4, 3-alpha-glycosyltransferase mutant for regulating and controlling product specificity

Abstract

The invention discloses a 4, 3-alpha-glycosyltransferase mutant for regulating and controlling product specificity, and belongs to the technical field of enzyme engineering and starch modification. According to the method, key amino acid residues are determined through a substrate channel and conservative analysis, glutamine at the 782nd position of Lf2970GtfB 4, 3-alpha-glycosyl transferase is mutated into proline, serine, aspartic acid, asparagine, leucine, phenylalanine or tryptophan, and then transglutaminase at the 782nd position of the Lf2970GtfB 4, 3-alpha-glycosyl transferase is mutated into glutamine at the 782nd position. By changing the interaction between the enzyme and the substrate, the regulation and control of the specificity such as the bond type proportion and the molecular weight of the product are realized. The mutant obtained by the invention can realize the preparation of alpha-glucan with multi-bond proportion and molecular weight distribution, and provides an effective enzyme tool for the preparation and structural regulation of functional alpha-glucan.

Description

TECHNICAL FIELD

[0001] The present application relates to a 4,3-alpha-glycosyltransferase mutant for regulating product specificity, belonging to the technical field of enzyme engineering and starch modification. BACKGROUND

[0002] Starch is a widely available and inexpensive carbohydrate, which is found in cereal grains, legumes, roots and tubers. As an indispensable energy source in human diet, starch is degraded by various amylases to generate glucose by hydrolyzing (α1→4) glycosidic bonds, providing calories. However, excessive intake of calories from starch has caused a series of health problems such as obesity, diabetes and cardiovascular and cerebrovascular diseases. With the improvement of living conditions and the enhancement of health awareness, the intake of calories from starch is no longer essential for life. Enzymatic conversion to regulate starch molecular structure and convert it into low-calorie products has become a research hotspot at home and abroad. GtfB-type 4,3 / 4,6-α-glucosyltransferases (4,3 / 4,6-α-GTases) belong to glycoside hydrolase family 70 (GH70), which can synthesize α-glucans by hydrolysis and transglycosylation reactions using starch or maltodextrin as a substrate. According to the specificity of the enzyme, products with different molecular structures can be obtained, including different molecular sizes, glycosidic bond arrangements and branching degrees. These factors determine their respective physical and chemical, biological properties, and thus determine their application range. For example, 4,6-α-glucosyltransferases represented by Lactobacillus reuteri 121 GtfB (Lr121 GtfB) catalyze the synthesis of iso-malto-malto-oligosaccharides (IMMPs) containing continuous (α1→6) bonds from starch substrates, which can resist the hydrolysis of digestive enzymes and reach the colon to serve as a carbon source for the microbial flora in the colon, and is a slowly fermentable dietary fiber. The reuteran structure obtained by Lr2613 GtfB enzymatic hydrolysis of starch contains (α1→4,6) branching structure and (α1→4) / (α1→6) bond alternating structure, which can limit the continuous attack of digestive enzymes on (α1→4) bonds that are prone to hydrolysis, and has a high content of slowly digestible starch (SDS) and resistant starch (RS), which is a slowly released energy carbohydrate. Subsequently, a GtfB enzyme with unique catalytic specificity, Lactobacillus fermentum 2970 GtfB (Lf2970 GtfB), was mined and characterized, and its product was an α-glucan formed by (α1→3) bonds connecting oligomaltose fragments in a linear chain or branching direction, which was the first time to find such a product structure in the GH70 family. The alternating glucosucroses from the GH70 family have a main chain structure with (α1→6) / (α1→3) bonds alternatingly connected, which endows them with low viscosity, high solubility and digestion resistance, and can be used as a low-calorie food additive to replace fat or oil. In the medical field, alternating glucosides also have potential application value. Studies have shown that the treatment of alternating glucosides can promote the proliferation and differentiation of human mesenchymal stem cells. In addition, the presence of (α1→3) bonds in glucans can endow α-glucans with the function of adsorbing heavy metal ions, or serve as good carriers for packaging and biological packaging.However, the synthesis mechanism of (α1→3) bond in Lf2970 GtfB product is still unclear, and the structure-activity relationship of 4,3-α-glucosyltransferase needs to be improved. Therefore, understanding the mechanism of 4,3 / 4,6-α-GTase product specificity is of great significance for developing structure-customized α-glucan products.

