Glucose isomerase mutants, uses thereof and methods for increasing the enzymatic activity of glucose isomerase

Abstract

The present application relates to the technical field of biological enzyme, disclose a kind of glucose isomerase mutant and its use and the method for improving the enzyme activity of glucose isomerase.The glucose isomerase mutant provided by the present application has similar or higher thermostability compared to wild-type glucose isomerase, while also having higher enzyme activity, providing new options for the production of food, medicine, biofuels and other fields.

Description

[0001] Divisional application This invention is a divisional application of Chinese patent application filed on September 28, 2025, entitled "Glucose isomerase mutant and its use and method for improving the enzyme activity of glucose isomerase", application number 202511399528.5. Technical Field

[0002] This invention relates to the field of bioenzyme technology, specifically to a glucose isomerase mutant and its uses, as well as a method for improving the enzyme activity of glucose isomerase. Background Technology

[0003] Glucose isomerases catalyze the isomerization of D-glucose to form D-fructose and have wide applications in food, pharmaceuticals, and biofuels. In industrial production, to improve reaction efficiency, glucose isomerization reactions are usually carried out at higher temperatures (such as 55-60℃). Therefore, high requirements are placed on the thermal stability and enzyme activity of glucose isomerases at high temperatures.

[0004] Currently, although some glucose isomerases capable of withstanding high-temperature conditions have been obtained through continuous screening of natural glucose isomerases and modification of existing glucose isomerases using mutagenesis and genetic engineering, the screening and modification of these glucose isomerases tend to focus on enzyme activity and adaptability to high-temperature reaction conditions, neglecting the enzyme's thermal stability (i.e., the time the enzyme retains its activity under high-temperature reaction conditions). However, in actual production, if the enzyme's thermal stability is insufficient, its activity will be lost in a short period of time, leading to problems such as short production time per batch, frequent enzyme replacement, and large enzyme consumption, which significantly impacts production efficiency and cost.

[0005] Therefore, there is an urgent need to develop glucose isomerases that possess advantages such as adaptability to high-temperature reaction conditions, high enzyme activity, and the ability to maintain high enzyme activity over a long period of time. Summary of the Invention

[0006] The purpose of this invention is to overcome the aforementioned problems in the prior art and to provide a glucose isomerase mutant, its uses, and a method for improving the enzyme activity of glucose isomerase. The glucose isomerase mutant provided by this invention is obtained by mutating a wild-type glucose isomerase with high thermostability. The mutant of this invention has higher enzyme activity and essentially retains the thermostability of the wild-type glucose isomerase, and even improves upon it.

[0007] To achieve the above objectives, the present invention provides a glucose isomerase mutant, which, relative to the wild-type glucose isomerase, has a mutation at least one of the amino acids at positions 89, 110, 136, 143, 171, 176, 180, and 214, and the amino acid sequence of the wild-type glucose isomerase is shown in SEQ ID NO:1.

[0008] A second aspect of the present invention provides a nucleic acid that encodes the glucose isomerase mutant described in the first aspect.

[0009] A third aspect of the present invention provides a construct comprising the nucleic acid described in the second aspect.

[0010] A fourth aspect of the present invention provides a host cell comprising the nucleic acid described in the second aspect or the construct described in the third aspect.

[0011] The fifth aspect of the present invention provides an immobilized enzyme, the immobilized enzyme comprising a carrier and an enzyme loaded on the carrier, the enzyme being selected from the glucose isomerase mutant described in the first aspect.

[0012] The sixth aspect of this invention provides the use of the glucose isomerase described in the first aspect, the nucleic acid described in the second aspect, the construct described in the third aspect, the host cell described in the fourth aspect, or the immobilized enzyme described in the fifth aspect in the preparation of food ingredients, pharmaceutical ingredients, or biofuel ingredients.

[0013] A seventh aspect of the present invention provides a method for improving the enzyme activity of glucose isomerase while maintaining its thermostability substantially undiminished, the method comprising causing a mutation in at least one of the amino acids at positions 89, 110, 136, 143, 171, 176, 180, and 214 of the wild-type glucose isomerase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0014] Through the above technical solution, the present invention can achieve at least the following beneficial effects: (1) The glucose isomerase mutant provided by the present invention has higher activity and good thermal stability. Its half-life is basically not reduced compared with wild type glucose isomerase, and some preferred enzymes even have a longer half-life than wild type.

[0015] (2) The glucose isomerase mutant provided by the present invention requires less dosage than wild-type glucose isomerase, which greatly reduces production costs.

[0016] (3) The mutant of the present invention can be used to make an immobilized enzyme, which further improves production efficiency and reduces production costs. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] In this invention, unless otherwise specified, "mutant" refers to the glucose isomerase mutant of this invention.

[0019] In this invention, the mutation site is described using the form "XnY", where n is a number representing the mutation site; X and Y are different uppercase letters representing the amino acids before and after the mutation. As is known in the art, this naming means that in the mutant, the amino acid at the mutation site (the nth position in the amino acid sequence) is mutated from X to Y. For example, "A89M" represents "the 89th amino acid in the mutant is mutated from alanine to methionine". The name of the mutant is consistent with the mutation site it contains. If the "XnY" form is used, it means that the mutant is a single-point mutant, that is, relative to the wild-type glucose isomerase, only the nth amino acid in the mutant is mutated; if the "X1n1Y1 / X2n2Y2 / ... / X" form is used, it means that the mutant is a single-point mutant, that is, relative to the wild-type glucose isomerase, the mutant has a mutation at only the nth amino acid. m n m Y m The "formal naming" indicates that the mutant is a multi-point mutant, meaning that relative to the wild-type glucose isomerase, the n1, n2, ..., nth points of this mutant are different. m All amino acids were mutated. In the mutants of this invention, no mutations were found at any sites not specified in the name.

