A key-type specific altered alpha-glucosyltransferase mutant

Abstract

The application discloses a key type specificity changed alpha-glucosyltransferase mutant, and belongs to the technical field of enzyme engineering and starch modification. The method is characterized in that, by analyzing the combination of Lf2970GtfB 4,3-alpha-glucosyltransferase in the active center donor / acceptor sub-site and the substrate, part of key amino acids of Lf2970GtfB are mutated into different types of amino acids, the mutant is prepared by changing the enzyme and substrate interaction force, space steric hindrance and other factors affecting the orientation of the substrate in the active center, the transglycosylation is changed, and the bond type composition of the product is changed. The application changes the bond type specificity of Lf2970GtfB 4,3-alpha-glucosyltransferase by mutation, the obtained mutant has novel 4,3 / 6-alpha-glucosyltransferase activity, and is helpful to synthesizing unique structure and more valuable alpha-glucan by using starch substrates.

Description

Technical Field

[0001] This invention relates to a mutant α-glucosyltransferase with bond type specific alteration, belonging to the fields of enzyme engineering and starch modification technology. Background Technology

[0002] The GH70 family of glycoside hydrolases can synthesize α-glucans with specific structures using starch or sucrose as substrates. Initially, it only included sucrase glucanases (GSs) derived from lactic acid bacteria. GSs can convert sucrose into α-glucans and oligosaccharides with different structures. In recent years, three subfamilies—GtfB, GtfC, and GtfD—have been discovered within the GH70 family. Among them, the GtfB subfamily derived from lactic acid bacteria is favored due to its "generally recognized as safe" (GRAS) status. Currently, 12 GtfB-type α-glucosyltransferases (α-GTases) have been characterized, exhibiting different reaction and product specificities. Lactobacillus reuteri 121 GtfB (Lr121 GtfB) was the first GtfB enzyme to be discovered and characterized. It can cleave the (α1→4) bonds of starch substrates and synthesize continuous (α1→6) bonds. The synthesized soluble starch derivative is called isomalt-maltose polysaccharide (IMMP). Lactobacillus reuteri 2613 GtfB (Lr2613 GtfB) can synthesize branched reuteri composed of linear (α1→4), (α1→6) bonds and (α1→4,6) branch points. Lactobacillus fermentum 2970 GtfB (Lf2970 GtfB) is the only GtfB enzyme reported to date with 4,3-α-glucosyltransferase activity. Its product is an α-glucan formed by oligomaltose fragments linked by (α1→3) bonds in a linear chain or branching direction. This structure is the first discovery of such a product in the GH70 family.

[0003] The bond diversity and structural complexity of α-glucans determine their unique physicochemical properties, leading to their widespread application in food, cosmetics, pharmaceuticals, and biotechnology. IMMP synthesized from Lr121 GtfB has a high (α1→6) bond content, resisting hydrolysis by digestive enzymes and reaching the colon to serve as a carbon source for the colonic microbiota, making it a slowly fermentable dietary fiber. Introducing (α1→2) and (α1→3) branches into the linear structure of IMMP revealed that these branches help regulate the abundance of beneficial colonic bacteria. The roitose structure obtained from starch hydrolysis by Lr2613 GtfB contains alternating (α1→4,6) branched structures and (α1→4) / (α1→6) bonds, limiting the continuous attack of easily hydrolyzed (α1→4) bonds by digestive enzymes. It exhibits a high content of slowly digestible starch (SDS) and resistant starch (RS), making it a slowly releasing carbohydrate. Therefore, understanding the mechanism of GtfB-type α-GTase bond synthesis and expanding the library of α-glucan products obtained after α-GTase conversion of starch substrates can increase the application value of starch in the food, healthcare and biomaterials industries.

[0004] Mutation studies on Lr121 GtfB have shown that mutations at key amino acid sites can alter the proportion of (α1→6) bonds in the product. Site-directed mutagenesis of the receptor subsite at the active site of Lr121 GtfB indicates that the direct interaction between H1056 at the +2 subsite and the ligand ensures α1,6-transglucosylation, resulting in varying degrees of decrease in the (α1→6) bond content of the product. Furthermore, no studies have shown that mutations can introduce new bond types into GtfB-type α-glucosyltransferases. As the only 4,3-α-glucosyltransferase among GtfB enzymes, the structural determinants of (α1→3) bond formation in Lf2970 GtfB remain unclear.