[0003] Current enzyme engineering methods for modifying GtfB-type 4,3 / 4,6-α-glucosyltransferase mainly target the active center site of the chemical reaction. According to the α-retaining double displacement catalytic mechanism of the GH70 family, the amino acid residues near the acceptor subsite are usually considered to play an important role in the transglycosylation half-reaction. The transglycosylation of the N1019 random mutant of the Lr121 GtfB+1 subsite is affected, and the enzyme activity and catalytic efficiency of the mutant N1019D are increased by 420% and 590% of the wild type, respectively. The D991 and G1028 near the acceptor subsite of the Lf2970 GtfB 4,3-α-glucosyltransferase 4,3-α-GTase active center make important contributions to the transglycosylation reaction and product specificity, and the mutants can introduce a certain proportion of (α1→6) bonds into the product, while only trace amounts are present in the wild type.

[0004] The substrate channel connects the active center and the surface region of the enzyme, affecting the process of substrate entering and / or product releasing, thereby indirectly affecting enzyme activity, substrate specificity and stereoselectivity. For example, by mutating the key amino acids in the channel of chitinase B of glycoside hydrolases, it was found that the mutation increased the conformational freedom of the channel wall, which was not conducive to substrate binding. The introduction of mutations in the channel of fatty aldehyde deformylating oxygenase made the conformation narrow, resulting in enhanced specific utilization of short-chain aldehyde by the enzyme. Currently, there is no report on the channel research of 4,3 / 4,6-α-glucosyltransferase, therefore, based on the resolved crystal structure, the substrate channel of Lf2970 GtfB is constructed in the present application, and the contribution of key amino acids in the channel to the specificity of product bond type is further explored, so as to realize the preparation of α-glucan with multiple bond type specificity. SUMMARY

[0005] In order to solve the above problems, the present application provides a 4,3-alpha-glucosyltransferase mutant for regulating product specificity, which is derived from Lactobacillus fermentum 2970, and the amino acid sequence thereof comprises, on the basis of the parent amino acid sequence Lf2970 GtfB 4,3-alpha-glucosyltransferase-ΔN, the amino acid sequence obtained by mutating the amino acid at position 782 of the parent amino acid sequence; the sequence of the parent amino acid Lf2970 GtfB 4,3-alpha-glucosyltransferase-ΔN is shown in SEQ ID NO. 2, or any sequence comprising the parent amino acid Lf2970 GtfB 4,3-alpha-glucosyltransferase-ΔN shown in SEQ ID NO. 2.

[0006] In an embodiment of the present application, any sequence comprising the parent amino acid Lf2970 GtfB 4,3-alpha-glucosyltransferase-ΔN shown in SEQ ID NO. 2 can be a GtfB-type alpha-glucosyltransferase with an amino acid sequence shown in SEQ ID NO. 1.

[0007] In an embodiment of the present application, the 4,3-alpha-glucosyltransferase mutant can be obtained by mutating the amino acid at position 1397 (corresponding to position 782 of Lf2970 GtfB 4,3-alpha-glucosyltransferase-ΔN shown in SEQ ID NO. 2) of the GtfB-type alpha-glucosyltransferase with an amino acid sequence shown in SEQ ID NO. 1. The parent amino acid Lf2970 GtfB 4,3-alpha-glucosyltransferase-ΔN shown in SEQ ID NO. 2 is obtained by truncating the N-terminal of the GtfB-type 4,3-alpha-glucosyltransferase with an amino acid sequence shown in SEQ ID NO. 1 at the 615th amino acid residue. After expression of the truncated amino acid residue in E. coli BL21, there is no significant difference in catalytic activity compared with that of the untruncated one.

[0008] 4,3-alpha-glucosyltransferase (SEQ ID NO. 1):

[0009]

[0010] Amino acid sequence of the parent amino acid Lf2970 GtfB 4,3-alpha-glycosyltransferase-ΔN (SEQ ID NO. 2):

[0011] MFGKDGRIATGLYKWDKNNQWYYFDPVTYLKVTNKWVDGNYYDEDGAQAISKLVTINNRLYYFDDQGKEISNQFRTIHGDKYYFGNDSAAVTGQQTIDGKVYKFSNYGYLLGNRYGKIENGKLNIYSLADHSLIKTVEAGPWENMAYSMDSNSINNIDGYISYTGWYRPYGTSQDGK TWYPTTVADWRPILMYVWPSKDVQVKFIQYFVNHGYENSNYGLTAGSVKDLSENTASINLNEVAQNLRYVIEQHIVAAKSTSQLANDINNFITTIPELSASSELPDESGSGQVIFVNNDNTSYADSKYRLMNRTVNNQTGNDNSDYCPEFVVGNDIDNSNPVVQAENLNWEYFLLNYGKLMGYNQDGNFDGFRIDAADDMDADVLDQIGQLMNDMYHMKGNPQNANNHLSYNEGYGPGAARMLNKKGNPQLFMDARECNTLENVLGRANNRDTISHLVTDSIVNRQNDVTENEATPNWSYVTNHDIRNNLINGLIIKDHPGMGSAYKAEYANQAWQEFYADQKKTDKQYAQYNVPAQYAILLSNKDTVPQIYYGDLYNETAQYMQEKSIYYDAITTLMKARKQFVSGGQTMTKLSDNLIASVRYGKGVANANSEGTDSLSRTSGMAVIVGNNPQMAEQTISINMGRAHANEQYRNLLDTTDNGLTYNADGAENPETLTTDDNGILKVTVKGYSNPYVSGYLGVWVPVASGNQDVTTNAATVSADSNKIFESNAALDSHMIYEDFSMYQPKPTSTENHAYNIIAQNAELFNNLGITDFWMAPAYTQAGTSRYNEGYSVADRYNLGTNANPTKYGSGEELANAIAALHSAGLKVQEDIVMNQMIGLPGQEAVTVTRADNRGMQTYVNGKTYANQMYFAYTTGGGNGQETYGGKYLSELQSKYPDLFTTRAISTGVAPDPTTHITKWSAKYENGTSLQNIGIGLAVKLANGDYAYLNDSNNKAFNTTLPETMSSTDYYANIEDN