[0020] Unless otherwise specified, thermal stability in this invention is measured by the half-life of enzyme activity at 60°C. "Half-life of enzyme activity at 60°C" refers to the time required for the remaining enzyme activity to decrease to 50% of its original activity after continuous incubation at 60°C. Enzyme activity in this invention is measured using specific enzyme activity, defined as the number of enzyme activity units per mg of enzyme protein, expressed in U / mg; wherein, one unit of glucose isomerase activity refers to the amount of enzyme required to produce 1 mg of fructose in 1 hour under specified reaction conditions, denoted as 1 U.

[0021] The enzyme activity testing method in this invention refers to the reaction system for immobilized glucose isomerase activity testing in the national standard GB / T 23533-2009: The 5 mL reaction system included: purified glucose isomerase molecules with a final concentration of 0.03 mg / mL, glucose with a final concentration of 210 mg / mL, magnesium sulfate with a final concentration of 6.1 mg / mL, 1.5 mL of phosphate buffer at pH 7.5 (1.96 g of sodium dihydrogen phosphate and 39.62 g of disodium hydrogen phosphate dodecahydrate were weighed, dissolved in water, and brought to a final volume of 500 mL, with the pH adjusted to 7.5 ± 0.05), and water was added to make up to 5 mL. The reaction was carried out at 70 °C for 1 h. After the reaction, the solution was diluted 5 times, boiled in a water bath for 5 min, and then used for sample preparation. The concentration of fructose in the reaction system was detected by HPLC, and the specific enzyme activity at 70 °C was calculated according to the following formula I: U / mg Formula I The inventors of this invention ingeniously discovered in their research that by making point mutations to amino acids at specific sites in wild-type glucose isomerase SmGI, the enzyme activity of the resulting mutant can be effectively improved while retaining or further enhancing its thermostability characteristics.

[0022] Based on this, the first aspect of the present invention provides a glucose isomerase mutant, which, relative to the wild-type glucose isomerase, has a mutation at least one of the amino acids at positions 89, 110, 136, 143, 171, 176, 180, and 214, and the amino acid sequence of the wild-type glucose isomerase is shown in SEQ ID NO:1.

[0023] MSFQPTPEDRFTFGLWTVGWQGRDPFGDATRPALDPVETVQRLAELGAYGVTFHDDDLIPFGSSDTERESHIKRFRQALDATGMTVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTIRNIDLAAELGAKTYVAWGGREGAESGGAKDVRDALDRMKEAFDLLGEYVIAQGYDLRFAIEPKPPNEPRGDILLP TVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGKLFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLETAGYEGPRHFDFKPPRTEDFDGVWASAAGCMRNYLILKDRAAAFRADPEVQEALRAARLDQLAQPTAADGLDALLADRAAFEDFDVDAAAARGMAFEHLDQLAMDHLLGARG (SEQ ID NO:1) The mutant provided by this invention can undergo arbitrary mutations at the above mutation sites, as long as the thermal stability of the mutant is not substantially reduced compared with the wild-type glucose isomerase mutant before mutation, and the enzyme activity is increased.

[0024] According to a preferred embodiment of the present invention, the mutation is a substitution mutation.

[0025] Preferably, the mutation includes at least one of the following: (1) The 89th amino acid is mutated from alanine (A) to methionine (M), valine (V) or isoleucine (I); (2) The 110th amino acid is mutated from aspartic acid (D) to serine (S) or alanine (A); (3) The amino acid at position 136 is mutated from alanine to phenylalanine (F), methionine (M), isoleucine (I), leucine (L) or valine (V); (4) The amino acid at position 143 is mutated from alanine (A) to serine (S); (5) The amino acid at position 171 is mutated from alanine (A) to serine (S) or aspartic acid (D); (6) The 176th amino acid is mutated from leucine (L) to isoleucine (I); (7) The 180th amino acid is mutated from isoleucine (I) to leucine (L); (8) The 214th amino acid was mutated from valine (V) to leucine (V).

[0026] The mutants provided by this invention may contain only one of the above-mentioned mutations (i.e., single-point mutants) or may contain a combination of any of the above-mentioned mutations (i.e., multi-point mutants).

[0027] According to some preferred embodiments of the present invention, the mutant is selected from any one of the following: A89M, A89V, A89I, D110S, D110A, A136F, A136M, A136I, A136L, A136V, A143S, A171S, A171D, L176I, I180L, V214L, D110S / A171S, A89V / D110S, A89V / L176I, A89V / A143S, A89V / A171S, A89V / A136M, A89V / I180L, A89V / V214L, A136M / A 143S / A171S, A89V / A143S / A171S, A89V / D110S / A136M, A89V / D110S / A143S, A89V / I180L / V214L, D110S / A136M / A171S / L176I, A136M / A143S / A171S / L176I, D110S / A136M / A143S / A171S / L176I, A89V / D110S / A143S / A171S / L176I and A89V / D110S / A143S / A171S / I180L / V214L.

[0028] A second aspect of the present invention provides a nucleic acid that encodes the glucose isomerase mutant described in the first aspect.

[0029] According to a preferred embodiment of the present invention, the nucleic acid may be DNA or RNA.

[0030] Those skilled in the art are familiar with the degeneracy of genes, and thus can obtain the encoded nucleic acid from the amino acid sequence of the aforementioned mutant.