[0005] To address the aforementioned issues, this invention proposes to mutate key amino acids at the donor and acceptor subsites of the Lf2970 GtfB active center to alter the proportion and types of glycosidic bonds in the product, thereby expanding the structural diversity of synthesizable α-glucans and providing a new approach for synthesizing novel α-glucans from starch. Summary of the Invention

[0006] To address the aforementioned issues, this invention employs site-directed mutagenesis to mutate key amino acids at the donor / acceptor subsites of the Lf2970 GtfB active site into residues at the corresponding positions of LrN1 GtfB, which possess 4,6-α-glucosyltransferase activity. This alters the microconformation of the active site or its interaction with the substrate, thereby affecting the type and proportion of glycosidic bonds in the product.

[0007] This invention provides a GtfB-type α-glucosyltransferase mutant derived from *Lactobacillus fermentum* 2970. Its amino acid sequence comprises an amino acid sequence obtained by mutating one or more amino acids at positions 783, 785, 790, 376, and 413 of the parent amino acid sequence GtfB-ΔN. The parent amino acid sequence is shown in SEQ ID NO.2, or any sequence containing the parent amino acid GtfB-ΔN shown in SEQ ID NO.2.

[0008] In one embodiment of the present invention, the sequence of the parent amino acid GtfB-ΔN is shown in SEQ ID NO.2.

[0009] In one embodiment of the present invention, the GtfB-type α-glucosyltransferase mutant can be obtained by mutating one or more amino acid positions from the GtfB-type α-glucosyltransferase shown in SEQ ID NO.1 (equivalent to position 783 of sequence 2), positions 1400 (equivalent to position 785 of sequence 2), positions 1405 (equivalent to position 790 of sequence 2), positions 991 (equivalent to position 376 of sequence 2), and positions 1028 (equivalent to position 413 of sequence 2). The parent amino acid GtfB-ΔN shown in SEQ ID NO.2 is obtained by truncating 615 amino acid residues from the N-terminus of the GtfB-type α-glucosyltransferase shown in SEQ ID NO.1.

[0010] GtfB type α-glucosyltransferase (SEQ ID NO.1):

[0011]

[0012] The amino acid sequence of the parent amino acid GtfB-ΔN (SEQ ID NO.2):

[0013] MFGKDGRIATGLYKWDKNNQWYYFDPVTYLKVTNKWVDGNYYDEDGAQAISKLVTINNRLYYFDDQGKEISNQFRTIHGDKYYFGNDSAAVTGQQTIDGKVYKFSNYGYLLGNRYGKIENGKLNIYSLADHSLIKTVEAGPWENMAYSMDSNSINNIDGYISYTGWYRPYGTSQDGKTWYPTTVADWRPILMYVWPSKDVQVKFIQYFVNHGYENSNYGLTAGSVKDLSENTASINLNEVAQNLRYVIEQHIVAAKSTSQLANDINNFITTIPELSASSELPDESGSGQVIFVNNDNTSYADSKYRLMNRTVNNQTGNDNSDYCPEFVVGNDIDNSNPVVQAENLNWEYFLLNYGKLMGYNQDGNFDGFRIDAADDMDADVLDQIGQLMNDMYHMKGNPQNANNHLSYNEGYGPGAARMLNKKGNPQLFMDARECNTLENVLGRANNRDTISHLVTDSIVNRQNDVTENEATPNWSYVTNHDIRNNLINGLIIKDHPGMGSAYKAEYANQAWQEFYADQKKTDKQYAQYNVPAQYAILLSNKDTVPQIYYGDLYNETAQYMQEKSIYYDAITTLMKARKQFVSGGQTMTKLSDNLIASVRYGKGVANANSEGTDSLSRTSGMAVIVGNNPQMAEQTISINMGRAHANEQYRNLLDTTDNGLTYNADGAENPETLTTDDNGILKVTVKGYSNPYVSGYLGVWVPVASGNQDVTTNAATVSADSNKIFESNAALDSHMIYEDFSMYQPKPTSTENHAYNIIAQNAELFNNLGITDFWMAPAYTQAGTSRYNEGYSVADRYNLGTNANPTKYGSGEELANAIAALHSAGLKVQEDIVMNQMIGLPGQEAVTVTRADNRGMQTYVNGKTYANQMYFAYTTGGGNGQETYGGKYLSELQSKYPDLFTTRAISTGVAPDPTTHITKWSAKYENGTSLQNIGIGLAVKLANGDYAYLNDSNNKAFNTTLPETMSSTDYYANIEDN

[0014] The nucleotide sequence encoding the parent amino acid GtfB-ΔN (SEQ ID NO.3)

[0015]

[0016] In one embodiment of the present invention, the mutant is:

[0017] The alanine at position 783 of the GtfB type α-glucosyltransferase, whose amino acid sequence is as shown in SEQ ID NO.2, is mutated to tyrosine and named: A783Y;

[0018] Alternatively, it can be obtained by mutating the threonine at position 785 of the GtfB type α-glucosyltransferase with an amino acid sequence as shown in SEQ ID NO.2 to glutamic acid, and named: T785E;

[0019] Alternatively, it can be obtained by mutating glutamic acid at position 790 of the GtfB type α-glucosyltransferase with an amino acid sequence as shown in SEQ ID NO.2 to aspartic acid, and named: E790D;

[0020] Alternatively, the aspartic acid at position 376 of the GtfB type α-glucosyltransferase with an amino acid sequence as shown in SEQ ID NO.2 can be mutated to histidine, leucine, asparagine, threonine, or tyrosine, and named as follows: D376H, D376L, D376N, D376T, D376Y, respectively.

[0021] Alternatively, the amino acid sequence of the GtfB type α-glucosyltransferase shown in SEQ ID NO.2 can be obtained by mutating the glycine at position 413 to arginine, alanine, aspartic acid, asparagine, tyrosine, or histidine, and named as follows: G413R, G413A, G413D, G413N, G413Y, G413H.

[0022] In one embodiment of the present invention, the GtfB type α-glucosyltransferase mutant may further be: obtained by mutating one or more amino acids from the following positions of GtfB type α-glucosyltransferase as shown in SEQ ID NO.1: A1398Y, T1400E, E1405D, D991H, D991L, D991N, D991T, D991Y, G1028R, G1028A, G1028D, G1028N, G1028Y, G1028H.

[0023] In one embodiment of the present invention, the nucleotide sequence encoding the parent amino acid GtfB-ΔN is shown in SEQ ID NO.3.

[0024] The present invention also provides a gene encoding the above-mentioned mutant.

[0025] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0026] In one embodiment of the present invention, the recombinant vector is pET15b as the expression vector.

[0027] The present invention also provides the above-mentioned mutants or recombinant cells carrying the above-mentioned genes or carrying the above-mentioned recombinant vectors.

[0028] In one embodiment of the present invention, the recombinant cells are expressed using bacteria or fungi as expression hosts.

[0029] In one embodiment of the present invention, the recombinant cells are expressed using Escherichia coli BL21(DE3) as the expression host.

[0030] This invention also provides a method for constructing Lf2970 GtfB active site mutants. The mutants are obtained by selecting mutations at donor subsites A783, T785, and E790, and acceptor subsites D376 and G413 through structural and sequence alignment. These mutations are then replaced with the corresponding amino acids in LrN1 GtfB, resulting in mutants A783Y, T785E, E790D, D376H, and G413R.

[0031] This invention also provides a method for preparing α-glucan using Lf2970 GtfB and the above-mentioned mutant. The method uses amylose as a substrate, first dissolving the amylose in sodium hydroxide solution, then neutralizing it with hydrochloric acid, adding buffer solution, and then adding an enzyme for modification to obtain the synthesized α-glucan. The synthesized product has different glycosidic bond compositions and molecular weight distributions. The α-glucan has (α1→4) glycosidic bonds, (α1→3) glycosidic bonds, and (α1→6) glycosidic bonds.

[0032] In one embodiment of the present invention, the concentration of amylose is 0.25%-20%.

[0033] In one embodiment of the present invention, the concentration of the buffer system used is 10-50 mM and the pH is 4-6.

[0034] In one embodiment of the present invention, the reaction temperature is controlled at 37-40°C and the reaction time is 4-48 hours.

[0035] In one embodiment of the present invention, the amount of enzyme added in the reaction is 10-50 U / g dry starch.

[0036] The present invention also provides a method for improving the activity of GtfB type α-glucosyltransferase, wherein the method comprises: mutating alanine at position 783 of the GtfB type α-glucosyltransferase as shown in SEQ ID NO.2 to tyrosine; or mutating threonine at position 785 of the GtfB type α-glucosyltransferase as shown in SEQ ID NO.2 to glutamic acid; or mutating glutamic acid at position 790 of the GtfB type α-glucosyltransferase as shown in SEQ ID NO.2 to aspartic acid; or mutating aspartic acid at position 376 of the GtfB type α-glucosyltransferase as shown in SEQ ID NO.2 to histidine, leucine, asparagine, threonine, or tyrosine; or mutating glycine at position 413 of the GtfB type α-glucosyltransferase as shown in SEQ ID NO.1 to arginine, alanine, aspartic acid, asparagine, tyrosine, or histidine.

