Glucosyltransferase mutant and its application

Abstract

The present invention belongs to the field of biotechnology and specifically relates to mutants of glucosyltransferases and their applications. The mutants of the glucosyltransferases are obtained by combining the amino acid sequence of the glucosyltransferase shown in SEQ ID NO.1 with only one or more of the following mutations: Q60S, Q60A, Q60M, Y452I, Y452V, Y452R, R455K, R455I, R455V, and R455H. Compared to the wild-type, the glucosyltransferase mutants obtained by the present invention have significantly improved catalytic activity and can catalyze maltose to produce maltooligosaccharides. They can be used in the preparation of modified starch to increase the content of slowly digestible starch and resistant starch. Therefore, the present invention has high application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a mutant of glucosyltransferase and application thereof. Background Art

[0002] Starch is one of nature's most abundant resources and a primary source of energy for humans. Consuming high levels of rapidly digestible starch can lead to metabolic diseases such as diabetes, obesity, and cardiovascular disease. Studies have shown that slowly digestible starch is slowly digested and absorbed in the small intestine, helping to maintain blood sugar homeostasis. Studies have shown that starch modification can significantly increase its content, suggesting broad application prospects for new functional foods in the future food industry.

[0003] Starch is a natural, renewable, and green resource. Products derived from starch have broad market applications in numerous industries, including food, pharmaceuticals, and papermaking. However, this widely used natural industrial raw material is no longer able to meet the demands of modern industry due to limitations such as cold water insolubility, susceptibility to aging and dehydration, and insufficient emulsification. To improve its natural quality, natural starch is currently modified primarily through physical, chemical, enzymatic, or combined methods. Enzymatic modification is safer and healthier than physical and chemical modification, offering advantages such as high specificity, high yield, low byproduct count, and minimal purification requirements. Research on the use of glucosyltransferases for starch modification is extensive, with applications such as replacing chemical gelatin, delaying starch aging and extending shelf life, and using them as fat substitutes and emulsion stabilizers in yogurt. However, research on the effects of glucosyltransferases on the fine structure and functional properties of starch remains limited.

[0004] However, the currently known glucosyltransferases have multiple functions including disproportionation, cyclization, coupling, and hydrolysis. By improving the disproportionation activity of glucosyltransferases and their modification effect on starch, we can not only open up new research directions for glucosyltransferases in starch modification, but also have important significance for the preparation and improvement of slowly digestible starch, laying the foundation for the industrialized production of functional foods in my country. Summary of the Invention

[0005] The present invention is based on Thermus filiformis A large number of glucosyltransferases were screened to obtain mutants with single-point mutations, and combined mutations were further obtained through saturation mutagenesis. Experiments showed that compared with the wild type, the thermal stability and catalytic activity were significantly improved, thus completing the present invention.

[0006] The present invention first provides a glucosyltransferase mutant, which is derived from Thermus filiformis The amino acid sequence of the glucosyltransferase shown in SEQ ID NO. 1 is based on the following mutations: one or more combinations of Q60S, Q60A, Q60M, Y452I, Y452V, Y452R, R455K, R455I, R455V, and R455H.

[0007] Preferably, the following mutations are present: Q60S / Y452I, Q60S / R455K, Q60S / Y452I / R455K, Q60S / Y452I / R455I, Q60S / Y452I / R455V, Q60S / Y452I / R455H, Q60S / R455K / Y452I, Q60S / R455K / Y452V, Q60S / R455K / Y452R, Q60S / R455K / Y452W.

[0008] The present invention provides a gene encoding the mutant, preferably, the nucleotide sequence of which is obtained by performing a mutation based on the nucleotide sequence shown in SEQ ID NO. 2.

[0009] The present invention provides a recombinant expression vector containing the gene, specifically a prokaryotic expression vector, such as an Escherichia coli expression vector.

[0010] The present invention also provides a recombinant bacterium containing the encoding gene or the recombinant expression vector, specifically a bacterium, such as Escherichia coli.