[0031] According to some preferred embodiments of the present invention, the nucleic acid may be DNA obtained by replacing the codons of the mutated amino acids at the corresponding mutation positions based on SEQ ID NO:2 (DNA encoding a wild-type glucose isomerase mutant) according to the amino acid mutation situation of the aforementioned mutant, or its corresponding RNA.

[0032] A third aspect of the present invention provides a construct comprising the nucleic acid described in the second aspect.

[0033] In this invention, "construction" refers to a nucleic acid used for in vitro expression of exogenous proteins, which usually exists in the form of plasmids.

[0034] According to a preferred embodiment of the present invention, the mutant further comprises a vector sequence. The vector sequence used in this invention can be derived from any expression vector in the art that can be used to carry and promote the expression of exogenous protein-coding genes in host cells cultured in vitro. For example, the vector sequence can be derived from commercially available and mature expression vectors such as pET28a, pET30a, pET21b, pET32a, pMA5, pHT01, and pHY300PLK.

[0035] A fourth aspect of the present invention provides a host cell comprising the nucleic acid described in the second aspect or the construct described in the third aspect.

[0036] The host cells provided by this invention can be used to preserve or express the mutants provided by this invention. Therefore, cells commonly used in the art for in vitro expression of exogenous proteins, especially enzymes, are applicable to this invention. These cells, after introducing the nucleic acid of the mutants provided by this invention or a construct containing that nucleic acid, become the host cells provided by this invention.

[0037] For example, the host cell is preferably selected from at least one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Streptomyces, Saccharomyces cerevisiae, methanolytrophic yeast, Hansenula polymorpha, Schizosoma, Kluyveromyces, Candida albicans, Pichia pastoris, and filamentous fungi.

[0038] The fifth aspect of the present invention provides an immobilized enzyme, the immobilized enzyme comprising a carrier and an enzyme loaded on the carrier, the enzyme being selected from the glucose isomerase mutant described in the first aspect.

[0039] This invention does not impose any particular limitation on the carrier used for the immobilized enzyme. For example, the carrier can be selected from silicon-based carriers such as diatomaceous earth, natural polymer materials such as chitosan and sodium alginate, synthetic polymers such as resins, magnetic nanoparticles, and novel nanomaterials that can be used as carriers for immobilized enzymes.

[0040] The sixth aspect of this invention provides the use of the glucose isomerase described in the first aspect, the nucleic acid described in the second aspect, the construct described in the third aspect, the host cell described in the fourth aspect, or the immobilized enzyme described in the fifth aspect in the preparation of food ingredients, pharmaceutical ingredients, or biofuel ingredients.

[0041] The mutant provided by this invention can convert glucose into fructose more efficiently and at a lower cost, thereby being used to prepare a series of food ingredients containing fructose (such as glucose syrup, high fructose syrup, fructose, etc.), and can also be used as a raw material for preparing medical materials such as fructooligosaccharides, and as a raw material for preparing biofuels (such as bioethanol) fermentation.

[0042] A seventh aspect of the present invention provides a method for improving the enzyme activity of glucose isomerase while maintaining its thermostability substantially undiminished, the method comprising causing a mutation in at least one of the amino acids at positions 89, 110, 136, 143, 171, 176, 180, and 214 of the wild-type glucose isomerase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0043] The method provided by this invention is to mutate the wild-type glucose isomerase of SEQ ID NO:1 (e.g., single-point or multi-point mutation) to obtain the aforementioned mutant. The specific mutation sites and mutation methods in the mutant are as described above and will not be repeated here.

[0044] In this invention, "the thermostability of glucose isomerase is not substantially reduced" means that the thermostability of the mutant of this invention is slightly reduced, the same, or improved compared with that of the wild-type glucose isomerase. In some preferred embodiments of this invention, the thermostability of the mutant of this invention is not less than 70% of that of the wild-type glucose isomerase (thermal stability is reflected by the half-life of enzyme activity at 60°C, that is, the half-life of the mutant of this invention is not less than 70% of that of the wild-type glucose isomerase).

[0045] For example, the thermostability of the mutant of the present invention can be 70-100% of the thermostability of wild-type glucose isomerase, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

[0046] Preferably, the thermostability of the mutant is not less than 80% of that of the wild-type glucose isomerase, the thermostability of the mutant is not less than 90% of that of the wild-type glucose isomerase, and the thermostability of the mutant is not less than 95% of that of the wild-type glucose isomerase.

[0047] In some preferred embodiments of the present invention, the thermostability of the mutant is also higher than that of the wild-type glucose isomerase. Preferably, the thermostability of the mutant is 100-300% of that of the wild-type glucose isomerase.

[0048] For example, the thermostability of the mutant of the present invention can be 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% of the thermostability of the wild-type glucose isomerase.

[0049] According to a preferred embodiment of the present invention, the method includes the step of constructing a mutant recombinant vector. Any method known to those skilled in the art for constructing a recombinant vector (i.e., the aforementioned construct) capable of expressing the mutant based on its amino acid sequence is applicable to the present invention.

[0050] Preferably, constructing the mutant recombinant vector involves in vitro amplification of a recombinant vector containing the wild-type glucose isomerase encoding gene using site-directed mutagenesis primers. The recombinant vector containing the wild-type glucose isomerase encoding gene is the construct obtained by inserting the wild-type glucose isomerase encoding gene into a vector. Preferably, the nucleotide sequence of the wild-type glucose isomerase encoding gene is shown in SEQ ID NO:2. The preferred vector is the vector used in the aforementioned construct, and will not be described further here.

