Thermostable enhanced glucose isomerase and use thereof

Abstract

The present application relates to the technical field of enzyme catalysis, and particularly relates to a glucose isomerase with enhanced heat resistance and application, and the amino acid sequence of the glucose isomerase is shown as SEQ ID NO:1. When used, D-glucose mother liquor is taken, PB and pure water are added, crude enzyme solution of the glucose isomerase is added, and reaction is carried out at 80 DEG C. The glucose isomerase with enhanced heat resistance of the present application can catalyze D-glucose to generate D-fructose at high temperature; compared with wild-type glucose isomerase, the glucose isomerase with enhanced heat resistance can be applied to high-temperature conversion of D-glucose to generate D-fructose, and shows stronger heat stability.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, specifically to a glucose isomerase with enhanced heat resistance. Background Technology

[0002] Glucose isomerase, also known as D-xylose isomerase, is an enzyme with significant industrial applications. In many microorganisms, it catalyzes the isomerization of D-xylose to xylulose in the first step of xylose metabolism. Glucose isomerase participates in sugar metabolism and is widely used in the industrial production of high-fructose corn syrup (HFCS) and bioethanol. This isomerization reaction is crucial in industrial processes, such as the production of HFCS in the food industry. HFCS itself is a popular sweetener in the United States and Japan, serving as an alternative source of sucrose or invert sugar in the pharmaceutical, food, and beverage industries. Driven by the growing demand for HFCS, glucose isomerase has been increasingly used in the production of HFCS from corn starch hydrolysate since the 1970s, ranking alongside amylase and protease as one of the three most consumed industrial enzymes. Because HFCS containing 55% or more fructose has a higher sweetness than sucrose, the food and beverage industry has a significant demand for such syrups, and as the HFCS market grows, the demand for more efficient glucose isomerase is also increasing.

[0003] In existing high-fructose corn syrup (HFCS) production processes, thermostable glucose isomerases from mesophilic microorganisms are used in immobilized enzyme reactors to produce 40% to 42% HFCS at operating temperatures of 50°C and 58°C, with residence times of less than one hour. Above these temperatures, unwanted browning products form due to non-enzymatic reactions between reducing sugars and proteins (e.g., Maillard reactions). However, this temperature limitation is detrimental to high glucose conversion rates. In fact, glucose isomerization reaches a reaction equilibrium that shifts towards fructose at higher temperatures. This process requires additional, costly chromatographic steps to obtain the desired 55% HFCS concentration. HFCS-55 is a more desirable HFCS variety for food and soft drink applications. During HFCS production, the final conversion of D-glucose to D-fructose is highly temperature-dependent, with higher temperatures generally favoring D-fructose yields. To avoid complex downstream concentration steps and to directly obtain HFCS-55 by promoting an isomerization equilibrium bias towards D-fructose formation, higher temperatures are required. In theory, isomerization needs to be carried out at high temperatures (around 95°C) and low pH (around 4.5–5.5) to achieve the desired fructose levels in the syrup, due to the equilibrium of the isomerization reaction. If this can be achieved, no additional concentration step is required.

[0004] In short, compared to currently used enzymes, industrially valuable glucose isomerases must have lower pH values, higher temperatures, and lower tolerance to Ca2+. 2+Resistance to inhibition and a high affinity for glucose are key factors. Therefore, a significant amount of research is currently focused on identifying thermostable and acid-stable glucose isomerases with higher affinity for glucose. Furthermore, advances in recombinant DNA technology and protein engineering have opened new possibilities for developing economically viable commercial processes. For this reason, researchers are attempting to isolate microorganisms capable of producing highly active and stable glucose isomerases at high temperatures.

[0005] US Patent WO2004 / 044129 identifies a need to discover glucose isomerases that maintain high activity levels at high temperatures and low pH. However, the peptides disclosed in that document exhibit optimal activity only above pH 5 and are unstable above 90°C. More importantly, to maintain high activity levels, these enzymes require cobalt (Co). 2+ ) as a cofactor. And Co 2+ It is not suitable for producing high-fructose corn syrup intended for human consumption. Not only is it associated with numerous potential health problems, but the disposal of waste media can also lead to environmental pollution. The most suitable ions are those commonly used in food-related applications, including Mn. 2+ and Mg 2+ However, these ions are generally only effective when they bind to enzymes from non-thermophilic organisms or microthermophilic organisms.