[0012] Nucleotide sequence encoding the amino acid Lf2970 GtfB 4,3-α-glycosyltransferase-ΔN of the mother strain (SEQ ID NO. 3)

[0013]

[0014] In an embodiment of the present application, the mutant is:

[0015] Q782P, obtained by mutating glutamine at position 782 of a 4,3-α-glucosyltransferase having an amino acid sequence as shown in SEQ ID NO. 2 to proline;

[0016] Q782S, obtained by mutating glutamine at position 782 of a 4,3-α-glucosyltransferase having an amino acid sequence as shown in SEQ ID NO. 2 to serine;

[0017] Q782D, Q782N, Q782L, Q782F, Q782W, obtained by mutating glutamine at position 782 of a 4,3-α-glucosyltransferase having an amino acid sequence as shown in SEQ ID NO. 2 to aspartic acid, asparagine, leucine, phenylalanine or tryptophan, respectively;

[0018] In an embodiment of the present application, the 4,3-α-glucosyltransferase mutant can also be: Q1397P, Q1397S, Q1397D, Q1397N, Q1397L, Q1397F, Q1397W, obtained by mutating the amino acid at position 1397 (corresponding to position 782 of Lf2970 GtfB 4,3-α-glucosyltransferase-ΔN shown in SEQ ID NO. 2) of a 4,3-α-glucosyltransferase having an amino acid sequence as shown in SEQ ID NO. 1.

[0019] In an embodiment of the present application, the nucleotide sequence encoding the parent amino acid GtfB-ΔN is shown in SEQ ID NO. 3.

[0020] The present application also provides a gene encoding the mutant.

[0021] The present application also provides a recombinant vector carrying the gene.

[0022] In an embodiment of the present application, the recombinant vector is pET15b as the expression vector.

[0023] The present application also provides a recombinant cell carrying the mutant, the gene or the recombinant vector.

[0024] In an embodiment of the present application, the recombinant cell is a bacterium or a fungus as the expression host.

[0025] In an embodiment of the present application, the recombinant cell is E. coli BL21 (DE3) as the expression host.

[0026] The application provides a method for constructing Lf2970 GtfB 4,3-alpha-glycosyltransferase substrate channel mutants, the mutants are obtained by sequence alignment, selecting a weak conservative site Q782, mutating the site into amino acids at corresponding positions of other enzymes, i.e. obtaining mutants Q782P and Q782S; or randomly mutating the Q782 to obtain mutants Q1397D, Q1397N, Q1397L, Q1397F, Q1397W.

[0027] The application also provides a method for preparing alpha-glucan by using Lf2970 GtfB 4,3-alpha-glycosyltransferase and the mutants, the method is to use linear dextrin as a substrate, first dissolving the linear dextrin with a sodium hydroxide solution, then neutralizing the linear dextrin with hydrochloric acid, adding a buffer, and then adding the enzyme for modification to obtain synthesized alpha-glucan, the synthesized product has different glycosidic bond compositions and molecular weight distributions. The alpha-glucan has (alpha1→4) glycosidic bonds, (alpha1→3) glycosidic bonds and (alpha1→6) glycosidic bonds.

[0028] In an embodiment of the application, the concentration of the linear dextrin is 0.25% to 20%.

[0029] In an embodiment of the application, the concentration of the buffer system used is 10 to 50 mM, and the pH is 4 to 6.

[0030] In an embodiment of the application, the reaction temperature is controlled to be 37 to 40 DEG C, and the reaction time is 4 to 48 h.

[0031] In an embodiment of the application, the amount of the enzyme used in the reaction is 10 to 50 U / g of dry basis linear dextrin.

