyme solution PsFFase-W405P-Y477N, which was stored in a refrigerator at 4°C for later use.

[0101] Test Example:

[0102] I. Wild-type and single-point mutant enzyme activity and thermal stability testing:

[0103] The enzyme activity was defined as 1 unit (U) of enzyme activity being the amount of enzyme required to convert 1 μmol of sucrose to f-RebA in 1 min.

[0104] Enzyme activity determination: 10 mM RebA, 30 mM sucrose and 10 μg / mL of the crude enzyme of Example 1 or Example 2 were added to a 1 mL assay system, and the remaining volume was made up with PBS buffer (pH 6.5), which was then incubated at 30°C and 200 rpm for 30 min. Finally, the reaction solution was inactivated in a 95°C water bath for 20 min. The inactivated sample was diluted ten-fold and then subjected to HPLC detection.

[0105] Enzyme thermal stability determination: the crude enzyme was placed in PBS (pH 6.5) buffer to a final concentration of 200 μg / mL. After incubation at 30°C for 1 h, the sample was measured for enzyme activity under the above experimental conditions. All operations were set up in triplicate.

[0106] The relative enzyme activity of the wild-type and mutants at different incubation times was calculated based on the activity of the wild-type PsFFase without incubation being 100%. The test results are shown in Table 1.

[0107] Table 1

[0108]

[0109]

[0110] As can be seen from the test results in Table 1, for the initial relative enzyme activity, the enzyme activity of the single mutants Q190A, Q347V, T365P, W405P, N446R and Y477N is obviously higher than that of the wild-type PsFFase, wherein the enzyme activity of the mutant W405P is 1.51 times that of the wild-type PsFFase. The enzyme activity of the mutants A87G, A275G, T297K, T361H and M388L is obviously reduced, and the enzyme activity of the mutant S128P is basically unchanged.

[0111] For the relative enzyme activity after 1h incubation, the mutants Q347V, T365P, W405P and Y477N are obviously higher than the wild-type PsFFase, i.e., better stability, wherein the mutant W405P has the highest thermal stability.

[0112] In summary, in combination with the test results of enzyme activity and thermal stability, it can be seen that the mutants Q347V, T365P, W405P and Y477N are positive mutation points for improving the performance of PsFFase.

[0113] II. Stability determination of wild-type and multi-point mutant:

[0114] The enzyme activity is defined as: the amount of enzyme required to convert 1 μmoL of f-RebA in 1 minute is 1 enzyme unit (U).

[0115] PsFFase and its multi-point mutant transglycosylase activity determination: 10 mM RebA, 30 mM sucrose and 10 μg / mL of crude enzyme were added to 1 mL of the determination system, and the remaining volume was supplemented with PBS buffer (pH 6.5), and the reaction was carried out at 30°C, 200 rpm for 30 min. Finally, the sample was placed in a 95°C water bath for inactivation for 20 min. The inactivated sample was diluted ten times for HPLC detection.

[0116] Thermal stability determination of PsFFase and its multi-point mutant under enzyme conversion conditions: the pure enzyme was placed in PBS (pH 6.5) buffer, and the final concentration was 200 μg / mL. After incubation at 40°C for 1h, the sample was measured for enzyme activity under the above experimental conditions. All operations were set up in triplicate experiments. The relative enzyme activity of the wild-type and mutants at different times during the incubation period was calculated based on the activity of the wild-type PsFFase without incubation as 100%. The test results are shown in Table 2.

[0117] Table 2

[0118]

[0119] From the test results in Table 2, it can be seen that the initial enzyme activity, 1h relative enzyme activity and residual enzyme activity retention rate of the multi-point mutant are all better than those of the wild type, wherein the 1h relative enzyme activity of the combined mutant W405P / Y477N is 145.01%, which is 9.33 times that of the wild type PsFFase.

[0120] Catalytic activity test:

[0121] wherein the conversion rate (%) = (initial amount of substrate - residual amount of substrate) / initial amount of substrate x 100%.

[0122] Application Example One

[0123] Catalytic synthesis of fructosyl steviol glycoside:

[0124] In a 5 mL catalytic reaction system, 100 mM RebA was added, 300 mM sucrose was added, 1 U / mL PsFFase-W405P prepared in Example 2 was added, and 50 mM PBS buffer solution (pH 6.0) was added, and incubated at 40°C for 4h. After the reaction was completed, the reaction solution was warmed and inactivated, diluted with pure water, and then detected by HPLC. The conversion rate of RebA was 61.23%, and the yield of f-RebA was 69.13 g / L.

