Glucosyltransferase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a mutant of glucosyltransferase and application of the mutant. The mutant of the glucosyltransferase is obtained by only having the following mutations on the basis of the glucosyltransferase of which the amino acid sequence is as shown in SEQ ID NO.1: one or a combination of more of Q60S, Q60A, Q60M, Y452I, Y452V, Y452R, R455K, R455I, R455V and R455H. Compared with a wild type, the obtained glucosyltransferase mutant has the advantages that the catalytic activity is obviously improved, and maltose can be catalyzed to generate maltooligosacharide; the method can be used for preparing modified starch and increasing the content of slowly digestible starch and resistant starch. Therefore, the method has a relatively high application value.

Description

Technical Field

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

[0002] Starch is one of the most abundant resources in nature and one of the main sources of energy for humans. Intake of high levels of fast-digestible starch can cause metabolic diseases such as diabetes, obesity and cardiovascular disease. Studies have shown that slowly digestible starch can be slowly digested and absorbed in the small intestine, which is beneficial for maintaining blood sugar homeostasis. Studies have shown that the content of slowly digestible starch can be significantly increased after starch modification, and the application of new functional foods in the future food industry has broad application prospects.

[0003] Starch is a natural renewable green resource. Products made from starch involve many industries such as food, medicine, and papermaking, and have broad market application prospects. This widely used natural industrial raw material can no longer meet the new requirements of modern industry due to its insolubility in cold water, easy aging and dehydration, and insufficient emulsification. In order to improve the natural quality of starch, natural starch is currently modified by physical, chemical, enzymatic or composite methods. Enzymatic modification is safer and healthier than physical and chemical modification, and has the advantages of high specificity, high yield, few by-products, and low purification requirements. At present, the research on starch modification by glucosyltransferase is very extensive, such as replacing chemical gelatin, delaying starch aging and extending shelf life, and being used in yogurt as a fat substitute and emulsifier stabilizer, etc. However, there are still few studies on the fine structure and functional properties of starch by glucosyltransferase.

[0004] However, the currently known glucosyltransferases have multiple functions such as disproportionation, cyclization, coupling, and hydrolysis. By enhancing 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 derived from Thermus filiformis A large number of glucosyltransferases were screened to obtain mutants with single-site mutations, and combined mutations were further obtained through saturation mutations. Experiments showed that compared with the wild type, the thermal stability and catalytic activity were significantly improved, thereby 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 thereof is obtained by performing a mutation on the basis of 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 coding 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 generating maltooligosaccharides, wherein the catalytic reaction system specifically comprises: maltose, 20 mg / mL; the mutant; acetate buffer, 20 mM, pH 6.0; reaction conditions: catalytic reaction at 50° C. for 12 hours.

[0013] The present invention particularly provides a method for preparing modified starch, using starch as a substrate and generating modified starch under the catalysis of a combination of enzymes, 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; The substrate is one or more of corn starch, potato starch, tapioca starch or pea starch.

[0014] Preferably, the glycogen branching enzyme is RpGBE derived from Rhodothermus erythroxysporum.

[0015] 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, 0.8 U / mL RpGBE; specifically, the reaction was carried out at 70° C. for 4 hours.

[0016] The glucosyltransferase mutant obtained by the present invention has significantly improved catalytic activity compared to the wild type, and can catalyze maltose to generate maltooligosaccharides; the preparation of modified starch established further, and studies have shown that the content of slowly digestible starch and resistant starch increased by 3.2% and 2.4% respectively. Therefore, the present invention has a high application value. DETAILED DESCRIPTION

[0017] 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 unless otherwise specified.

[0018] 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).

[0019] Materials or reagents with the same names used in the examples are the same unless otherwise specified. The methods for obtaining various biological materials described in the examples are only to provide an experimental method for obtaining them to achieve the specific disclosed purpose, and should not be a limitation on the source of biological materials when implementing the present invention. In fact, the sources of biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0020] The primers and genes used in the present invention were synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd.

[0021] The present invention is further described in detail below in conjunction with examples. The examples are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes 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.

[0022] Example 1 Expression and purification of glucosyltransferase TfGT (1) We searched for 4-α-glucosyltransferase in the UniProt database (www.uniprot.org), screened for 4GT from thermophilic sources by keywords and functions, and used clustW for sequence alignment and HHfilter to control homology. We obtained Thermus filiformis The original protein sequence of the glucosyltransferase from Thermus filiformis The glucosyltransferase (whose amino acid sequence is shown in SEQ ID NO.1) and its nucleotide sequence after codon optimization had a nucleotide sequence after the preferred codon of Escherichia coli (SEQ ID NO.2) and was sent to Jiangsu Jinweizhi Biotechnology Co., Ltd. for gene synthesis and constructed into the expression vector pET-21a.