[0032] In an embodiment of the application, the (alpha1→3) bond proportion of the alpha-glucan is 20.6% to 26.3%, the (alpha1→6) bond proportion is 1.5% to 7.9%, and the weight average molecular weight ranges from 38.43 kDa to 1724.68 kDa.

[0033] In an embodiment of the application, the method is to use starch or starch derivatives as a substrate, the starch includes amylopectin and amylose, and the starch derivatives include malt dextrin and malt oligosaccharide (polymerization degree is greater than or equal to 3).

[0034] In an embodiment of the application, when linear starch / dextrin is used as a substrate, the starch is first dissolved with an alkali, then neutralized with an acid, a buffer is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain synthesized alpha-glucan.

[0035] In an embodiment of the present application, when using amylopectin as a substrate, a buffer is added to the starch, and then the mutant or the recombinant mutant enzyme is added for modification, to obtain synthetic alpha-glucan.

[0036] In an embodiment of the present application, when using amylopectin as a substrate, a buffer is added, and then the mutant or the recombinant mutant enzyme is added for modification, to obtain synthetic alpha-glucan.

[0037] The present application also provides a use of the alpha-glucan synthesized by the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant cell, the above-mentioned mutant or the above-mentioned method in the fields of food, health products, beverages, medicine and additives.

[0038] The present application also provides a use of the above-mentioned mutant or the above-mentioned recombinant cell in the preparation of alpha-glucan, wherein the mutant or the recombinant cell or the recombinant mutant enzyme expressed by the recombinant cell is used as a catalyst to react with starch or starch derivatives as a substrate to prepare alpha-glucan; the alpha-glucan has (α1→4) glycosidic bond, (α1→3) glycosidic bond and (α1→6) glycosidic bond.

[0039] Advantages

[0040] (1) The Lf2970 GtfB 4,3-alpha-glycosyltransferase mutant provided by the present application can effectively convert substrates, and the alpha-glucan / oligosaccharide produced by the mutant introduces (α1→6) bond and (α1→4,6) branch on the basis of the wild type, and the total (α1→6) bond ratio increases from 1.5% to 7.9%, and the total (α1→3) bond ratio increases from 21.9% to 26.3%, which is an increase of 48% compared with the unmutated Lf2970 GtfB 4,3-alpha-glycosyltransferase. Through these mutations, the ratio of (α1→6) bond and (α1→3) bond in the Lf2970 GtfB product is adjusted, so that the anti-digestion ability of the product is improved, and the product has great application potential as a new type of soluble dietary fiber and prebiotic in the fields of food and medicine.

[0041] (2) The Lf2970 GtfB 4,3-alpha-glycosyltransferase mutant provided by the present application does not significantly affect the expression amount and enzyme activity, and has good industrial application potential. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a schematic diagram of the channel of Lf2970 GtfB 4,3-alpha-glycosyltransferase obtained by software simulation;

[0043] Figure 2 is a multiple sequence alignment of representative GH70 family enzymes in the channel region;

[0044] Figure 3 are protein purification results for wild type and mutants;

[0045] Figure 4 are one-dimensional nuclear magnetic resonance hydrogen spectra of wild type and representative mutants; DETAILED DESCRIPTION

[0046] The technical solutions of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the scope of protection of the present application.

[0047] Part of the definitions or terms involved in the present application:

[0048] Mutant: As used herein, the term "mutant" or "polypeptide variant" or "polypeptide" or "RNA polymerase mutant" when referring to the use of variants of the present application means a polypeptide having RNA polymerase enzyme activity that comprises an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "parent" RNA polymerase. Substitution means replacement of the amino acid occupying a position with a different amino acid; deletion means removal of the amino acid occupying a position; and insertion means addition of an amino acid after abutting and immediately following the amino acid occupying a position. In describing the variants of the present application, the nomenclature described below has been adapted for ease of reference. The accepted IUPAC one-letter or three-letter amino acid abbreviations have been used. Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, amino acid substituted for. Thus, substitution of glutamine at position 782 with proline is denoted "Q782P".

[0049] Wild-type enzyme: The term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance (e.g., recombinant nucleic acid and protein sequences produced in the laboratory or modifications of wild-type sequences) that is not found in nature. The terms "wild-type enzyme" and "parent enzyme" are used interchangeably when the parent enzyme is not a variant enzyme.

[0050] Expression: The term "expression" as used herein refers to any step involved in the production of a variant, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0051] Expression vector: The term "expression vector" as used herein refers to a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

[0052] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present application. The term "host cell" encompasses any progeny of the parent cell which is not identical to the parent cell (e.g., by way of example, a host cell which comprises a mutation not found in the parent cell), as well as recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), heterologous host cells.

