Fructosidase mutant, preparation method and application

Abstract

The invention discloses a fructosidase mutant. The mutant is obtained by carrying out mutation on any one or more sites of the 347 site, the 365 site, the 405 site and the 477 site of fructosidase PsFFase with the amino acid sequence as shown in SEQ ID NO: 1. The invention also discloses a preparation method of the fructosidase mutant and application of the fructosidase mutant in catalytic synthesis of fructosyl rebaudioside A and fructosyl rubusoside. According to the invention, the PsFFase mutant is utilized to realize glycosylation of RebA and Rub to prepare f-RebA and f-Rub, and the PsFFase mutant is high in catalytic activity and good in thermal stability.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a fructosidase mutant, a preparation method and an application thereof. Background Art

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

[0003] SGs belong to the diterpenoid group of plant secondary metabolites. Their chemical structures are all centered on a glycoside called steviol, which is composed of different numbers and types of sugar groups connected at the C19 and C13 positions of the structure. Relevant studies have been conducted on the relationship between these different types and numbers of sugar groups and the sensory properties and physicochemical properties of SGs. It was 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 part is an efficient method.

[0004] By screening fructosidases in the NCBI database, it was found that a fructosidase (PsFFase) from Pseudoalteromonas can 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 generally loses its enzyme activity significantly after catalysis at 40°C for 1 hour, which seriously limits the efficient application of this fructosidase. Summary of the invention

[0005] The technical problem to be solved by the present invention is: in view of the shortcomings of the prior art, a fructosidase mutant, a preparation method and an application are provided. The present invention utilizes the PsFFase mutant to realize the glycosylation of RebA and Rub to prepare f-RebA and f-Rub. The PsFFase mutant has high catalytic activity and good thermal stability.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A fructosidase mutant is obtained by mutating any one or more of the 87th, 128th, 190th, 275th, 297th, 347th, 361st, 365th, 388th, 405th, 446th and 477th positions of the fructosidase PsFFase as shown in the amino acid sequence of 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, and 477. More preferably, the mutation site is selected from at least one of positions 405 and 477.

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

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

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

[0012] Preferably, the W405P / Y477N mutant is a mutant in which the amino acid at position 405 in the fructosidase PsFFase 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 shown in SEQ ID NO:3.

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

[0014] A recombinant plasmid is connected with the above-mentioned expression gene.

[0015] A recombinant cell, characterized in that: the recombinant cell contains the above-mentioned expression gene and / or the above-mentioned recombinant plasmid, and is an empty vector bacterium.

[0016] Furthermore, the empty vector bacteria is preferably Escherichia coli BL21 (DE3) strain.

[0017] The invention discloses an application of a fructosidase mutant for catalyzing the synthesis of fructosyl steviol glycoside.

[0018] A method for synthesizing fructosyl steviol glycoside by catalysis of a fructosidase mutant comprises the following steps:

[0019] (1) Construction of recombinant strains expressing enzymes:

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

[0021] (2) Induced enzyme production of recombinant strains: The recombinant strain PsFFase was activated and transferred to LB induction medium at a volume concentration of 1-2%, and cultured at 25-40°C until OD600 reached 0.6-0.8, and then the inducer IPTG was added to a final concentration of 0.02-1 mM, and induced at 20-37°C, 150-300 r / min for 24 h. After the culture was completed, the cells were centrifuged at 8000 rpm and collected, and the cells were resuspended in a buffer solution, ultrasonically disrupted, and the supernatant was collected by centrifugation to obtain a crude enzyme solution PsFFase;

[0022] (3) Dissolve stevioside in water, add the crude enzyme solution PsFFase and sucrose, react at pH 6.0 and 20-60° C. for 1-10 h, terminate the reaction at high temperature after the reaction is completed, and centrifuge to obtain the supernatant, which is the 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 amount of crude enzyme added is 0.1-10 U / ml.