[0051] More preferably, the site-directed mutagenesis primers are selected from at least one group of SEQ ID NO:5 and 6, SEQ ID NO:44 and 54, SEQ ID NO:46 and 54, SEQ ID NO:60 and 61, SEQ ID NO:15 and 16, SEQ ID NO:62 and 71, SEQ ID NO:63 and 71, SEQ ID NO:65 and 71, SEQ ID NO:66 and 71, SEQ ID NO:19 and 20, SEQ ID NO:23 and 24, SEQ ID NO:87 and 88, SEQ ID NO:27 and 28, SEQ ID NO:29 and 30, SEQ ID NO:33 and 34, SEQ ID NO:7 and 8, SEQ ID NO:117 and 118, and SEQ ID NO:7+19+23 and 8+20+24.

[0052] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0053] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0054] In the following examples, the enzyme activity testing method refers to the reaction system for immobilized glucose isomerase activity testing in the national standard GB / T 23533-2009: The 5 mL reaction system included: purified glucose isomerase molecules with a final concentration of 0.03 mg / mL, glucose with a final concentration of 210 mg / mL, magnesium sulfate with a final concentration of 6.1 mg / mL, 1.5 mL of phosphate buffer at pH 7.5 (1.96 g of sodium dihydrogen phosphate and 39.62 g of disodium hydrogen phosphate dodecahydrate were weighed, dissolved in water, and brought to a final volume of 500 mL, with the pH adjusted to 7.5 ± 0.05), and water was added to make up to 5 mL. The reaction was carried out at 70 °C for 1 h. After the reaction, the solution was diluted 5 times, boiled in a water bath for 5 min, and then used for sample preparation. The concentration of fructose in the reaction system was detected by HPLC, and the specific enzyme activity at 70 °C was calculated according to the following formula I: U / mg Formula I Example 1 This example illustrates the construction of a wild-type glucose isomerase expression system and the results of its enzyme activity and half-life determination.

[0055] (I) Construction of wild-type glucose isomerase expression system SmGI derived from *Streptomyces griseus* (the amino acid sequence of SmGI is shown in SEQ ID NO:1; in the experiment, a 6×His tag was attached to its C-terminus for ease of operation, facilitating protein purification via metal chelate chromatography) was selected as the wild-type glucose isomerase. Jiangsu Saisofe Biotechnology Co., Ltd. was commissioned to introduce the SmGI encoding gene (nucleotide sequence shown in SEQ ID NO:2) into the pET28a expression vector using gene synthesis technology to obtain the wild-type construct pET28a-SmGI.

[0056] The wild-type construct pET28a-SmGI was transformed into Escherichia coli Transetta competent cells (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain the Escherichia coli expression strain Transetta-pET28a-SmGI. The next day, colony PCR was performed using the universal primers T7 (nucleotide sequence TAATACGACTCACTATAGG, SEQ ID NO:41) and T7 ter (nucleotide sequence CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAG, SEQ ID NO:42) of the pET28a vector. Positive bacteria were then amplified using LB liquid medium.

[0057] (II) Determination of wild-type enzyme activity and half-life (1) Enzyme activity test (enzyme activity is measured by specific enzyme activity) The E. coli expression system Transetta-pET28a-SmGI was subjected to shake-flask fermentation and protein induction expression as follows: Transetta-pET28a-SmGI was inoculated at a rate of 1% (v / v) into 100 mL of LB liquid medium (containing 50 μg / mL Kana antibiotic), and cultured on a shaker at 37°C and 200 rpm until OD500. 600 =0.75±0.25. Then, IPTG (final concentration 0.5mM) was added as an inducer, and the mixture was induced and cultured for 18h at 25℃ and 200rpm to allow the E. coli expression system to express wild-type glucose isomerase SmGI.

[0058] After expression, the bacterial culture concentration was adjusted to OD. 600 =10, take 1.5 mL of the bacterial culture after adjusting the concentration, centrifuge at 12000 rpm for 5 min, discard the supernatant, resuspend in 1.5 mL of 50 mM Tris-HCl buffer (pH=8.0), and perform ultrasonic disruption (amplitude bar 2, ultrasonic for 2 seconds / interval for 2 seconds, total time 5 min). The supernatant obtained after centrifugation of the disrupted solution is the crude enzyme solution.

[0059] A portion of the crude enzyme solution was filtered through a 0.22 μm filter membrane to obtain the sample to be purified. The sample was then purified using a 1 mL pre-packed Ni column (Cytiva, HiTrap FF 1 mL), followed by desalting using a 5 mL pre-packed desalting column (Cytiva, HiTrap Desalting 5 mL) (replacing the protein with 50 mM pH=8.0 Tris-HCl buffer) to obtain the purified SmGI enzyme solution. The protein concentration in the purified enzyme solution was determined using a BCA kit (Thermo, 23227).

[0060] The purified SmGI was tested for enzyme activity at 70℃ according to the reaction system for immobilized glucose isomerase activity assay in GBT23533-2009 national standard (referred to as "initial enzyme activity").

[0061] (2) 24h equilibrium conversion rate A glucose isomerase reaction system was formed by adding purified SmGI to a final concentration of 0.03 mg / mL using a glucose aqueous solution with a final concentration of 50 g / L as the substrate. The reaction was carried out at 60℃, and samples were taken after 24 h. The reaction was terminated by boiling in a water bath for 5 min. The samples were then centrifuged and filtered to prepare HPLC samples, which were then analyzed by HPLC. The detection conditions are shown in Table 1. The equilibrium conversion rate was calculated based on the detection results of the HPLC samples.