[0053] Recombinant: The term "recombinant" when used with reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified in the form found in nature. For example, a recombinant cell expresses genes that are not found in an identical form in nature, or expresses native genes at different levels, or under different conditions than found in nature. A recombinant nucleic acid differs from a native sequence in that one or more nucleotides and / or is operably linked to a heterologous sequence (e.g., a heterologous promoter in an expression vector). A recombinant protein differs from a native sequence in that one or more amino acids and / or is fused to a heterologous sequence. A vector comprising a nucleic acid encoding a polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "transgenic".

[0054] It is noted that the abbreviations for amino acid residues herein are in accordance with the generally accepted notations used in the art, and the full names of the abbreviations are indicated in parentheses following the abbreviations in some instances herein. The terms "amino acid residue" and "amino acid" are used interchangeably herein. The amino acid sequences herein are presented starting from the N-terminus.

[0055] The detection methods involved in the following examples are as follows:

[0056] Enzyme activity detection method:

[0057] The activities of wild-type and mutant enzymes were determined using amylose-iodine staining method. The reaction was carried out at 40°C with 0.25% (w / v) amylose as substrate, 9 μg / mL enzyme, and 25 mM NaAC, 5 mM CaCl2, pH 5.5 as buffer system. One enzyme activity unit (U) was defined as the amount of enzyme that catalyzed the conversion of 1 mg amylose per minute. The hydrolysis activity was determined using 3,5-dinitrosalicylic acid method (DNS), and the reaction system and enzyme addition amount were the same as those for total enzyme activity. One enzyme activity unit (U) was defined as the amount of enzyme that released 1 mg reducing sugar from amylose per minute under optimal conditions.

[0058] Kinetic parameter test:

[0059] Total enzyme activity was measured at different amylose concentrations (0.025%, 0.03%, 0.04, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%) and the kinetic parameters Km and kcat were calculated according to the Michaelis-Menten equation: where Vmax is the maximum reaction rate, [S] is the substrate concentration, and [E] is the enzyme concentration. Finally, the catalytic efficiency kcat / Km of the enzyme was calculated.

[0060] Product glycosidic bond composition test:

[0061] The Lf2970 GtfB 4,3-a-glycosyltransferase-ΔN wild type and mutant modified amylose products were subjected to NMR and methylation analysis to determine the type, content and branching degree of glycosidic bonds.

[0062] 1 H nuclear magnetic resonance:

[0063] Take 20 mg of lyophilized sample, add 1 mL D2O, boil for 1 h, and immediately lyophilize. After lyophilization, add 1 mL D2O, boil for 1 h, and immediately lyophilize for standby use. Finally, dissolve the sample in D2O, boil in a boiling water bath for 1 h, and then record the one-dimensional nuclear magnetic resonance hydrogen spectrum on an AVANCE-600 MHz spectrometer (Bruker, Germany). The test probe temperature is 25°C.

[0064] Methylation analysis:

[0065] Take the lyophilized oligosaccharide / polysaccharide (60 mg) and dissolve it in 20 mL DMSO, then add 20 mL NaOH solution dissolved in DMSO and react for 1 h, then add CH3I liquid drop by drop under ice bath for 2 h of methylation reaction. The methylation product is dialyzed with a 1000 Da dialysis bag, then extracted with chloroform, dehydrated with sodium sulfite, and then nitrogen-blow dried. Then, hydrolyze with trifluoroacetic acid solution (2M), reduce with NaBD4, and acetylate with acetic anhydride and pyridine (1:1, v / v). The partially methylated sugar alcohol acetate (PMAAs) is analyzed using a gas chromatography-mass spectrometry (GC-MS) system (Agilent Technologies, Santa Clara, USA). The obtained EI-MS spectrum is compared with the CCRC spectral database-PMAAs to determine the type and proportion of glycosidic bonds.

[0066] Product molecular weight test: High performance gel filtration chromatography (HPGFC) is used to determine the relative molecular weight distribution of enzyme modified amylose products. Lyophilized reaction samples are dissolved in pure water to a final concentration of 5 mg / mL and filtered with a 0.45 μm cellulose ester microporous filter. 30 μL of sample is injected into a gel filtration chromatograph equipped with a refractive index detector to monitor the molecular weight distribution of the sample.

[0067] Example 1: Simulation of Lf2970 GtfB 4,3-a-glycosyltransferase substrate channel and determination of key sites

[0068] 1. Construction of substrate channel

[0069] Using the crystal structure of LrN1 GtfB-acarbose complex (PDB: 8HW3) as a template, the structure model of Lf2970 GtfB 4,3-a-glycosyltransferase-acarbose complex was obtained by homology modeling, in which two molecules of acarbose were bound at similar positions in the active site and on the surface. A substrate channel was calculated using CAVER Web software as shown in Figure 1 , which is located on the donor side, with the ligands at both ends connecting the surface and the active site, respectively, reflecting the credibility of the simulation.