[0037] The present invention also provides a method for preparing α-glucan, wherein the method comprises using the above-mentioned mutant or the above-mentioned recombinant cell or the recombinant mutant enzyme expressed by the above-mentioned recombinant cell as a catalyst, and using starch or starch derivative as a substrate, to prepare α-glucan by fermentation; wherein the α-glucan has (α1→4) glycosidic bonds, (α1→3) glycosidic bonds, and (α1→6) glycosidic bonds.

[0038] In one embodiment of the present invention, the α-glucan has a (α1→3) bond ratio of 6.3% to 23.2% and a (α1→6) bond ratio of 1% to 10.6%.

[0039] In one embodiment of the present invention, the method is to use starch or starch derivatives as a substrate, wherein the starch includes amylopectin and amylose, and the starch derivatives include maltodextrin and maltodextrin (degree of polymerization ≥3).

[0040] In one embodiment of the present invention, when using amylose as a substrate, the starch is first dissolved with alkali, then neutralized with acid, buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

[0041] In one embodiment of the present invention, when amylopectin is used as a substrate, a buffer solution is added to the starch, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

[0042] In one embodiment of the present invention, when dextrin is used as a substrate, a buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

[0043] In one embodiment of the present invention, the buffer system is a buffer solution with a concentration of 10-50 mM and a pH of 4-6.

[0044] In one embodiment of the present invention, the amount of the mutant or the recombinant mutant enzyme added is 10-50 U / g dry starch.

[0045] In one embodiment of the present invention, the reaction temperature is 37-40°C and the reaction time is 4-48 hours.

[0046] The present invention also provides the application of the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned mutant, or the above-mentioned method synthesized α-glucan in the fields of food, health products, beverages, medicine and additives.

[0047] The present invention also provides the application of the above-mentioned mutant or recombinant cell in the preparation of α-glucan. The application is to use the above-mentioned mutant or recombinant cell or recombinant mutant enzyme expressed by the above-mentioned recombinant cell as a catalyst and starch or starch derivative as a substrate to prepare α-glucan by reaction. The α-glucan has (α1→4) glycosidic bonds, (α1→3) glycosidic bonds, and (α1→6) glycosidic bonds.

[0048] In one embodiment of the present invention, starch or starch derivatives are used as substrates, wherein the starch includes amylopectin and amylose, and the starch derivatives include maltodextrin and maltodextrin (degree of polymerization ≥3).

[0049] In one embodiment of the present invention, when using amylose as a substrate, the starch is first dissolved with alkali, then neutralized with acid, buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

[0050] In one embodiment of the present invention, when amylopectin is used as a substrate, a buffer solution is added to the starch, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan; preferably, when dextrin is used as a substrate, a buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

[0051] In one embodiment of the present invention, the substrate concentration is 0.25% to 20% (w / v), the buffer system concentration is a 10 to 50 mM buffer solution with a pH of 4 to 6, the amount of mutant or recombinant mutant enzyme added is 10-50 U / g dry starch, the reaction temperature is 37 to 40°C, and the reaction time is 4 to 48 h.

[0052] Beneficial effects

[0053] (1) The Lf2970 GtfB mutant provided by this invention can effectively transform substrates, and the resulting α-glucan / oligosaccharide introduces new (α1→6) bonds and (α1→4,6) branches on the basis of the wild type, increasing the total (α1→6) bond ratio from <1% to 10.6%. Through these mutations, a novel enzyme with 4,3 / 6-α-glucosyltransferase activity is generated, expanding the GH70 family enzyme library, and proposing key sites affecting the bond type specificity of GtfB-type α-glucosyltransferases at the molecular structure level. α-glucans with multiple bond types are not only structurally unique, but also have promising applications in the food, healthcare, and biomaterials industries.

[0054] (2) The Lf2970 GtfB mutant provided by this invention can effectively improve enzyme activity, thereby enabling efficient conversion of starch substrates. Among them, the total enzyme activity of mutants T785E, G413N and G413D is increased to about 4 times that of the wild type.

[0055] (3) The mutant-modified amylose products provided by this invention have diverse molecular weight distributions. The smallest product has a molecular weight of only 0.46 kDa, produced by mutant G413R; while mutant D376Y can produce α-glucan with a molecular weight of 4710.68 kDa, which is three times the molecular weight of the substrate. These α-oligosaccharides / polysaccharides of different sizes have broad application value. Attached Figure Description

[0056] Figure 1 This is a partial structural diagram of the active centers of Lf2970 GtfB (left) and LrN1 GtfB (right) with acarbose.