[0062] Balanced conversion rate (%) = (Fructose content / (Glucose content + Fructose content)) × 100% Table 1

[0063] (3) Half-life Wild-type glucose isomerase SmGI purified by Ni column was incubated at 60 °C, and enzyme activity was measured every 24 hours. The half-life of the enzyme was defined as the point at which the enzyme activity decreased to 50% of the initial enzyme activity (in days; if the enzyme activity on day n is 50-54% (excluding 54%) of the initial enzyme activity, and the enzyme activity on day n+1 is 45-50% (excluding 50%) of the initial enzyme activity).

[0064] The equilibrium conversion rate of SmGI was calculated to be 47.2%, the initial enzyme activity was 142 U / mg, and the half-life of the reaction at 60℃ was 192 h (8 days).

[0065] Example 2 This example illustrates the construction of glucose isomerase mutants and the results of enzyme activity and half-life determination.

[0066] (a) Single-point mutant (1) Construction of mutants Using the wild-type construct pET28a-SmGI from Example 1 as a template, PCR amplification was performed using the site-directed mutagenesis primers in Table 2 and the PCR system and conditions in Table 3 to obtain the recombinant constructs of each single-point mutant.

[0067] Table 2

[0068] Table 3

[0069] Take 10 μL of PCR product and digest it with 0.3 μL of DMT enzyme (TransGen Biotech, Beijing, GD111-01) at 37°C for 1 h to remove the template. Add the digested product to 50 μL of DMT competent cells (TransGen Biotech, Beijing, CD511-01), mix well, and incubate on ice for 30 min. Then heat shock at 42°C for 45 s. After heat shock, quickly transfer to ice and incubate for 2 min. Then add 500 μL of sterile LB liquid medium and incubate at 37°C with shaking at 200 rpm for 1 h to revive the cells. Finally, centrifuge the bacterial culture at 4000 rpm for 30 s, remove 400 μL of supernatant, mix the remaining bacterial culture, and spread it onto LB agar plates containing 50 μg / mL Kana antibiotic. Invert the plates and incubate overnight at 37°C.

[0070] On the second day, colony PCR was performed using universal primers T7 (SEQ ID NO:41) and T7 ter (SEQ ID NO:42) for the pET28a vector. Positive bacteria from the colony PCR were further verified by sequencing to determine if mutations had occurred. The mutant plasmid, verified by sequencing, was transformed into Transetta competent cells according to Example 1. The following day, colony PCR was performed using universal primers T7 (SEQ ID NO:41) and T7 ter (SEQ ID NO:42) for the pET28a vector, and the mutant positive bacteria were amplified using LB liquid medium.

[0071] (2) Determination of mutant enzyme activity and half-life The equilibrium conversion rate, initial enzyme activity and half-life of each mutant enzyme were tested in the manner described in Example 1. The results are detailed in Table 4.

[0072] Table 4*

[0073] *In Table 4, “—” indicates that enzyme activity and half-life were not tested because the conversion rate of the mutant was lower than that of SmGI.

[0074] As can be seen from the results in Table 4, the enzyme activities of mutants A89M, D110S, A136F, A143S, A171S, L176I, I180L, and V214L are higher than those of wild-type SmGI, and their half-lives are the same as those of SmGI. This indicates that these mutants have improved enzyme activity while retaining the thermostability of wild-type enzymes.

[0075] (3) Half-saturation mutation Based on the results of experiment (2), sites A89, D110, A136, A143, A171, L176, I180 and V214 were selected for further half-saturation mutation in order to obtain mutants with further enhanced enzyme activity.

[0076] Using the wild-type construct pET28a-SmGI from Example 1 as a template, PCR amplification was performed using the site-directed mutagenesis primers in Table 5 and the PCR system and conditions in Table 3 to obtain recombinant constructs of each half-saturated single-point mutant. Then, mutant positive bacteria were obtained using the method in Experiment (1) and amplified separately.

[0077] Table 5

[0078] *In Table 5, except for the A143 mutants (A143L, A143I, A143M, A143F, A143T, A143C, A143Y, A143D) where primer 1 is the downstream primer and primer 2 is the upstream primer, all other mutants use primer 1 as the upstream primer and primer 2 as the downstream primer.

[0079] The equilibrium conversion rate, initial enzyme activity and half-life of each half-saturated single-point mutant were tested in the manner described in Example 1. The results are detailed in Table 6.

[0080] Table 6

[0081] (ii) Multipoint mutants To further enhance enzyme activity, we will conduct combinatorial mutations on the mutants (A89V, D110S, A136M, A143S, A171S, L176I, I180L, and V214L) in Table 6 that maintain the same half-life as the wild type while showing increased enzyme activity. The construction method is similar to that for constructing single-point mutants, except that the templates (constructors of single-point mutants) and primers in Table 7 are used for amplification of the constructs.

[0082] Table 7

[0083] Each multi-point mutant was tested for initial enzyme activity and half-life using the method described in Example 1. The results are detailed in Table 8.

[0084] Table 8

[0085] *In Table 8, the remaining enzyme activity after incubation at 60℃ for 8 days refers to the percentage of enzyme activity on the 8th day relative to the initial enzyme activity.