[0011] The present invention also provides the use of the mutant or the encoding gene in catalyzing the production of maltooligosaccharides.

[0012] The present invention also provides a method for catalytically producing maltooligosaccharides, wherein the catalytic reaction system specifically comprises: 20 mg / mL maltose; the mutant; 20 mM acetate buffer, pH 6.0; and reaction conditions: catalytic reaction at 50° C. for 12 hours.

[0013] The present invention particularly provides a method for preparing modified starch, which uses starch as a substrate and produces modified starch under the catalysis of an enzyme combination, wherein the enzyme combination is: the mutant and glycogen branching enzyme, or the mutant and saccharifying enzyme, isoamylase and pullulanase, or the mutant and amylosucrase;

[0014] The substrate is one or more of corn starch, potato starch, tapioca starch or pea starch.

[0015] Preferably, the glycogen branching enzyme is RpGBE derived from Rhodothermus erythroxysporum.

[0016] The reaction system during catalysis was as follows: 20 mM acetate buffer, pH 6.0, 40 mg / mL starch slurry, 0.4 U / mL TfGT, and 0.8 U / mL RpGBE; specifically, the reaction was carried out at 70° C. for 4 h.

[0017] The glucosyltransferase mutant obtained in this invention has significantly improved catalytic activity compared to the wild type, and can catalyze maltose to produce maltooligosaccharides. Furthermore, studies have established a method for preparing modified starch, and studies have shown that the content of slowly digestible starch and resistant starch increased by 3.2% and 2.4%, respectively. Therefore, this invention has high application value. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the percentage concentrations mentioned in the present invention and the examples are all mass / mass (W / W, unit: g / 100 g) percentage concentrations, mass / volume (W / V, unit: g / 100 mL) percentage concentrations, or volume / volume (V / V, unit: mL / 100 mL) percentage concentrations.

[0019] The methods used in the following examples are conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0020] Unless otherwise specified, materials or reagents with the same names used in the examples are identical. The methods for obtaining the various biological materials described in the examples merely provide an experimental approach to achieve the disclosed objectives and should not be construed as limiting the sources of biological materials used in the practice of the present invention. In fact, the sources of biological materials used are diverse; any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0021] The primers and genes used in the present invention were synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd.

[0022] The present invention is further described in detail below in conjunction with examples. The examples are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are given. The examples will help to understand the present invention, but the protection scope of the present invention is not limited to the following examples. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0023] Example 1 Expression and purification of glucosyltransferase TfGT

[0024] (1) 4-α-glucosyltransferase was searched in the UniProt database (www.uniprot.org). 4GT from thermophilic sources was screened by keywords and functions. Sequence alignment was performed using clustW. Homology was controlled using HHfilter. Thermus filiformis The original protein sequence of the glucosyltransferase from Thermus filiformis The glucosyltransferase (its amino acid sequence is shown in SEQ ID NO.1) was codon-optimized, and the gene sequence had a nucleotide sequence after the Escherichia coli preferred codon (SEQ ID NO.2). The gene was sent to Jiangsu Jinweizhi Biotechnology Co., Ltd. for gene synthesis and constructed into the expression vector pET-21a.

[0025] (2) The recombinant plasmid pET-TfGT containing the above-mentioned glucosyltransferase gene (the optimized sequence SEQ ID NO. 2) was transformed into Escherichia coli BL21 (DE3) for exogenous expression and purification.

[0026] Example 2 Protein expression and purification

[0027] The recombinant plasmid containing the above-mentioned glucosyltransferase gene (the optimized sequence SEQ ID NO. 2) was transformed into Escherichia coli for exogenous expression and purification.

[0028] (1) The E. coli expression recombinant plasmid pET-TfGT was transferred into E. coli BL21 (DE3) to obtain recombinant bacteria.

[0029] (2) Pick a single clone and transfer it to 5 mL of LB liquid medium and culture until OD 600 When the concentration of IPTG was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM and the expression was induced for 16 h.