[0057] Figure 2 This is a schematic diagram of the subsite loop structure of the Lf2970 GtfB (left) and LrN1 GtfB (right) donors and the structure of maltopentose.

[0058] Figure 3 The results are polyacrylamide gel electrophoresis of Lf2970 GtfB wild type and its mutants.

[0059] Figure 4 It is Lf2970 GtfB wild type and mutant. 1 H nuclear magnetic resonance spectrum. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0061] The amylose used in the following examples was purchased from Sigma Aldrich.

[0062] The culture media involved in the following examples are as follows:

[0063] LB medium: 1% (w / v) peptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl.

[0064] The detection methods involved in the following embodiments are as follows:

[0065] Methods for detecting α-glucosyltransferase activity: The activity of wild-type and mutant enzymes was determined using the amylose iodine staining method. Using 0.25% (w / v) amylose as the substrate, the enzyme concentration was 9 μg / mL, and the reaction was carried out at 40℃. The buffer system was 25 mM NaAC, 5 mM CaCl2, pH 5.5. One unit of enzyme activity (U) was defined as the amount of enzyme required to catalyze the conversion of 1 mg of amylose per minute. Hydrolytic activity was determined using the 3,5-dinitrosalicylic acid method (DNS), with the same reaction system and enzyme concentration as for total enzyme activity. One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 mg of reducing sugar from amylose per minute under optimal conditions.

[0066] Glycosidic bond composition test of the product:

[0067] This invention uses NMR and methylation analysis on Lf2970 GtfB-ΔN wild-type and mutant modified amylose products to determine the type, content and degree of glycosidic bonds.

[0068] 1 H nuclear magnetic resonance:

[0069] Take 20 mg of the lyophilized sample, add 1 mL of D2O, boil for 1 h, and immediately lyophilize. After lyophilization, add another 1 mL of D2O, boil for 1 h, and immediately lyophilize for later use. Finally, dissolve the sample in D2O, boil in a water bath for 1 h, and then record the one-dimensional proton NMR spectrum on an AVANCE-600 MHz spectrometer (Bruker, Germany) with a probe temperature of 25 °C.

[0070] Methylation:

[0071] 60 mg of lyophilized oligosaccharides / polysaccharides were dissolved in 20 mL of DMSO, followed by the addition of 20 mL of NaOH solution dissolved in DMSO and reacted for 1 h. Then, CH3I liquid was added dropwise and the reaction was carried out under ice bath conditions for 2 h for methylation. The methylated products were dialyzed using a 1000 Da dialysis bag, extracted with chloroform, dehydrated with sodium sulfite, and dried under nitrogen. The products were then hydrolyzed with trifluoroacetic acid solution (2 M), reduced with NaBD4, and acetylated with acetic anhydride and pyridine (1:1, v / v). Partially methylated sugar alcohol acetates (PMAAs) were analyzed using a gas chromatography-mass spectrometry (GC-MS) system (Agilent Technologies, Santa Clara, USA). The obtained EI-MS spectra were compared with the CCRC spectral database - PMAAs to determine the type and ratio of glycosidic bonds.

[0072] Product molecular weight test:

[0073] High-performance gel filtration chromatography (HPGFC) was used to determine the relative molecular weight distribution of enzyme-modified amylose products. The lyophilized sample was dissolved in purified water to a final concentration of 5 mg / mL and filtered through a 0.45 μm cellulose ester microporous membrane. 30 μL of the sample was injected into a gel filtration chromatograph equipped with a refractive index detector to monitor the molecular weight distribution.

[0074] Example 1: Preparation of GtfB type α-glucosyltransferase active site mutant

[0075] The specific steps are as follows:

[0076] 1. Selecting mutation sites

[0077] The binding of GtfB from Lf2970 (amino acid sequence shown in SEQ ID NO.2) and GtfB from LrN1 to the substrate at the donor and acceptor subsites was compared. Based on the crystal structure of GtfB from LrN1 and acarbose (PDB: 8HW3), the AlphaFold structure of GtfB from Lf2970 was superimposed to obtain the active sites of the two enzymes and the structure of acarbose, identifying the key amino-terminal G413 and D376 that interact with acarbose in both enzymes.

[0078] Based on the crystal structure (PDB: 5JBF) of GtfB from Lr121 binding to maltpentose (G5) at the donor subsite, binding models of G5 at the donor subsites of LrN1 GtfB and Lf2970 GtfB were obtained by Pymol superposition. Key amino-terminal sites A783, T785, and E790 on loop A2 were selected through multiple sequence alignment. The sequence alignments of the characterized GtfB-type α-glucosyltransferases at loop A2 are shown in Table 1.