[0070] 2. Selection of mutation sites

[0071] As a "bridge" between the surrounding solvent and the active site, the substrate channel has an important influence on enzyme activity, catalytic specificity and stability, and the role of key amino acids in the channel on enzyme catalytic process has not been reported in GH70 family enzymes. Analysis of the amino acids around the Lf2970 GtfB 4,3-a-glycosyltransferase channel found that the Q782 site was located in a relatively narrow region of the channel, and had weak conservation, so modification of this site could affect the orientation of the substrate in the active site by changing the spatial conformation of the channel, indirectly leading to changes in product specificity.

[0072] Example 2: Design and construction of Lf2970 GtfB 4,3-a-glycosyltransferase channel Q782 site mutants

[0073] Sequence alignment of representative GH70 family glucosucrase (GSs) and GtfB enzymes showed that the glutamine at position 782 in Lf2970 GtfB 4,3-a-glycosyltransferase was replaced by proline or serine in other GH70 enzymes, as shown in Figure 2 , so the glutamine at position 782 was mutated to proline and serine, respectively, to construct the site-directed mutants Q782P, Q782S. In addition, to further understand the contribution of this site, it was randomly mutated. The primer design of the above mutants is shown in Table 1.

[0074] Table 1. Mutant primer sequence table

[0075]

[0076]

[0077] Note: Underlined is the mutated base

[0078] Using the pET15b-Lf2970 GtfB 4,3-α-glycosyltransferase-ΔN as a template, the target amino acid was mutated using the whole plasmid PCR method.

[0079] The PCR program was as follows: pre-denaturation at 98°C for 3 min, followed by 30 cycles of denaturation at 98°C for 20 s, annealing at 55°C for 30 s, and extension at 68°C for 9 min, and further extension at 68°C for 10 min.

[0080] The reacted PCR fragments were digested using Dpn I enzyme, and the plasmid was cloned into the E. coli DH5α competent cells by heat shock method. The plasmid with successful sequencing was introduced into the E. coli BL21 competent cells, and the recombinant E. coli containing the mutant was prepared, i.e., BL21 / pET15b-Q782P-ΔN, BL21 / pET15b-Q782S-ΔN, BL21 / pET15b-Q782D-ΔN, BL21 / pET15b-Q782N-ΔN, BL21 / pET15b-Q782L-ΔN, BL21 / pET15b-Q782F-ΔN, and BL21 / pET15b-Q782W-ΔN.

[0081] Meanwhile, the recombinant E. coli containing the wild-type enzyme was obtained, i.e., BL21 / pET15b-Lf2970 GtfB 4,3-α-glycosyltransferase-ΔN.

[0082] Example 3: Expression and purification of Lf2970 GtfB 4,3-α-glycosyltransferase-ΔN and mutants

[0083] The specific steps were as follows:

[0084] (1) The recombinant E. coli containing the wild-type and mutant genes obtained in Example 2 was inoculated into LB medium containing ampicillin (100 μg / ml), and cultured at 37°C and 200 rpm. When the OD600 nm reached 0.4-0.6, the cells were treated in ice bath for 15 min, and then isopropyl-β-thiogalactoside (IPTG, 0.1 mM) was added, and the enzyme production was induced at 16°C and 160 r / min for 20 h. The fermentation broth was prepared.

[0085] (2) The obtained fermentation broth was centrifuged at 4°C and 8000 g for 30 min to collect the cells, which were resuspended in 10 mL 25 mM Tris-HCl (250 mM NaCl, pH 7.5) according to 1 g of bacterial body, and then ultrasonically broken in ice bath for 20 min (30% power, crushing for 2 s, and interval for 3 s). The supernatant obtained by centrifuging the broken bacterial liquid at 4°C and 8000 g for 40 min was the crude enzyme liquid, and the following was prepared:

[0086] Pure enzyme solution containing wild type Lf2970 GtfB 4,3-alpha-glycosyltransferase-ΔN, crude enzyme solution containing Q782P, Q782S, Q782D, Q782N, Q782L, Q782F and Q782W.

[0087] (3) The crude enzyme solution obtained in step (2) was purified by nickel affinity chromatography, eluted with 20 mM Tris-HCl (250 mM NaCl, pH 7.5) and 20 mM Tris-HCl (250 mM NaCl, pH 7.5) containing different concentrations of imidazole in sequence, and the flow-through of each part was collected. The purity was verified by polyacrylamide gel electrophoresis, and the results are shown in Figure 3

[0088] The results show that the wild type enzyme and mutant enzymes of the application are expressed.