[0030] (3) The above culture solution was centrifuged to collect the bacterial cells.

[0031] (4) Bacterial disruption: Use an ultrasonic disruptor to break the cell wall and centrifuge to obtain the supernatant.

[0032] (5) Heat purification: Heat-treat the supernatant in a 70°C water bath for 1 hour, and centrifuge to obtain the heat-treated protein.

[0033] (6) Ni purification: The supernatant obtained after crushing and centrifugation is bound to a nickel column, eluted, and purified to obtain pure protein.

[0034] Example 3 Mutant Construction and Verification

[0035] (1) Construction of TfGT glucosyltransferase mutants and enzyme activity determination

[0036] Will come from Thermus filiformis Molecular docking of the glucosyltransferase (TfGT) with the substrate maltose was performed to obtain the complex structure. Subsequently, Rosseta was used, combined with free energy analysis and other analyses to obtain sites 60, 452, and 455 that may be related to catalytic activity. Saturation mutagenesis was carried out around the above sites, that is, the above sites were mutated into 20 different amino acid residues, thereby constructing a mutant library and measuring its dismutation activity towards maltose. An enzyme activity assay system for the mutant library was established: including maltose, 20 mg / mL; acetate buffer, 20 mM, pH 6.0; glucosyltransferase TfGT, 2 μg / mL;

[0037] The glucosyltransferase is selected from Thermus filiformis , which is labeled A0A0A2X7D9 on UniProt. The expression and preparation method of the enzyme is the same as that of Example 1 and Example 2. The catalytic reaction was carried out at 50°C for 30 minutes, and the final reaction sample was subjected to liquid chromatography detection. Definition of specific enzyme activity (1U / mg): the amount of 1 micromole of glucose catalyzed by each milligram of TfGT per minute. Assuming that TfGT has good catalytic activity for maltose, the glucose concentration in the reaction system is high. Therefore, by calculating the specific enzyme activity of the glucosyltransferase TfGT and the relative enzyme activity of the mutant relative to the wild type, mutants with high catalytic activity were screened. The enzyme activities obtained by calculation and analysis are shown in Table 1.

[0038] The experimental results showed that the wild-type TfGT had a specific enzyme activity of approximately 18.2 U / mg; when Q60 mutated to S, A, and M, the specific enzyme activity of TfGT was significantly increased, reaching 5.9, 2.1, and 4.3 times that of the wild type, respectively, confirming that this site may have a strong interaction with the substrate; when the mutation Y452 became I, V, and R, the specific enzyme activity was significantly increased to 4.8, 4.8, and 4.2 times that of the wild type, proving that the mutant may enhance the catalytic activity of TfGT; when the mutation R455 was I, V, K, and H, the specific enzyme activity was significantly increased to 3.3, 3.4, 2.6, and 1.3 times that of the wild type.

[0039] Table 1. Mutants and relative enzyme activities

[0040]

[0041] (2) Dominant mutant combinations

[0042] Subsequently, using Q60S as a template, the above-mentioned dominant mutants were combined, sequentially mutating Y452 to I, V, R, and W to obtain Q60S / Y452I, Q60S / Y452V, and Q60S / Y452R. R455 was sequentially mutated to I, V, K, and H to obtain double mutants Q60S / R455I, Q60S / R455V, Q60S / R455K, and Q60S / R455H. Enzyme solutions of these double mutants were prepared using the method of Example 2 and screened in the system of Example 3. Glucose content was measured, and relative enzyme activity was calculated. The results are shown in Table 2. The results showed that the combined mutants Q60S / Y452I Q60S / Y452V, and Q60S / Y452R increased the specific enzyme activity to 1.31, 1.12, and 1.22 times that of Q60S, and Q60S / R455I, Q60S / R455V, Q60S / R455K, and Q60S / R455H increased the specific enzyme activity to 0.28, 1.12, 1.27, and 1.24 times that of Q60S.