[0079] Table 1: Sequence alignment of characterized GtfB type α-glucosyltransferases at loop A2

[0080]

[0081] 2. Design mutation primers to mutate the selected sites to the corresponding amino acids of LrN1 GtfB, namely D376H, G413R, A783Y, T785E and E790D. The primer designs are shown in Table 2 below.

[0082] The aspartic acid at position D376 was mutated to leucine, asparagine, threonine, and tyrosine; the glycine at position G413 was mutated to alanine, aspartic acid, asparagine, tyrosine, and histidine. The primer designs are shown in Table 2 below.

[0083] Table 2: Primer sequence listing for mutants

[0084]

[0085] Note: Underlined bases are mutant bases.

[0086] 3. Construction of pET15b-Lf2970 GtfB-ΔN

[0087] The gene fragment of the Lf2970 GtfB-ΔN enzyme, with an artificially synthesized nucleotide sequence as shown in SEQ ID NO.3, was used as an expression vector to construct the recombinant plasmid pET15b-Lf2970GtfB-ΔN, which contains XholⅠ and BamHI restriction enzyme sites. The plasmid was then expressed using E. coli BL21(DE3) expression strain.

[0088] 4. Using pET15b-Lf2970 GtfB-ΔN as a template, the target amino acid was mutated using whole plasmid PCR.

[0089] The PCR program was as follows: pre-denaturation at 98℃ for 3 min, followed by 30 cycles (denaturation at 98℃ for 20 s; annealing at 55℃ for 30 s; extension at 68℃ for 9 min), and extension at 68℃ for 10 min.

[0090] The PCR fragments after the reaction were digested with DpnI enzyme and then introduced into *E. coli* DH5α competent cell clone plasmids using the heat shock method. The successfully sequenced plasmids were then introduced into *E. coli* BL21 competent cells; recombinant *E. coli* cells containing mutants were prepared as follows: BL21 / pET15b-D376H-ΔN, BL21 / pET15b-G413R-ΔN, BL21 / pET15b-A783Y-ΔN, BL21 / pET15b-T785E-ΔN, BL21 / pET15b-E790D-ΔN, BL21 / pET15b-D376L-ΔN, and BL21 / pET15... b-D376N-ΔN, BL21 / pET15b-D376T-ΔN, BL21 / pET15b-D376Y-ΔN, BL21 / pET15b-G413R-ΔN, BL21 / pET15b-G41 3A-ΔN, BL21 / pET15b-G413D-ΔN, BL21 / pET15b-G413N-ΔN, BL21 / pET15b-G413Y-ΔN, BL21 / pET15b-G413H-ΔN.

[0091] At the same time, recombinant Escherichia coli containing wild-type enzymes was obtained: BL21 / pET15b-Lf2970 GtfB-ΔN.

[0092] Example 2: Expression and purification of Lf2970 GtfB-ΔN and mutants

[0093] The specific steps are as follows:

[0094] (1) The recombinant Escherichia coli containing wild-type and mutant genes obtained in Example 1 were inoculated into LB medium containing ampicillin (100 μg / ml) and cultured at 37°C and 200 rpm. When OD 600 nm When the enzyme concentration reached 0.4–0.6, after ice bath treatment for 15 minutes, isopropyl-β-thiogalactoside (IPTG, 0.1 mM) was added, and enzyme production was induced at 16 °C and 160 r / min for 20 h; fermentation broths were then prepared.

[0095] (2) The obtained fermentation broth was centrifuged at 4℃ and 8000g for 30 min to collect cells. The cells were resuspended in 10 mL of 25 mM Tris-HCl (250 mM NaCl, pH 7.5) at a concentration of 1 g of cell. The cells were then sonicated in an ice bath for 20 min (30% power, 2 s pulverization, 3 s interval). The supernatant obtained after centrifuging the disrupted broth at 4℃ and 8000g for 40 min was the crude enzyme solution, which was then prepared as follows:

[0096] Pure enzyme solution containing wild-type Lf2970 GtfB-ΔN, crude enzyme solution containing D376H, crude enzyme solution containing G413R, crude enzyme solution containing A783Y, crude enzyme solution containing T785E, crude enzyme solution containing E790D, crude enzyme solution containing D376H, crude enzyme solution containing D376L, crude enzyme solution containing D376N, crude enzyme solution containing D376T, crude enzyme solution containing D376Y, crude enzyme solution containing G413R, crude enzyme solution containing G413A, crude enzyme solution containing G413D, crude enzyme solution containing G413N, crude enzyme solution containing G413Y, and crude enzyme solution containing G413H.