[0024] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] The invention discloses a fructosidase enzyme mutant. Based on fructosidase PsFFase, single-point and combined mutations are performed on predicted residues. By comparing the relative activity and thermal stability of the mutant strains, the catalytic ability of the fructosidase mutant is investigated, and a fructosidase mutant strain with good catalytic effect and good thermal stability is obtained. The mutant is easy to prepare. Under the same conditions, the mutant maintains enzyme activity within 6 hours, while the wild type loses enzyme activity seriously after 1 hour.

[0026] The present invention utilizes fructosidase PsFFase to catalyze substrate RebA or Rub, adds an appropriate amount of sucrose, and catalyzes the synthesis of fructosyl steviol glycosides. The catalytic effect of the mutant system is better than that of the non-mutated system. The conversion rate of RebA reaches 70.56% in 4 hours, and the concentration of f-RebA is 79.66 g / L in 4 hours of reaction; the conversion rate of Rub reaches 53.49% in 4 hours, and the concentration of f-Rub is 43.06 g / L in 4 hours of reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[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 understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0031] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

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

[0033] The amino acid sequence of the fructosidase PsFFase is shown in SEQ ID NO:1, and the nucleotide sequence is 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, and 477. More preferably, the mutation site is selected from at least one of positions 405 and 477.

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

[0036] Preferably, the amino acid at position 365 in the fructosidase PsFFase 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 shown in SEQ ID NO:1 is mutated from tryptophan (W) to proline (P), and the amino acid sequence of the mutant is shown in SEQ ID NO:2.

[0038] Preferably, the amino acid at position 405 in the fructosidase PsFFase 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 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 (250mm×4.6mm), UV 210nm, UltiMate3000 was used to determine the concentration of fructosyl stevioside and substrate. The flow rate was 1.0mL / min, and the column temperature was 55℃. The mobile phase was acetonitrile + 0.1% formic acid (A) and water + 0.1% formic acid (B). The gradient program was that the content of mobile phase A increased from 25% to 43% at 0.0-15.0min; at 15.0-25.0min, the content of mobile phase A increased to 100%; at 25.0-30.0min, the content of mobile phase A decreased to 25%.

[0041] Example 1

[0042] The construction and induction expression of the wild enzyme system includes the following steps:

[0043] (1) Construction of recombinant strains expressing enzymes:

[0044] The gene fragment encoding fructosidase was codon optimized and cloned between the NcoⅠ and XhoⅠ restriction sites of pET28a (Novagen). The resulting recombinant plasmid was named pET-PsFFase (the whole plasmid was synthesized by GenScript Biotech Co., Ltd. in this step); 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 competent cells were mixed by flicking, and then placed on ice for 30 minutes, and then placed in a 42°C water bath for heat shock for 90 seconds, and placed on ice for 2 minutes, and activated in a 37°C shaker for 60 minutes. Finally, the recombinant bacterial solution was spread on an LB solid plate (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing 50 mg / L kanamycin resistance, and cultured in a 37°C incubator for 12 hours to obtain the recombinant strain PsFFase;

[0046] (2) Induced enzyme production of recombinant strains:

[0047] Select a single colony from the solid plate and place it in 5 mL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 mg / L kanamycin, and place it in a 37°C incubator for 12 hours. Inoculate 1% by volume into 200 mL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 mg / L kanamycin resistance and culture at 37°C and 200 rpm until the absorbance (OD600) reaches 0.6, add IPTG to the medium to a final concentration of 0.1 mM, and finally incubate at 20°C for 24 hours;

[0048] Collect the bacterial liquid PsFFase and refrigerate centrifuge (4°C, 8000rpm, 5min), discard the supernatant to obtain the bacterial mud, rinse twice with PBS buffer, add 5mL PBS buffer for every 1g of bacterial mud to resuspend, place in an ice-water mixture, use an ultrasonic disruptor to ultrasonically disrupt the bacteria with the parameters set to Ф6, 300W, 15min, use a refrigerated centrifuge with the parameters set to 4°C, 8000rpm, 20min, take the supernatant as the crude enzyme liquid PsFFase, and store it in a 4°C refrigerator for use.