[0023] (2) The recombinant plasmid pET-TfGT containing the above-mentioned glucosyltransferase gene (the optimized sequence SEQ ID NO.2) was transferred into Escherichia coli BL21 (DE3) for exogenous expression and purification.

[0024] Example 2 Protein expression and purification The recombinant plasmid containing the above-mentioned glucosyltransferase gene (the optimized sequence SEQ ID NO.2) was transferred into Escherichia coli for exogenous expression and purification.

[0025] (1) The E. coli expression recombinant plasmid pET-TfGT was transferred into E. coli BL21 (DE3) to obtain recombinant bacteria.

[0026] (2) Pick a single clone and place it in 5 mL LB liquid medium and culture until OD 600 was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced for 16 h.

[0027] (3) The above culture solution was centrifuged to collect the bacterial cells.

[0028] (4) Bacterial disruption: Use an ultrasonic disruptor to break the cell wall and centrifuge to obtain the supernatant.

[0029] (5) Heat purification: The supernatant was heat treated in a water bath at 70°C for 1 hour and the supernatant was collected by centrifugation to obtain the heat-treated protein.

[0030] (6) Ni purification: The supernatant obtained after crushing and centrifugation is bound to a nickel column, eluted, and purified to obtain pure protein.

[0031] Example 3 Mutant Construction and Verification (1) Construction of TfGT glucosyltransferase mutants and enzyme activity determination Will come from Thermus filiformis The glucosyltransferase (TfGT) of the substrate maltose was molecularly docked to obtain the complex structure, and then Rosseta was used to obtain sites 60, 452 and 455 that may be related to catalytic activity in combination with free energy analysis. Saturation mutations were carried out around the above sites, that is, the above sites were mutated into 20 different amino acid residues, so as to construct a mutant library and measure its dismutation activity on 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; 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 sample of the reaction was subjected to liquid chromatography detection. Definition of specific enzyme activity (1U / mg): the amount of 1 micromole of glucose catalyzed per minute per milligram of TfGT. 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 glucosyltransferase TfGT, the relative enzyme activity of the mutant relative to the wild type, the mutant with high catalytic activity was screened. The enzyme activity obtained by calculation and analysis is shown in Table 1.

[0032] 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, to 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.

[0033] Table 1. Mutants and relative enzyme activities

[0034] (2) Advantageous mutant combination Then, using Q60S as a template, the above-mentioned advantageous mutants were combined, and Y452 was sequentially mutated 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 Q60S / R455I, Q60S / R455V, Q60S / R455K, and Q60S / R455H double mutants. For the constructed double mutants, the enzyme solution was prepared by the method of Example 2, and screened in the system of Example 3, the glucose content was determined, and the 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 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 enzyme activity to 0.28, 1.12, 1.27, and 1.24 times that of Q60S.

[0035] Table 2. Double mutants and relative enzyme activities

[0036] Subsequently, Q60S / Y452I was used as a template for combination, and R455 was sequentially mutated to I, V, K, and H to obtain double mutants of Q60S / Y452I / R455I, Q60S / Y452I / R455V, Q60S / Y452I / R455K, and Q60S / Y452I / R455H. For the constructed double mutants, the enzyme solution was prepared by the method of Example 2, and screened in the above system, and the content of glucose was determined. 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 to 0.90, 1.01, 1.34, and 1.07 times that of Q60S / Y452I.

[0037] Table 3. Triple mutants and relative enzyme activities

[0038] Example 4 Preparation of maltooligosaccharides The TfGT triple mutant Q60S / Y452I / R455K was used to obtain pure protein through the expression preparation method as in Example 2.

[0039] The reaction system was established as follows: maltose, 20 mg / mL, 20 mM acetate buffer (pH 6.0), TfGT 2 mg / mL, reaction conditions at 50 ° C, reaction for 12 hours, and boiling water bath for 10 minutes to terminate the reaction. After the reaction, the reaction solution samples were analyzed by high performance anion chromatography.

[0040] 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.

[0041] Example 5 Preparation of modified starch Using red hot bacteria from deep sea Rhodothermus profundi ) glycogen glycogen branching enzyme (RpGBE) to increase the branching degree of starch. The amino acid sequence of RpGBE was codon-optimized to obtain a nucleotide sequence with Escherichia coli codon preference, which was constructed into the expression vector pET21 by gene synthesis, and these recombinant vectors were transferred into Escherichia coli BL21 (DE3), and the pure protein was obtained by induction and expression as shown in Example 2. The TfGT triple mutant Q60S / Y452I / R455K was expressed and prepared by the method shown in Example 2 to obtain a pure protein.

[0042] The reaction system was established as follows: 20 mM acetate buffer pH 6.0, 40 mg / mL starch slurry, 0.4 U / mL TfGT, 0.8 U / mL RpGBE; reaction conditions were 70 ° C, reaction time was 4 h, and the reaction was terminated by boiling water bath for 10 minutes. After the reaction, the reaction liquid samples were used to simulate the in vitro determination of starch digestibility using the Englys method.