[0089] (4) Detection of enzyme activity

[0090] The enzyme activity of the pure enzyme solution of step (3) was detected, and the results are shown in Table 2. The total enzyme activity of Q782P and Q782S increased significantly, but the transglycosylation ability was maintained, and the relative hydrolysis activity did not change significantly compared with the wild type. The total enzyme activity of Q782W was also higher than that of the wild type, but the hydrolysis activity also increased significantly, resulting in an increase in the relative hydrolysis activity, showing strong hydrolysis. Q782N / F / D also showed strong hydrolysis, and the total enzyme activity of Q782F and Q782D decreased significantly. The hydrolysis activity of mutant Q782L decreased significantly to 0.29 U / mg, which reduced the relative hydrolysis activity, and showed relatively strong transglycosylation activity.

[0091] Table 2 Lf2970 GtfB 4,3-alpha-glycosyltransferase wild type and mutant enzyme activitya

[0092]

[0093] a Each data represents the average value ± standard deviation of three independent repeated experiments, and different lowercase letters in the same column represent significant difference (p<0.05).

[0094] b

[0095] Example 4: Preparation of alpha-glucan using Lf2970 GtfB 4,3-alpha-glycosyltransferase-ΔN wild type and mutants

[0096] The specific steps are as follows:

[0097] (1) The potato starch was debranched to obtain amylose; specifically: ​

[0098] Potato starch was dissolved in buffer (20 mM, pH 4.6 sodium acetate buffer) to a final concentration of 2% (w / v) and dispersed evenly in a boiling water bath for 1 h. After cooling to room temperature, commercial pullulanase (enzyme activity > 1000 U / mL) was added at a volume of 1% of the solution, and the reaction was carried out at 60°C for 24 h, after which the enzyme was inactivated in a boiling water bath for 15 min. The supernatant was collected by centrifugation (8000 x g, 20 min) and dried at 40°C. The product was ground, sieved, washed with 90% methanol three times, and dried again at 40°C to obtain the debranched potato starch.

[0099] (2) The debranched potato starch obtained in step (1) was dissolved in 1 M sodium hydroxide solution to a final concentration of 4% (w / v), and then neutralized with an equal amount of 1 M hydrochloric acid. Buffer (25 mM NaAc, 5 mM CaCl2, pH 5.5) was added to a final concentration of 1% (w / v) of potato starch, and after cooling to room temperature, the Lf2970 GtfB 4,3-α-glucosyltransferase-ΔN and mutant pure enzyme solution prepared in Example 2 was added at an amount of 50 U / g, and the reaction was carried out at 40°C for 24 h. The reaction was terminated by boiling in a water bath for 15 min, and then the product was freeze-dried to obtain a freeze-dried powder.

[0100] Example 5: Analysis of the products of Lf2970 GtfB 4,3-α-glucosyltransferase-ΔN wild type and mutant enzymes

[0101] 1. Analysis of product molecular weight and glycosidic bond type

[0102] The products prepared in Example 4 were analyzed by one-dimensional nuclear magnetic hydrogen spectrum and molecular weight analysis, respectively, and the results are shown in Table 3 and Figure 4 .

[0103] Table 3 Glycosidic bond composition and molecular weight distribution of Lf2970 GtfB 4,3-α-glucosyltransferase wild type and mutant products

[0104]

[0105] ND: not detected

[0106] The results show that:

[0107] (1) In terms of molecular weight distribution, the molecular weight of the products of Lf2970 GtfB 4,3-alpha-glycosyltransferase wild type and mutants all showed bimodal distribution. The product molecular weight of the Q782P mutant decreased significantly to 38.43 kDa, and the product molecular weight of the mutants Q782D / N / F / W decreased slightly, but was in the same order of magnitude as the size of the wild type product. The product molecular weight of Q782S was similar to that of the wild type, and the product molecular weight of Q782L was significantly higher than that of the wild type, which reflected strong reaction continuity.

[0108] (2) In terms of glycosidic bond composition, the product bond type specificity of the mutant Q782P changed significantly, the proportion of (alpha 1 to 4) bond decreased to 65.8%, the proportions of (alpha 1 to 3) bond and (alpha 1 to 6) bond increased significantly to 26.3% and 7.9% respectively, which increased by 20% and 558% respectively compared with the wild type, showing strong transglycosylation ability; the non-polar mutants Q782L / F all showed strong alpha 1,6-transglycosylation, but Q782P significantly improved alpha 1,3-transglycosylation and alpha 1,6-transglycosylation at the same time, which indicated that the rigidity of the site had a stronger impact on catalytic specificity. The product bond type specificity of Q782D / W was similar to that of the wild type.