[0049] Example 2

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

[0051] (1) Selection of mutation sites:

[0052] The spatial structure of fructosidase PsFFase was simulated by homology modeling, and the potential thermal stability-related residues were predicted in combination with the consensus sequence, including key positions 87, 128, 190, 275, 297, 347, 361, 365, 388, 405, 446, and 477. The amino acid at position 365 of the amino acid sequence was mutated from threonine (T) to proline (P), the amino acid at position 405 of the amino acid sequence was mutated from tryptophan (W) to proline (P), and the amino acid at position 477 of the amino acid sequence was mutated from tyrosine (Y) to asparagine (N). These amino acid residues were simulated and mutated to obtain the following mutants that are beneficial to improving the enzymatic activity and stability of PsFFase, so as to continue experimental verification.

[0053] The following are single mutation primers for PsFFase:

[0054] The primers for site-directed mutagenesis of A87G are:

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

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

[0057] The primers for S128P site-directed mutagenesis are:

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

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

[0060] The primers for site-directed mutagenesis of Q190A are:

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

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

[0063] The primers for site-directed mutagenesis of A275G are:

[0064] Forward primer: 5'-ATGGATCGTGgcACGGCTCAGTGTGATGCG-3', reverse primer: 5'-CTGAGCCGTgcCACGATCCATCGCGGTGTT-3'; primers for T297K site-directed mutagenesis are:

[0065] Forward primer: 5'-GAGACAGTGAaaCAAGTTAACGCCAGCGGC-3', reverse primer: 5'-GTTAACTTGttTCACTGTCTCCGCGTACGG-3'; primer for Q347V site-directed mutagenesis:

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

[0067] The primers for T361H site-directed mutagenesis are:

[0068] Forward primer: 5'-AGCCACAGCcatACCTACGCGACGGGCATC-3', reverse primer: 5'-CGCGTAGGTatgGCTGTGGCTAATCGTGAA-3'; primer for T365P site-directed mutagenesis:

[0069] Forward primer: 5'-ACCTACGCGcCGGGCATCACCGGTCCGGAA-3', reverse primer: 5'-GGTGATGCCCGgCGCGTAGGTGGTGCTGTG-3'; primers for site-directed mutagenesis of M388L are:

[0070] Forward primer: 5'-TACCAACCGcTGAATCAAGGTAGTGGCTTA-3', reverse primer: 5'-ACCTTGATTCAgCGGTTGGTAATCGGAACG-3'; primers for W405P site-directed mutagenesis are:

[0071] Forward primer: 5'-TTGAATTATccGCCGGGTTCGCCGTTCGCC-3', reverse primer: 5'-CGAACCCGGCggATAATTCAAATTCGTCGG-3'; primers for N446R site-directed mutagenesis are:

[0072] Forward primer: 5'-ACCAAAGAAcgcTTCCGCCGTGGTGGTACT-3', reverse primer: 5'-ACGGCGGAAgcgTTCTTTGGTGCCAATGGT-3'; primers for Y477N site-directed mutagenesis are:

[0073] Forward primer: 5′-GTCAACGGTaatGGGTTGGGAGGTTGGGCA-3′, reverse primer: 5′-TCCCAACCCattACCGTTGACGCCGTAGGA-3′;

[0074] (2) Construction of a recombinant strain expressing a single mutant enzyme:

[0075] Using the recombinant plasmid pET-PsFFase as a template, the above-mentioned single mutation primers and PrimeSTAR DNA polymerase (TaKaRA) were used for PCR amplification (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 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, 5 g / L yeast powder, 10 g / L NaCl), cultured at 37 ° C, 200 rpm for 12 h, then extracted the plasmid, sent for identification, and obtained 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, and pET-PsFFase-Y477N;

[0076] (3) Inducible expression of single mutant enzyme:

[0077] The identified recombinant plasmids were transformed into E. coli BL21 (DE3) according to the method in Example 1. The next day, single colonies were selected from the solid plate and placed in 5 mL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 mg / L kanamycin, placed in a 37°C incubator for 12 h, and then inoculated with 1% volume into 200 mL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 mg / L kanamycin resistance, cultured at 37°C and 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;

[0078] The fermentation liquid was collected and centrifuged at 4°C, 8000rpm, 5min, and the supernatant was discarded to obtain the bacterial sludge, which was then rinsed with PBS buffer. 5mL of PBS buffer was added to each 1g of bacterial sludge to resuspend it, and it was placed in an ice-water mixture. The bacterial cells were ultrasonically disrupted with an ultrasonic disruptor, and the parameters were set to Ф6, 300W, and 15min. Then, the supernatant was taken as the mutant crude enzyme solution, and it was stored in a 4°C refrigerator for later use.

[0079] Example 3

[0080] Construction and inducible expression of multiple mutant enzyme system:

[0081] (1) Selection of mutation sites:

[0082] The spatial structure of fructosidase PsFFase was simulated by homology modeling, and the potential thermal stability-related residues were predicted in combination with the consensus sequence. At the key sites 365, 405, and 477, the amino acid at position 365 of the amino acid sequence was mutated from threonine (T) to proline (P), the amino acid at position 405 of the amino acid sequence was mutated from tryptophan (W) to proline (P), and the amino acid at position 477 of the amino acid sequence was mutated from tyrosine (Y) to asparagine (N); these amino acid residues were simulated and combined for mutation, and the following mutants that were beneficial to improving the enzymatic activity and stability of PsFFase were obtained for further experimental verification.

[0083] Primers for the Q347V / W405P mutation are as follows:

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

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

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

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

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

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

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

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

[0092] The mutation 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 expressing multiple mutant enzymes:

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

[0097] The combined mutation of Q347V, T365P or Y477N was performed using the mutation method in step (2) of Example 2, and finally sent for testing and identification. Specifically, the following steps were included: using the recombinant plasmid pET-PsFFase-W405P as a template, using the above-mentioned W405P / Y477N mutation primers and PrimeSTAR DNA polymerase (TaKaRA) for 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 ddH2O, 25 μL PrimeSTAR DNA polymerase); after verification by agarose gel electrophoresis, adding 1 μL of Dpn I restriction enzyme (TaKaRA) to the PCR reaction product and incubating at 37°C for 30 minutes to digest the template, and then transforming the digested PCR reaction product into E. coli according to the method in Example 1. BL21 (DE3), finally pick a single colony on the plate, inoculate it into LB medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl) containing 50mg / L kanamycin, culture at 37°C, 200rpm for 12h, extract the plasmid, send it for testing and identification, and obtain the recombinant plasmid pET-PsFFase-W405P-Y477N. The nucleotide sequence of the PsFFase mutant W405P-Y477N is shown in SEQ ID NO: 5;

[0098] (3) Inducible expression of multiple mutant enzymes:

[0099] The 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 placed in 5 mL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 mg / L kanamycin, and cultured in a 37°C incubator for 12 h, and then inoculated with 1% volume into 200 mL LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 mg / L kanamycin resistance at 37°C and 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] Collect the fermentation liquid and centrifuge it at 4°C, 8000rpm, 5min, discard the supernatant to get the bacterial sludge, and rinse it twice with PBS buffer. Add 5mL of PBS buffer to each 1g of bacterial sludge and resuspend it. Place it in an ice-water mixture and use an ultrasonic disruptor to ultrasonically disrupt the bacteria. The parameters are set to Ф6, 300W, and 15min. Centrifuge it again with a refrigerated centrifuge at 4°C, 8000rpm, and 20min. Take the supernatant as the crude enzyme solution PsFFase-W405P-Y477N and store it in a 4°C refrigerator for later use.

[0101] Test example:

[0102] 1. Wild-type and single-point mutant enzyme activity and thermal stability test:

[0103] The enzyme activity was defined as: the amount of enzyme required to convert sucrose to produce 1 μmol f-RebA within 1 minute was 1 enzyme activity unit (U).