[0086] As can be seen from the data comparison in Table 8, the enzyme activities of the multi-point mutants D110S / A171S, A136M / A143S / A171S, A136M / A143S / A171S / L176I, A89V / D110S, A89V / A136M, A89V / D110S / A136M, A89V / D110S / A143S, A89V / I180L, and A89V / V214L are significantly improved while maintaining a half-life that is basically the same as that of wild-type glucose isomerase. The enzymes A89V / A143S, A89V / A171S, A89V / A143S / A171S, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L not only show improved enzyme activity but also a further extended half-life. Although the half-life of A89V / D110S / A143S / A171S / L176I, A89V / L176I, D110S / A136M / A143S / A171S / L176I, and D110S / A136M / A171S / L176I is slightly reduced, their enzyme activity is significantly improved, thus resulting in a substantial increase in reaction efficiency in practical applications.

[0087] Example 3 This embodiment is used to illustrate the performance effects of mutant enzymes expressed by different expression hosts, as well as the preparation and effects of immobilized mutant enzymes.

[0088] (1) Mutant enzymes obtained from different expression hosts The wild-type SmGI and the mutant sequences A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L from Example 2 were constructed into the pMA5 expression vector and transformed into expression hosts Bacillus subtilis 168, WB600N, WB800N, and 1012. The introduced expression hosts were then cultured and protein expression was performed. The specific procedures for constructing the Bacillus subtilis expression vector, introducing the expression hosts, and culturing for expression are as follows: 1) Construction of pMA5 expression vector: Using pET28-SmGI and its mutants A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L plasmids as templates, primers pMA5- Bam HI-SmGI-F (aagtgaaatcagggggatccATGAGCTTCCAGCCGACAC(SEQ ID NO:120), containing a partial fragment of the pMA5 vector, Bam HI restriction site and N-terminal fragment of SmGI) and pMA5- Mlu I-SmGI-R (gacctctagaacgcgtTCAgtggtggtggtggtggtgTCCTCT(SEQ ID NO:121), containing a partial fragment of the pMA5 vector, Mlu The fragments were amplified using the I restriction site and the C-terminal fragment of SmGI to obtain fragments of the pMA5 vector at both ends. Bam HI、 Mlu Gene fragments of pMA5-SmGI and its mutants were extracted from the I restriction site. The PCR reaction system and amplification conditions are shown in Table 9. After amplification, the PCR products were recovered by gel excision. Bam HI and Mlu I. The pMA5 plasmid was double-digested with enzymes. The double-digestion system is shown in Table 10. The pMA5 vector was then recovered by gel excision.

[0089] Table 9. PCR amplification system and procedure for the pMA5-SmGI gene fragment.

[0090] Table 10. Reaction system and conditions for vector restriction endonuclease digestion.

[0091] Following the system described in Table 11, the pMA5-SmGI gene fragment and its mutant gene fragment were ligated to the pMA5 vector using the homologous recombination kit (pEASY®-Uni Seamless Cloning and Assembly Kit), transformed into *E. coli* TransT1 competent cells, plated on LB agar plates containing 50 mg / L Amp, and incubated overnight at 37 °C. The next day, single clones were picked and colony PCR was performed using pMA5-seq-F (GAGCACACACTTTATGAATA, SEQ ID NO:122) and pMA5-seq-R (AAGGAGCCTTTAATTGTATC, SEQ ID NO:123) primers. Positive single clones were activated, plasmids were extracted, and sequencing analysis was performed.

[0092] Table 11 Homologous Recombination System and Conditions

[0093] 2) Construction of recombinant Bacillus subtilis: Bacillus subtilis 168, WB600N, WB800N and 1012 glycerol bacteria were taken from the -80 °C freezer, streaked on antibiotic-free LB agar plates, and incubated overnight at 37 °C. The next day, single colonies were picked and activated overnight in 30 mL of antibiotic-free LB liquid medium.

[0094] The activated bacterial culture was transferred to 40 mL of HS medium (preparation method shown in Table 12) to allow its initial OD to reach its maximum. 600 Incubate at 0.1°C, 37°C, and 200 rpm on a shaker until OD reaches zero. 600 The value reached 3.7±0.2 (after approximately 4-5 hours of incubation). Then, 10 mL of bacterial culture was added to 1 mL of 87% glycerol aqueous solution, placed on ice for 15 minutes, and then 1 mL was aliquoted into 1.5 mL EP tubes. These are the competent cells of Bacillus subtilis and stored at -80 °C.

[0095] Remove competent Bacillus subtilis cells from a -80 °C freezer and incubate at 37 °C for approximately 10 min until completely thawed. Add the thawed cells to 20 mL of LS medium (preparation method shown in Table 12) and incubate at 30 °C and 150 rpm for 2 h. Then, remove 1 mL of the bacterial culture, add 10 μL of 0.1 MEGTA solution, and incubate at room temperature for 5 min. Add approximately 1 μg of pMA5-SmGI and its mutant plasmid, and incubate at 37 °C and 200 rpm for 2 h to allow the cells to recover. After incubation, spread the bacterial culture on LB agar plates containing 25 mg / L Kana and incubate overnight at 37 °C. The next day, pick single colonies and perform colony PCR verification using pMA5-seq-F and pMA5-seq-R primers to obtain recombinant Bacillus subtilis containing SmGI and its mutant.

[0096] Table 12 Formulations and preparation methods of HS and LS media

[0097] 3) Expression of recombinant Bacillus subtilis protein: The recombinant Bacillus subtilis clones obtained above were picked and cultured overnight at 37 °C and 200 rpm in 5 mL LB medium containing 25 mg / L Kana. The next day, 1 mL of the bacterial culture was transferred to 50 mL SR medium containing 25 mg / L Kana (preparation method shown in Table 13) using a 250 mL shake flask with baffles. The culture was fermented at 37 °C and 200 rpm for 24 h, and the cells were collected by centrifugation at 4 °C and 8,000 rpm for 10 min. The transformation rate, enzyme activity, and half-life of the mutants expressed in each host were tested using the method in Example 1. The results showed that the enzyme activity and half-life of each mutant expressed in different hosts were basically the same as those tested in Example 2.