[0043] The experimental results showed that the content of slowly digestible starch in starch modified by 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%.

[0044] Example 6 Preparation of Dayuan Cyclodextrin The TfGT triple mutant Q60S / Y452I / R455K was used to obtain pure protein through the expression preparation method as in Example 2.

[0045] The reaction system was established as follows: 1% (w / v) potato starch solution was prepared with 20mM pH 6.0 acetic acid-sodium acetate buffer, and the starch was fully gelatinized by heating in a boiling water bath for 10 minutes; the gelatinized starch solution was adjusted to pH 6.0 with dilute hydrochloric acid or dilute sodium hydroxide solution, 1 U / mL TfGT was added, and the reaction was kept constant at 50℃ in a water bath shaker for 2 hours, and the reaction was terminated by boiling in a water bath for 10 minutes. 50 U / g saccharifying enzyme, isoamylase and pullulanase were added to the reaction solution, mixed thoroughly, and reacted in a water bath shaker at 40℃ for 10 hours; the reaction was terminated by boiling in a water bath for 30 minutes.

[0046] The enzyme reaction solution was centrifuged at 14000r / min for 30min, the supernatant was taken, anhydrous ethanol was added for precipitation, the precipitate was taken after centrifugation and placed in a 60°C oven overnight to obtain Dayuan cyclodextrin.

[0047] 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.

[0048] Example 7 Preparation of Thermoreversible Starch Gel Using the Deinococcus geothermalus ( Deinococcus geothermalis DSM 11300 ) was used to prepare thermoreversible starch gel with TfGT wild type or triple mutant Q60S / Y452I / R455K. The reaction system was established as follows: 0.2 mol / L sucrose and corn starch were suspended in 20mM pH 6.0 acetic acid-sodium acetate buffer at a ratio of 3:1, and heated in boiling water for 1h to fully gelatinize. Cool to 35℃, keep warm for 10min, add 5 U / g TfGT wild type or triple mutant Q60S / Y452I / R455K and 1 U / g DgAS to react for 3h, and inactivate the enzyme in a boiling water bath for 30min.

[0049] The experimental results showed that after the starch was treated with TfGT wild type and DgAS, the content of amylose decreased from 10.7% to 7.3%, the proportion of 6<DP<12 chain length in amylopectin decreased by 7.6%, and the proportion of 13<DP<36 increased by 12.2%. After the triple mutant Q60S / Y452I / R455K and DgAS were treated, the content of amylose decreased to 4.9%, the proportion of 6<DP<12 in amylopectin decreased by 5.1%, and the proportion of 13<DP<36 increased by 15.4%. The decrease in the content of amylose in the product inhibited the retrogradation of amylopectin, and the extension of amylopectin chain length helped to improve the strength of starch gel.

Claims

1. A mutant of a glucosyltransferase, characterized in that: Based on the amino acid sequence of the glucosyltransferase shown in SEQ ID NO.1, only the following mutations exist: one or more combinations of Q60S, Q60A, Q60M, Y452I, Y452V, Y452R, R455K, R455I, R455V, and R455H.

2. The mutant of glucosyltransferase according to claim 1, characterized in that Based on the glucosyltransferase with an amino acid sequence as shown in SEQ ID NO.1, only the following mutations exist: 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.

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

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

2.

5. A recombinant expression vector containing the coding gene as claimed in claim 3 or 4.

6. The recombinant expression vector according to claim 5, characterized in that It is a prokaryotic expression vector.

7. The recombinant expression vector according to claim 6, characterized in that It is an E. coli expression vector.

8. A recombinant bacterium containing the coding gene according to claim 3 or 4 or the recombinant expression vector according to claim 5 or 6.

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

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

11. Use of the mutant of glucosyltransferase according to claim 1 or 2, or the gene encoding the mutant in catalyzing the production of maltooligosaccharides.

12. 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 or 2, and acetate buffer; the reaction conditions are: the catalytic reaction is carried out at 40-60°C.

13. A method for preparing modified starch, characterized in that: With starch as substrate, modified starch is generated under the catalysis of an enzyme combination, wherein the enzyme combination is: the mutant as claimed in claim 1 or 2 and glycogen branching enzyme, or the mutant as claimed in claim 1 or 2 and saccharifying enzyme, isoamylase and pullulanase, or the mutant as claimed in claim 1 or 2 and amylosucrase.

14. The preparation method according to claim 13, characterized in that: The substrate is one or more of corn starch, potato starch, tapioca starch, or pea starch.

15. The preparation method according to claim 14, characterized in that: The catalytic reaction system is 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 is carried out at 70°C.

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