[0043] Table 2. Double mutants and relative enzyme activities

[0044]

[0045] Subsequently, using Q60S / Y452I as a template, combinations were performed, sequentially mutating R455 to I, V, K, and H, to obtain the double mutants Q60S / Y452I / R455I, Q60S / Y452I / R455V, Q60S / Y452I / R455K, and Q60S / Y452I / R455H. Enzyme solutions of these constructed double mutants were prepared using the method of Example 2, and screening was performed in the above-described system. The glucose content was measured, and the results are shown in Table 3. The results showed that Q60S / Y452I / R455I, Q60S / Y452I / R455V, Q60S / Y452I / R455K, and Q60S / Y452I / R455H increased the specific enzyme activity by 0.90, 1.01, 1.34, and 1.07 times that of Q60S / Y452I, respectively.

[0046] Table 3. Triple mutants and relative enzyme activities

[0047]

[0048] Example 4 Preparation of Maltooligosaccharides

[0049] The TfGT triple mutant Q60S / Y452I / R455K was used to obtain pure protein by the expression preparation method as in Example 2.

[0050] The reaction system was established as follows: 20 mg / mL maltose, 20 mM acetate buffer (pH 6.0), and 2 mg / mL TfGT. The reaction was incubated at 50°C for 12 hours and terminated in a boiling water bath for 10 minutes. After completion of the reaction, the reaction solution was sampled and analyzed by high-performance anion chromatography.

[0051] The experimental results showed that when the reaction time reached 12 hours, the chain length of maltooligosaccharide was distributed between DP1-DP9, and the content gradually decreased with the increase of DP value.

[0052] Example 5 Preparation of modified starch

[0053] Using the deep sea red heat bacteria ( Rhodothermus profundi ) to increase starch branching degree. The amino acid sequence of RpGBE was codon-optimized to obtain a nucleotide sequence with Escherichia coli codon preference. This sequence was then synthesized and incorporated into the expression vector pET21. These recombinant vectors were then transformed into Escherichia coli BL21 (DE3) and induced to express the purified protein using the protein expression method described in Example 2. The TfGT triple mutant, Q60S / Y452I / R455K, was also expressed using the method described in Example 2 to obtain a purified protein.

[0054] The reaction system was established as follows: 20 mM acetate buffer, pH 6.0, 40 mg / mL starch slurry, 0.4 U / mL TfGT, and 0.8 U / mL RpGBE. The reaction was conducted at 70°C for 4 hours and terminated in a boiling water bath for 10 minutes. After the reaction, the reaction solution samples were sampled and the starch digestibility was simulated in vitro using the Englys method.

[0055] The experimental results showed that the content of slowly digestible starch in starch modified with TfGT and RpGBE increased from 10.5% of the original starch to 13.7%, an increase of 3.2%, and the content of resistant starch increased from 11.2% of the original starch to 13.6%, an increase of 2.4%.

[0056] Example 6 Preparation of Dayuan Cyclodextrin

[0057] The TfGT triple mutant Q60S / Y452I / R455K was used to obtain pure protein by the expression preparation method as in Example 2.

[0058] The reaction system was established as follows: a 1% (w / v) potato starch solution was prepared in 20 mM acetic acid-sodium acetate buffer (pH 6.0) and heated in a boiling water bath for 10 minutes to fully gelatinize the starch. The gelatinized starch solution was adjusted to pH 6.0 with dilute hydrochloric acid or sodium hydroxide solution, and 1 U / mL TfGT was added. The solution was incubated in a 50°C shaker water bath for 2 hours and then terminated by boiling in a 10-minute water bath. 50 U / g of saccharifying enzyme, isoamylase, and pullulanase were added to the reaction solution, mixed thoroughly, and incubated in a 40°C shaker water bath for 10 hours. The reaction was terminated by boiling in a 30-minute water bath.

[0059] The enzyme reaction solution was centrifuged at 14000 r / min for 30 minutes, and the supernatant was taken out. Anhydrous ethanol was added to precipitate. After centrifugation, the precipitate was taken out and placed in a 60°C oven overnight to obtain Dayuan cyclodextrin.