[0098] Table 13 SR liquid culture medium formulation and preparation method

[0099] (2) Immobilized mutant enzymes The immobilized glucose isomerase was prepared using the method described in Industrial Use of Immobilized Enzymes (DOI: 10.1039 / c3cs35506c). Wild-type SmGI and mutant enzymes A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L obtained by fermentation with Bacillus subtilis in Example 3 were used to prepare the corresponding immobilized mutant enzymes.

[0100] The conversion rate, enzyme activity, and half-life of the obtained immobilized mutant enzyme were detected using the method described in Example 1. Since immobilization significantly increases the half-life of the enzyme, in order to shorten the testing time, this example measured the half-life of the immobilized enzyme at 70°C. That is, the enzyme activity was tested at 70°C according to the national standard method, and the incubation time was measured when the remaining enzyme activity was 50%.

[0101] The results showed that the immobilized SmGI enzyme activity was 3500 U / g, the equilibrium conversion rate was 48%, and the half-life at 70℃ was 2.5 days. The mutants A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L all achieved equilibrium conversion rates above 50%, with enzyme activity increased to 7000-10000 U / g and a half-life at 70℃ increased to 3-4 days.

[0102] Example 4 This embodiment illustrates the application effect of the mutant enzyme of the present invention in the production of fructose syrup.

[0103] Using the wild-type SmGI and its mutants A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L immobilized enzymes obtained in Example 3, fructose syrup production was simulated on a small scale according to the following steps: 1. Prepare a 500g / L glucose reaction solution and adjust its pH to 7.1-8.5; 2. Weigh 20g of immobilized enzyme and soak it in 500g / L glucose reaction solution for about 5 hours to activate the immobilized enzyme; 3. Rinse the chromatography column with distilled water, and after sterilization, use water to remove air bubbles from the bottom of the chromatography column. 4. Add approximately 1 / 3 column volume of 500 g / L glucose reaction solution to the column, open the outlet valve, and adjust the flow rate to the target value so that the reaction solution flows out slowly until the liquid level in the column is 2-3 cm above the bottom sieve plate; 5. Slowly pour the pretreated immobilized enzyme particles (suspended in a small amount of reaction solution) into the chromatography column, avoiding direct pouring which may cause particle accumulation. 6. After the column is packed, a uniform immobilized enzyme layer will form inside the column. The top of the column should contain about 3±2 cm of reaction solution to prevent the column from drying out. 7. Connect the chromatography column to the feed pump and the glucose reaction solution feed tank, and seal the entire chromatography column; 8. Start the feed pump and flush the column with glucose reaction solution at the target flow rate. The amount of reaction solution used should be 10-15 times the column volume (BV). Observe whether the column pressure is stable (no significant increase) and whether the effluent is clear (no enzyme particle leakage). 9. The laboratory column test simulating the production process officially began. The reaction temperature was maintained at 60±2℃ using circulating water. The conversion rate was measured daily and the flow rate was adjusted in real time to ensure that the conversion rate was above 42%. The amount of dry fructose converted per unit mass of enzyme was recorded throughout the process. 10. Calculate the conversion rate using the following formula: Conversion rate = Product fructose concentration / Initial substrate glucose concentration × 100%; 11. Statistically analyze the test results of different enzymes in simulated production, and calculate the amount of oven-dry fructose converted and the amount of oven-dry fructose converted per unit mass of enzyme according to Formulas II and III below.

[0104] Conversion of dry fructose (kg) = Total sugar content (L) × 0.5 (kg / L) × Average conversion rate (Formula II) The amount of oven-dry fructose converted by enzyme per unit mass (kg / g) = the amount of oven-dry fructose converted (kg) / the enzyme loading mass (g) Equation III The amount of fructose converted by the wild-type SmGI and its mutants A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A143S / A171S / I180L / V214L immobilized enzymes prepared in Example 3 during a 180-day simulated production test in the laboratory is shown in Table 14. The amount of oven-dry fructose converted from 20g of wild-type SmGI immobilized enzyme in 180 days was 110kg, with a unit mass enzyme conversion of 5.5kg / g of oven-dry fructose. The same mass (20g) of mutants A89V, A89V / A143S, A89V / A171S, A89V / D110S / A143S / A171S / L176I, A89V / I180L / V214L, and A89V / D110S / A1 The amount of oven-dry fructose converted by the 43S / A171S / I180L / V214L immobilized enzyme after 180 days was 132 kg, 146 kg, 144 kg, 149 kg, 145 kg, and 152 kg, respectively, with oven-dry fructose conversion per unit mass of enzyme being 6.6 kg / g, 7.3 kg / g, 7.2 kg / g, 7.5 kg / g, 7.3 kg / g, and 7.6 kg / g, respectively, all higher than that of the wild-type SmGI immobilized enzyme. Among them, the mutant immobilized enzyme A89V / D110S / A143S / A171S / I180L / V214L showed a 38% increase in yield compared to the wild-type SmGI immobilized enzyme.

[0105] Table 14

[0106] *Table 14 shows how to adjust the conversion rate by adjusting the flow rate to maintain it at 42±1%. The average conversion rate is calculated as a weighted average of the daily conversion rates.

[0107] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A glucose isomerase mutant, characterized in that, Compared to the wild-type glucose isomerase, the mutant has a mutation at least one of the amino acids at positions 89, 110, 136, 143, 171, 176, 180, and 214, and the amino acid sequence of the wild-type glucose isomerase is shown in SEQ ID NO:

1.