[0104] Enzyme activity assay: 10 mM RebA, 30 mM sucrose and 10 μg / mL crude enzyme in Example 1 or Example 2 were added to 1 mL of the assay system, and the remaining volume was made up with PBS buffer (pH 6.5), and the reaction was carried out 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 times and then subjected to HPLC detection.

[0105] Enzyme thermal stability assay: The crude enzyme was placed in PBS (pH 6.5) buffer at a final concentration of 200 μg / mL. After incubation at 30°C for 1 h, samples were taken to measure enzyme activity under the above experimental conditions. Three parallel experiments were set for all operations.

[0106] The wild-type PsFFase activity without incubation was taken as 100%, and the relative enzyme activities of the wild-type and mutant at different times during the incubation period were calculated. The test results are shown in Table 1.

[0107] Table 1

[0108]

[0109]

[0110] From the test results in Table 1, it can be seen that for the initial relative enzyme activity, the enzyme activities of the single mutants Q190A, Q347V, T365P, W405P, N446R and Y477N were significantly higher than that of the wild-type PsFFase, among which the enzyme activity of the mutant W405P was 1.51 times that of the wild-type PsFFase. The enzyme activities of the mutants A87G, A275G, T297K, T361H and M388L were significantly reduced, while the enzyme activity of the mutant S128P remained basically unchanged.

[0111] For the relative enzyme activity after incubation for 1 h, the mutants Q347V, T365P, W405P and Y477N were significantly higher than the wild-type PsFFase, that is, they had better stability, among which the mutant W405P had the highest thermal stability.

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

[0113] 2. Stability determination of wild type and multiple point mutants:

[0114] The enzyme activity was defined as: the amount of enzyme required to convert sucrose to produce 1 μmol f-RebA within 1 minute was 1 enzyme activity unit (U).

[0115] PsFFase and its multi-point mutant transglycosylase activity assay: 10mM RebA, 30mM sucrose and 10μg / mL crude enzyme were added to 1mL of the assay system, and the remaining volume was made up with PBS buffer (pH6.5), and the reaction was carried out at 30℃ and 200rpm for 30min. Finally, the sample was inactivated in a 95℃ water bath for 20min. The inactivated sample was diluted ten times for HPLC detection.

[0116] Determination of thermal stability of PsFFase and its multi-point mutants under enzyme conversion conditions: The pure enzyme was placed in PBS (pH 6.5) buffer at a final concentration of 200 μg / mL. After incubation at a temperature range of 40°C for 1 hour, samples were taken to measure the enzyme activity under the above experimental conditions. Three parallel experiments were set up for all operations. The relative enzyme activity of the wild type and mutants at different times during the incubation stage was calculated, taking the unincubated wild type PsFFase activity 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 mutants are better than those of the wild type. Among them, 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, conversion rate (%) = (initial amount of substrate - residual amount of substrate) / initial amount of substrate × 100%.

[0122] Application Example 1

[0123] Catalytic synthesis of fructosyl steviol glycosides:

[0124] 100 mM RebA was added to 5 mL of the catalytic reaction system, and 300 mM sucrose, 1 U / mL PsFFase-W405P prepared in Example 2 and 50 mM PBS buffer solution (pH 6.0) were added, and incubated at 40° C. for 4 h. After the reaction was completed, the reaction solution was heated and inactivated, and 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 2

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

[0127] Application Example 3

[0128] Compared with Application Example 2, the difference is that an equal amount of Rub is used to replace RebA, and other conditions are the same as those of Application Example 5. Finally, after 4 hours of reaction, the conversion rate of Rub is 53.49%, and the yield of f-Rub is 43.06 g / L.