[0097] (3) The crude enzyme solution obtained in step (2) was purified by nickel affinity chromatography. Elution was performed sequentially 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. The flow-through fractions were collected, and purity was verified by polyacrylamide gel electrophoresis. The results are as follows: Figure 3 As shown.

[0098] The results showed that both the wild-type enzyme and the mutant enzyme of the present invention were expressed.

[0099] (4) Detection of enzyme activity

[0100] The enzyme activity of the pure enzyme solution from step (3) was tested, and the results are shown in Table 3.

[0101] Table 3: Enzyme activity of Lf2970 GtfB wild-type and mutant strains

[0102]

[0103]

[0104] a is

[0105] As shown in Table 3, the enzyme activities of the mutants showed varying degrees of change compared to the wild type. Except for mutant G413R, the total enzyme activity of all mutants at position 413 was significantly increased, with the activity increasing approximately 3.5-fold after mutation to asparagine. However, the total enzyme activity of mutants at position 376 showed different changes; the activity of mutant D376N increased to about 3 times that of the wild type, while the activity of D376Y decreased significantly. Mutating T785 on LoopA2 to glutamate also increased the total enzyme activity of the mutants by about 4-fold.

[0106] Example 3: Preparation of α-glucan using Lf2970 GtfB-ΔN wild-type and mutant

[0107] The specific steps are as follows:

[0108] (1) Debranching of potato starch to obtain amylose; specifically:

[0109] Potato starch was dissolved in buffer (20 mM, pH 4.6 sodium acetate buffer) to a final concentration of 2% (w / v), and then boiled in a water bath for 1 h to ensure uniform dispersion. After cooling to room temperature, 1% (w / v) of commercial pullulanase (enzyme activity ≥1000 U / mL) was added, and the mixture was reacted at 60°C for 24 h, followed by boiling in a water bath for 15 min to inactivate the enzyme. The mixture was centrifuged, the supernatant was collected, and dried at 40°C. The product was ground, sieved, washed three times with 90% methanol, and dried again at 40°C to obtain debranched potato starch.

[0110] (2) The debranched potato starch obtained in step (1) was first dissolved in 1M sodium hydroxide solution to make the final starch concentration 4% (w / v), then neutralized with an equal amount of 1M hydrochloric acid, and then buffer solution (25mM NaAc, 5mM CaCl2, pH 5.5) was added to make the final concentration of potato starch 1% (w / v). After cooling to room temperature, Lf2970 GtfB-ΔN and mutant pure enzyme solution prepared in Example 2 were added at a concentration of 50U / g. The reaction was carried out at 40℃ for 24h, and the reaction was terminated by boiling water bath for 15min. The product was then freeze-dried to obtain freeze-dried powder.

[0111] Example 4: Analysis of enzyme action products of Lf2970 GtfB-ΔN wild-type and mutant

[0112] 1. Analysis of molecular weight and structure of the product

[0113] The products prepared in Example 3 were subjected to one-dimensional 1H NMR spectroscopy and molecular weight analysis, and the results are shown in Tables 4 and 5.

[0114] Table 4: Structures of Lf2970 GtfB-ΔN wild-type and site-directed mutant products

[0115]

[0116] a blank represents the substrate amylose.

[0117] Table 5: Molecular weight and glycosidic bond composition of products from D376,G413 site mutants

[0118]

[0119] product 1 H nuclear magnetic resonance spectrum as follows Figure 4 As shown.

[0120] The results show:

[0121] (1) Regarding the molecular weight distribution of the products, the molecular weight of mutants G413R and D376H was significantly reduced, showing a single-peak distribution, indicating that the mutation at the recipient subsite enhanced the hydrolytic activity of the enzyme. The molecular weight distribution of the products of the donor side mutants showed a similar situation to that of the wild type, showing a bimodal distribution. Among them, the molecular weight of the E790D product increased to 3101.49 kDa, which is about 4.8 times that of the wild type, indicating that the key site of the donor subsite can affect the enzyme's continuous synthesis ability.

[0122] (2) The wild-type product contains almost no (α1→6) bonds, while the product bond type of the receptor subsite mutants G413R and D376H has undergone structural changes. The proportion of (α1→6) bonds is significantly increased while the proportion of (α1→3) bonds decreases. This indicates that the mutation changes the binding orientation of the receptor substrate at the active site, causing the transglycosylation to switch between forming (α1→6) bonds and (α1→3) bonds.