[0109] 2. Methylation analysis of polysaccharide product

[0110] For the mutant Q782P with large changes in product glycosidic bond ratio, further methylation analysis was performed to test the type and proportion of linear / branched glycosidic bonds in the polysaccharide, and the wild type results were used as a control. The results are shown in Table 4. The linear (alpha 1 to 4) bond and branched (alpha 1 to 3,4) bond proportions in the Q782P product were similar to those of the wild type, but the linear (alpha 1 to 3) bond decreased significantly from 20% to 11%, and the branched (alpha 1 to 4,6) bond content increased significantly to 9%. Combined with the nuclear magnetic and methylation results, it can be seen that the Q782P mutation changes the product bond type specificity by weakening (alpha 1 to 3) bond synthesis and promoting (alpha 1 to 4,6) bond synthesis.

[0111] Table 4 Methylation analysis

[0112]

[0113] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A mutant of a 4,3-a-glycosyltransferase enzyme that modulates product specificity, characterized in that, The amino acid sequence of the 4,3-alpha-glucosyltransferase mutant includes, on the basis of the parent amino acid sequence, the amino acid sequence obtained by mutating the amino acid at position 782 of the parent amino acid sequence; the sequence of the parent amino acid is shown in SEQ ID NO. 2, or any sequence containing the parent amino acid shown in SEQ ID NO.

2.

2. The 4,3-a-glucosyltransferase mutant of claim 1, wherein, The amino acid sequence of the 4,3-alpha-glucosyltransferase mutant includes any one of the following amino acid sequences: (1) the amino acid sequence obtained by mutating glutamine at position 782 of the 4,3-alpha-glucosyltransferase having the amino acid sequence shown in SEQ ID NO. 2 to proline; (2) the amino acid sequence obtained by mutating glutamine at position 782 of the 4,3-alpha-glucosyltransferase having the amino acid sequence shown in SEQ ID NO. 2 to serine; (3) the amino acid sequence obtained by mutating glutamine at position 782 of the 4,3-alpha-glucosyltransferase having the amino acid sequence shown in SEQ ID NO. 2 to aspartic acid, asparagine, leucine, phenylalanine or tryptophan.

3. A gene encoding the mutant of claim 1.

4. A recombinant vector carrying the gene of claim 3.

5. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 3 or the recombinant vector of claim 4.

6. The recombinant cell of claim 5, wherein, The recombinant cell is a bacterial or fungal expression host.

7. A method for preparing a mutant of a 4,3-α-glycosyltransferase with modulated product specificity, characterized in that, The method comprises the following steps: (1) surface binding structure analysis of the enzyme determines the surface binding amino acid of Lactobacillus fermentum 2970 4,3-alpha-glucosyltransferase, and a channel for the substrate to enter the active center of the enzyme is simulated accordingly, and the key amino acid sites in the channel are determined by sequence alignment; (2) design mutation primers for key amino acids, and use the mutated plasmid as a template to mutate the plasmid carrying the gene encoding Lactobacillus fermentum 2970 4,3-alpha-glucosyltransferase to obtain the mutated plasmid; (3) transform the mutated plasmid into a host bacterium, select positive monoclonal fermentation, obtain Lactobacillus fermentum 2970 4,3-alpha-glucosyltransferase mutant crude enzyme, and purify to obtain the Lactobacillus fermentum 2970 4,3-alpha-glucosyltransferase mutant pure enzyme.

8. A method of preparing alpha-glucans, characterized in that, The method is to use the mutant of claim 1 or 2 or the recombinant cell of claim 5 or 6 or the recombinant mutant enzyme expressed by the recombinant cell as a catalyst, and use starch or starch derivatives as a substrate to prepare alpha-glucan; the alpha-glucan has (α1→4) glycosidic bond, (α1→3) glycosidic bond and (α1→6) glycosidic bond.

9. The method of claim 8, wherein, The method is, taking starch or starch derivatives as substrate, the starch includes amylopectin, amylose, the starch derivatives includes maltodextrin, maltoligosaccharide (polymerization degree ≥3); Preferred, taking amylose as substrate, first dissolving starch with alkali, then neutralizing with acid, adding buffer, then adding the recombinant mutant enzyme to modify, obtaining synthesized α-glucan; Preferred, taking amylopectin as substrate, adding buffer to starch, then adding the recombinant mutant enzyme to modify, obtaining synthesized α-glucan; Preferred, taking dextrin as substrate, adding buffer, then adding the recombinant mutant enzyme to modify, obtaining synthesized α-glucan; Preferred, the concentration of substrate is 0.25% to 30% (w / v), the concentration of buffer system is 10 to 50 mM, pH 4 to 6 buffer, the addition amount of mutant or the recombinant mutant enzyme is 10 to 50 U / g dry base starch, the temperature of reaction is 37 to 40 DEG C, and the time is 4 h to 48 h.

10. The use of α-glucan prepared by the mutant of claim 1 or the gene of claim 3 or the recombinant carrier of claim 4 or the recombinant cell of claim 5 or 6 or the method of any one of claims 8 to 9 in the field of food, biomaterial, medicine, additive or cosmetics.

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