2. The mutant according to claim 1, wherein, The mutation is a substitution mutation; Preferably, the mutation includes at least one of the following: (1) The 89th amino acid is mutated from alanine to methionine, valine, or isoleucine; (2) The 110th amino acid is mutated from aspartic acid to serine or alanine; (3) The amino acid at position 136 is mutated from alanine to phenylalanine, methionine, isoleucine, leucine or valine; (4) The amino acid at position 143 is mutated from alanine to serine; (5) The amino acid at position 171 is mutated from alanine to serine or aspartic acid; (6) The amino acid at position 176 is mutated from leucine to isoleucine; (7) The 180th amino acid is mutated from isoleucine to leucine; (8) The 214th amino acid was mutated from valine to leucine; More preferably, the mutant is selected from any one of the following: A89M, A89V, A89I, D110S, D110A, A136F, A136M, A136I, A136L, A136V, A143S, A171S, A171D, L176I, I180L, V214L, D110S / A171S, A89V / D110S, A89V / L176I, A89V / A143S, A89V / A171S, A89V / A136M, A89V / I180L, A89V / V214L, A136M / A143S / A 171S, A89V / A143S / A171S, A89V / D110S / A136M, A89V / D110S / A143S, A89V / I180L / V214L, D110S / A136M / A171S / L176I, A136M / A143S / A171S / L176I, D110S / A136M / A143S / A171S / L176I, A89V / D110S / A143S / A171S / L176I and A89V / D110S / A143S / A171S / I180L / V214L.

3. A nucleic acid, characterized in that, The nucleic acid encodes the glucose isomerase mutant as described in claim 1 or 2.

4. A construct, characterized in that, The construct comprises the nucleic acid as described in claim 3.

5. A host cell, characterized in that, The host cell comprises the nucleic acid of claim 3 or the construct of claim 4, preferably the host cell is selected from at least one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Streptomyces, Saccharomyces cerevisiae, methanolytrophic yeast, Hansenula polymorpha, fissile yeast, Kluyveromyces kusnezoffii, Candida albicans, Pichia pastoris, and filamentous fungi.

6. An immobilized enzyme, said immobilized enzyme comprising a carrier and an enzyme loaded on the carrier, characterized in that, The enzyme is selected from the glucose isomerase mutant described in claim 1 or 2.

7. Use of the glucose isomerase of claim 1 or 2, the nucleic acid of claim 3, the construct of claim 4, the host cell of claim 5, or the immobilized enzyme of claim 6 in the preparation of food ingredients, pharmaceutical ingredients or biofuel ingredients.

8. A method for increasing the enzyme activity of glucose isomerase while maintaining its thermostability substantially, characterized in that, The method includes causing a mutation in at least one of the amino acids at positions 89, 110, 136, 143, 171, 176, 180, and 214 of the wild-type glucose isomerase, the amino acid sequence of which is shown in SEQ ID NO:

1.

9. The method according to claim 8, wherein, The mutation is a substitution mutation; Preferably, the mutation includes at least one of the following: (1) The 89th amino acid is mutated from alanine to methionine, valine, or isoleucine; (2) The 110th amino acid is mutated from aspartic acid to serine or alanine; (3) The amino acid at position 136 is mutated from alanine to phenylalanine, methionine, isoleucine, leucine or valine; (4) The amino acid at position 143 is mutated from alanine to serine; (5) The amino acid at position 171 is mutated from alanine to serine or aspartic acid; (6) The amino acid at position 176 is mutated from leucine to isoleucine; (7) The 180th amino acid is mutated from isoleucine to leucine; (8) The 214th amino acid was mutated from valine to leucine; More preferably, the mutation can yield any of the following mutants: A89M, A89V, A89I, D110S, D110A, A136F, A136M, A136I, A136L, A136V, A143S, A171S, A171D, L176I, I180L, V214L, D110S / A171S, A89V / D110S, A89V / L176I, A89V / A143S, A89V / A171S, A89V / A136M, A89V / I180L, A89V / V214L, A136M / A143S / A171S, A89V / A143S / A171S, A89V / D110S / A136M, A89V / D110S / A143S, A89V / I180L / V214L, D110S / A136M / A171S / L176I, A136M / A143S / A171S / L176I, D110S / A136M / A143S / A171S / L176I, A89V / D110S / A143S / A171S / L176I and A89V / D110S / A143S / A171S / I180L / V214L.

10. The method according to claim 8 or 9, wherein, The method includes the step of constructing a mutant recombinant vector; Preferably, constructing the mutant recombinant vector includes in vitro amplification of the recombinant vector containing the wild-type glucose isomerase encoding gene using site-directed mutagenesis primers; More preferably, the site-directed mutagenesis primers are selected from at least one group of SEQ ID NO:5 and 6, SEQ ID NO:44 and 54, SEQ ID NO:46 and 54, SEQ ID NO:60 and 61, SEQ ID NO:15 and 16, SEQ ID NO:62 and 71, SEQ ID NO:63 and 71, SEQ ID NO:65 and 71, SEQ ID NO:66 and 71, SEQ ID NO:19 and 20, SEQ ID NO:23 and 24, SEQ ID NO:87 and 88, SEQ ID NO:27 and 28, SEQ ID NO:29 and 30, SEQ ID NO:33 and 34, SEQ ID NO:7 and 8, SEQ ID NO:117 and 118, and SEQ ID NO:7+19+23 and 8+20+24.

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