[0060] The experimental results showed that the yield of cyclodextrin produced by the reaction of TfGT triple mutant Q60S / Y452I / R455K with potato starch was 80%-90%, and the degree of polymerization was concentrated around DP27.

[0061] Example 7 Preparation of Thermoreversible Starch Gel

[0062] Using the bacteria from geothermal Deinococcus Deinococcus geothermalis DSM 11300 Thermoreversible starch gels were prepared using amylosucrase (DgAS) derived from wild-type TfGT or the triple mutant Q60S / Y452I / R455K. The reaction system was established as follows: 0.2 mol / L sucrose and corn starch were suspended in a 3:1 ratio in 20 mM acetic acid-sodium acetate buffer, pH 6.0, and heated in boiling water for 1 hour to achieve full gelatinization. The mixture was then cooled to 35°C and incubated for 10 minutes. Then, 5 U / g of wild-type TfGT or the triple mutant Q60S / Y452I / R455K and 1 U / g of DgAS were added and allowed to react for 3 hours. The enzymes were then inactivated in a boiling water bath for 30 minutes.

[0063] Experimental results showed that after simultaneous treatment with wild-type TfGT and DgAS, the amylose content of starch decreased from 10.7% to 7.3%, the proportion of amylopectin chains with a chain length of 6 < DP < 12 decreased by 7.6%, and the proportion of amylopectin chains with a chain length of 13 < DP < 36 increased by 12.2%. In contrast, treatment with the triple mutant Q60S / Y452I / R455K and DgAS decreased the amylose content to 4.9%, decreased the proportion of amylopectin chains with a chain length of 6 < DP < 12 by 5.1%, and increased the proportion of amylopectin chains with a chain length of 13 < DP < 36 by 15.4%. This decrease in amylose content inhibited the retrogradation of amylopectin, and the extension of amylopectin chain length helped improve starch gel strength.

Claims

1. A mutant of glucosyltransferase, characterized in that Based on the amino acid sequence of the glucosyltransferase shown in SEQ ID NO. 1, only the following mutations exist: Q60S, Q60A, Q60M, Q60S / Y452R, Q60S / Y452I, Q60S / Y452V, Q60S / R455K or Q60S / R455H.

2. A gene encoding a mutant of the glucosyltransferase according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence is obtained by mutation based on the nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector containing the coding gene according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, wherein It is a prokaryotic expression vector.

6. The recombinant expression vector according to claim 5, wherein It is an E. coli expression vector.

7. A recombinant bacterium containing the coding gene according to claim 2 or 3 or the recombinant expression vector according to claim 4 or 5.

8. The recombinant bacterium according to claim 7, characterized in that It is bacteria.

9. The recombinant bacterium according to claim 8, characterized in that It is Escherichia coli.

10. Use of the mutant of glucosyltransferase according to claim 1, or the gene encoding the mutant in catalyzing the production of maltooligosaccharides.

11. A method for catalytically producing maltooligosaccharides, characterized in that: The catalytic reaction is carried out using the following reaction system: maltose, the mutant according to claim 1, and acetate buffer; the reaction conditions are: the catalytic reaction is carried out at 40-60°C.

12. A method for preparing modified starch, characterized in that: Starch is used as a substrate and modified starch is generated under the catalysis of an enzyme combination, wherein the enzyme combination is: the mutant according to claim 1 and glycogen branching enzyme, or the mutant according to claim 1 and saccharifying enzyme, isoamylase and pullulanase, or the mutant according to claim 1 and amylosucrase.

13. The preparation method according to claim 12, wherein The substrate is one or more of corn starch, potato starch, tapioca starch, or pea starch.

14. The preparation method according to claim 13, wherein The catalytic reaction system was as follows: 20 mM acetate buffer pH 6.0, 40 mg / mL starch slurry, 0.4 U / mL TfGT, 0.8 U / mL RpGBE; and the reaction was carried out at 70°C.

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