[0129] Application Comparative Example 1

[0130] Catalytic synthesis of fructosyl steviol glycosides:

[0131] 100 mM RebA was added to 5 mL of the catalytic reaction system, and 300 mM sucrose, 1 U / mL crude enzyme PsFFase prepared in Example 1 and 50 mM PBS buffer solution (pH 6.0) were added, and incubated at 40°C for 4 h. After the reaction was completed, the reaction solution was heated and inactivated, diluted with pure water, and then detected by HPLC. (The supernatant obtained by centrifugation is the fructosylated 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 is used to replace RebA, and other conditions are the same as those of Application Example 2. The conversion rate of Rub is 33.54%, and the f-Rub yield is 26.99 g / L.

[0134] The liquid phase diagram of the catalytic reaction of PsFFase is shown in 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 fructosidase mutant prepared in the present invention has high catalytic activity when catalyzing the synthesis of rebaudioside A and rubusoside, thereby greatly improving the yield of the target product.

[0136] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas, including the best mode, and also enable any technician in the field to practice the present invention, including making and using any device or system, and implementing any combined method. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the literal expression of the claims, or if they include equivalent structural elements that are not substantially different from the literal expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A fructosidase mutant, characterized in that: The mutant is obtained by mutating any one or more of the 347th, 365th, 405th and 477th positions of the fructosidase PsFFase in the amino acid sequence as shown in SEQ ID NO:

1.

2. A fructosidase mutant according to claim 1, characterized in that: The mutant is obtained by mutating any one or more of the 365th, 405th and 477th positions of the fructosidase PsFFase as shown in the amino acid sequence of SEQ ID NO:

1.

3. A fructosidase mutant according to claim 2, characterized in that: The amino acid at position 365 in the fructosidase PsFFase sequence as shown in SEQ ID NO: 1 is mutated from threonine (T) to proline (P).

4. A fructosidase mutant according to claim 2, characterized in that: The amino acid at position 405 in the fructosidase PsFFase sequence shown in SEQ ID NO:1 is mutated from tryptophan (W) to proline (P). The amino acid sequence of the mutant is shown in SEQ ID NO:

2.

5. A fructosidase mutant according to claim 2, characterized in that: The amino acid at position 405 of the fructosidase PsFFase sequence 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). The amino acid sequence of the mutant is shown in SEQ ID NO:

3.

6. An expression gene, characterized in that: The gene encodes the fructosidase mutant according to claim 2.

7. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the expression gene according to claim 6.

8. A recombinant bacterium, characterized in that: The recombinant cell comprises the expression gene according to claim 6 and / or the recombinant plasmid according to claim 7, and an empty vector bacterium, wherein the empty vector bacterium is Escherichia coli.

9. A use of the fructosidase mutant according to claim 1, characterized in that: Used for catalyzing the synthesis of fructosyl steviol glycoside, specifically comprising the following steps: (1) Construction of recombinant strains expressing enzymes: The gene of the fructosidase mutant is constructed into a plasmid to obtain a recombinant plasmid; the recombinant plasmid is transformed into competent cells of Escherichia coli BL21 (DE3) to obtain a recombinant strain containing an enzyme expression system; (2) Induced enzyme production of recombinant strains: The recombinant strain PsFFase was activated and transferred to LB induction medium, and cultured at 25-40°C until OD600 reached 0.6-0.8, and then the inducer IPTG was added for induction culture. After the culture was completed, the cells were centrifuged at 8000 rpm and collected, and the cells were resuspended in a buffer solution, ultrasonically disrupted, and the supernatant was collected by centrifugation to obtain a crude enzyme solution; (3) Dissolve stevioside in water, add the crude enzyme solution and sucrose, and react. After the reaction is completed, terminate the reaction at high temperature and centrifuge to obtain the supernatant, which is the fructosylated derivative.

10. The method for synthesizing fructosyl steviol glycoside catalyzed by a fructosidase mutant according to claim 9, characterized in that: In step (2), the final concentration of the inducer IPTG is 0.02-1 mM, and the induction culture conditions are: temperature 20-37°C, rotation speed 150-300 r / min, and induction time 24 h; Or in step (3), the reaction pH is 6.0, the temperature is 20-60°C, and the time is 1-10h; Or 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 amount of crude enzyme added is 0.1-10 U / ml.

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