[0123] (3) To further investigate the role of the G413 and D376 sites in bond-forming specificity, they were mutated to different types of amino acids, including mutating aspartic acid at the D376 site to amino acids, asparagine, threonine, and tyrosine; and mutating glycine at the G413 site to alanine, aspartic acid, asparagine, tyrosine, and histidine. The structural properties of the products are shown in Table 5. After mutating the D376 site to asparagine, the (α1→6) bond ratio increased to 10.6%, nearly 7 times that of the wild type, while the (α1→3) bond ratio decreased from 21.5% to 13.6%. Other mutants of the G413 site all showed a higher (α1→4) bond ratio and a weaker transglycosylation ability.

[0124] 2. Methylation analysis of the product

[0125] Based on the data of the above mutants, methylation analysis was performed on the mutants G413R, D376H and D376N of Lf2970 GtfB-ΔN to further determine the proportion and branching of each glycosidic bond in the product.

[0126] The methylated structures are shown in Table 6.

[0127] Table 6: Methylation Analysis

[0128]

[0129] The results showed that the proportion of (α1→3,4) branches in mutant D376N was reduced, while the newly synthesized (α1→6) bonds were mainly presented in the form of branches.

[0130] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A GtfB-type α-glucosyltransferase mutant, characterized in that, The GtfB-type α-glucosyltransferase mutant is obtained by mutating threonine at position 785 of the GtfB-type α-glucosyltransferase, as shown in SEQ ID NO.2, to glutamic acid.

2. The gene encoding the mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector.

4. The recombinant cell according to claim 3, characterized in that, The recombinant cells use bacteria or fungi as expression hosts.

5. A method for increasing the activity of GtfB type α-glucosyltransferase, characterized in that, The method involves mutating threonine at position 785 of the GtfB type α-glucosyltransferase, whose amino acid sequence is shown in SEQ ID NO.2, to glutamic acid.

6. A method for preparing α-glucan, characterized in that, The method involves using the mutant described in claim 1, the recombinant cell described in claim 3 or 4, or the recombinant mutant enzyme expressed by the aforementioned recombinant cell as a catalyst, and starch or a starch derivative as a substrate to prepare α-glucan; the α-glucan has α1→4 glycosidic bonds, α1→3 glycosidic bonds, or α1→6 glycosidic bonds; the starch is amylopectin or amylose, and the starch derivative is maltodextrin or maltodextrin with a degree of polymerization ≥3.

7. The method according to claim 6, characterized in that, When using amylose as a substrate, the starch is first dissolved with alkali, then neutralized with acid, buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

8. The method according to claim 6, characterized in that, When amylopectin is used as a substrate, a buffer solution is added to the starch, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

9. The method according to claim 6, characterized in that, When maltodextrin is used as a substrate, a buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

10. The method according to claim 6, characterized in that, The substrate concentration is 0.25%~20% (w / v), the buffer system concentration is 10~50 mM, pH 4~6, the amount of mutant or recombinant mutant enzyme added is 10-50 U / g dry starch, the reaction temperature is 37~40 ℃, and the time is 4 h~48 h.

11. The use of the mutant of claim 1 or the recombinant cell of claim 3 or 4 in the preparation of α-glucan, characterized in that, Using the mutant of claim 1, the recombinant cell of claim 3 or 4, or the recombinant mutant enzyme obtained by expressing the above-mentioned recombinant cells as a catalyst, and starch or starch derivatives as a substrate, α-glucan is prepared by reaction; the α-glucan has α1→4 glycosidic bonds, α1→3 glycosidic bonds, or α1→6 glycosidic bonds; the starch is amylopectin or amylose, and the starch derivative is maltodextrin or maltodextrin with a degree of polymerization ≥3.

12. The application according to claim 11, characterized in that, When using amylose as a substrate, the starch is first dissolved with alkali, then neutralized with acid, buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

13. The application according to claim 11, characterized in that, When amylopectin is used as a substrate, a buffer solution is added to the starch, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

14. The application according to claim 11, characterized in that, When maltodextrin is used as a substrate, a buffer solution is added, and then the mutant or the recombinant mutant enzyme is added for modification to obtain the synthesized α-glucan.

15. The application according to claim 11, characterized in that, The substrate concentration is 0.25%~20% (w / v), the buffer system concentration is 10~50 mM, pH 4~6, the amount of mutant or recombinant mutant enzyme added is 10-50 U / g dry starch, the reaction temperature is 37~40 ℃, and the time is 4 h~48 h.

Images