[0125] Application Example Two

[0126] In a catalytic reaction system (5 mL), 100 mM RebA was added, 300 mM sucrose was added, 1 U / mL PsFFase-W405P-Y477N prepared in Example 3 was added, and 100 mM PBS buffer solution (pH 6.0) was added, and reacted at 40°C for 4h. After high-temperature inactivation, the supernatant obtained by centrifugation was f-RebA. Finally, at the reaction time of 4h, the conversion rate of RebA was 70.56%, and the yield of f-RebA was 79.66 g / L.

[0127] Application Example Three

[0128] Compared with Application Example Two, the difference is that an equal amount of Rub is used instead of RebA, and the other conditions are the same as those in Application Example Five. Finally, at the reaction time of 4h, the conversion rate of Rub was 53.49%, and the yield of f-Rub was 43.06 g / L.

[0129] Application Comparative Example One

[0130] Catalytic synthesis of fructosyl steviol glycoside:

[0131] 100 mM RebA, 300 mM sucrose, 1 U / mL of the crude enzyme PsFFase prepared in Example 1, and 50 mM PBS buffer solution (pH 6.0) were added to a 5 mL catalytic reaction system. The mixture was incubated at 40 °C for 4 h. After the reaction was completed, the reaction solution was heated to inactivate the enzyme, diluted with pure water, and detected by HPLC. (The supernatant obtained by centrifugation is the fructose-based derivative). The conversion rate of RebA was 36.27%, and the yield of f-RebA was 40.95 g / L.

[0132] Application Comparative Example 2

[0133] Compared with Application Example 1, the difference is that an equal amount of Rub was used to replace RebA, and other conditions were the same as in Application Example 2. The conversion rate of Rub was 33.54%, and the f-Rub yield was 26.99 g / L.

[0134] The liquid phase diagram of the catalytic reaction of PsFFase is as follows: Figure 1 As shown, from Figure 1 It can be seen that the retention time of the catalytic product f-RebA is around 8.80 min; the retention time of the catalytic product f-Rub is around 11.19 min.

[0135] In summary, the fructosylase mutant prepared by this invention exhibits high catalytic activity in the synthesis of rebaudioside A and stevia, significantly improving the yield of the target products.

[0136] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely to aid in understanding the method and core ideas of the present invention, including the best mode, and to enable any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements similar to those expressed in the claims, or if they include equivalent structural elements that are not substantially different from those expressed in the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A fructosylase mutant, characterized in that: The mutant is formed by any one of the following mutations in the fructosylase PsFFase, as shown in the amino acid sequence of SEQ ID NO:1: Q347V, T365P, W405P, Y477N, Q347V / W405P, T365P / W405P, W405P / Y477N, or T365P / W405P / Y477N.

2. An expressed gene, characterized in that: This gene encodes the fructosylase mutant as described in claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid is linked to the expression gene as described in claim 2.

4. A recombinant bacterium, characterized in that: The recombinant bacteria contains the expression gene as described in claim 2 and / or the recombinant plasmid as described in claim 3; the recombinant bacteria also includes an empty vector bacteria, wherein the empty vector bacteria is Escherichia coli.

5. The application of the fructosylase mutant as described in claim 1, characterized in that: The method for catalyzing the synthesis of fructosylsteviosides specifically includes the following steps: (1) Constructing a recombinant strain for enzyme expression: The gene of the fructosylase mutant was constructed into a plasmid to obtain a recombinant plasmid; the recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain containing an enzyme expression system; (2) Induction of enzyme production by recombinant strains: The recombinant strain PsFFase was activated and transferred to LB induction medium. It was cultured at 25-40℃ until the OD600 reached 0.6-0.

8. Then, IPTG was added as an inducer for induction culture. After the culture was completed, the cells were collected by centrifugation at 8000 rpm. The cells were resuspended in buffer, sonicated, and the supernatant was collected by centrifugation to obtain crude enzyme solution. (3) Dissolve steviol glycosides in water, add the above crude enzyme solution and sucrose, react, terminate the reaction at high temperature after the reaction is completed, centrifuge to obtain the supernatant, which is the fructose-based derivative.

6. The method for synthesizing fructosylsteviosides catalyzed by the fructosylase mutant according to claim 5, characterized in that: In step (2), the final concentration of the inducer IPTG is 0.02-1mM, and the induction culture conditions are: temperature 20-37℃, rotation speed 150-300r / min, and induction time 24h. Or in step (3), the pH of the reaction is 6.0, the temperature is 20-60℃, and the time is 1-10h; In step (3), the concentration of steviol glycosides in the reaction system is 1-500 mM; the concentration of sucrose is 1-3000 mM; and the amount of crude enzyme added is 0.1-10 U / ml.

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