[0006] Therefore, the optimal glucose isomerase for the food industry should meet the following conditions: it should function efficiently under acidic conditions (i.e., pH 6 or lower) to avoid browning; it should be tolerant of high substrate and product inhibition (e.g., greater than 200 g / L glucose) to improve production efficiency per unit volume; it should remain stable at high reaction temperatures (i.e., 80°C or higher) to promote high fructose conversion; it should have high catalytic activity, reaching the reaction equilibrium point in a short time to reduce energy and labor consumption; and it should not use harmful metal ions, such as Co. 2+ It can be efficiently and massively recombined and prepared in common microorganisms such as Escherichia coli or Bacillus, so as to facilitate production and reduce costs.

[0007] Therefore, developing a new glucose isomerase or mutant to improve its high-temperature tolerance and catalytic efficiency under high-temperature reaction conditions has great practical value. Summary of the Invention

[0008] The purpose of this invention is to provide a glucose isomerase with enhanced heat resistance and its application.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A thermostable glucose isomerase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0011] The heat-resistant glucose isomerase of the present invention can be used to catalyze the production of D-glucose from D-glucose. The method of use is as follows: take D-glucose mother liquor, add PB and pure water, add crude enzyme solution of glucose isomerase, and react at 80°C.

[0012] The method for preparing the crude enzyme solution includes the following steps:

[0013] (1) The corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO: 1 was assembled by primer splicing and cloned into a prokaryotic expression vector to achieve high expression in Escherichia coli;

[0014] (2) Shake flask fermentation or fed-batch fermentation are adopted.

[0015] ① Shake flask fermentation

[0016] A single colony of Escherichia coli containing the expression vector was picked and inoculated into 10 mL of autoclaved medium A and cultured overnight at 30°C and 250 rpm.

[0017] The next day, take a 1L Erlenmeyer flask and inoculate it into 100mL of autoclaved culture medium B at an inoculation ratio of 1:100. Incubate at 30°C until the bacterial OD reaches 5-6. Immediately place the Erlenmeyer flask in a 25°C shaker and incubate at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue incubating at 25°C and 250rpm for 16 hours.

[0018] After the culture was completed, the culture medium was centrifuged at 12000g for 20 minutes at 4℃ to collect the wet bacterial cells; then the bacterial precipitate was washed twice with distilled water, the bacterial cells were collected and stored at -70℃; at the same time, a small amount of bacterial cells were taken for SDS-PAGE detection.

[0019] ② Batch feeding fermentation

[0020] Fed-batch fermentation was carried out in a computer-controlled bioreactor. A single colony of *E. coli* containing the expression vector was prepared in a 200ml seed shake flask. When the culture in the seed shake flask reached an OD of 2.0, it was inoculated into the bioreactor. Throughout the fermentation process, the temperature was maintained at 37°C. The dissolved oxygen concentration was automatically controlled at 30% by a cascade of stirring and aeration, while the pH of the culture medium was maintained at 7.0 using 50% v / v orthophosphate and 30% v / v ammonia. During fermentation, when a significant increase in dissolved oxygen occurred, feeding was initiated. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When the OD600 reached 50.0, the temperature was controlled at 25°C, and expression was induced with 0.1mM IPTG for 16 hours. The cells were then centrifuged, collected, and stored at -25°C. For use, 2 kg of pure water was added per kg of wet cells.

[0021] The culture medium A consists of: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.8 g / L glucose, with kanamycin added to bring the concentration to 50 mg / L.

[0022] The culture medium B consists of: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.3 g / L glucose, with kanamycin added to a concentration of 50 mg / L.

[0023] The culture medium used for fed-batch fermentation consisted of 24 g / L yeast extract, 12 g / L peptone, 0.4% glucose, 2.31 g / L dihydrogen phosphate enzyme, and 12.54 g / L dipotassium hydrogen phosphate, with a pH of 7.0.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The thermostable glucose isomerase of this invention can catalyze the conversion of D-glucose to D-fructose at high temperatures. Compared with wild-type glucose isomerase, it can be applied to the high-temperature conversion of D-glucose to D-fructose, exhibiting stronger thermal stability and achieving better social and economic benefits. Attached Figure Description

[0026] Figure 1 The results are from a high-performance liquid chromatography (HPLC) study of 20 g / L fructose. The fructose peak is observed at 8.4 minutes.

[0027] Figure 2 These are the results of high-performance liquid chromatography (HPLC) for 20 g / L glucose. The glucose peak is observed at 10 minutes.

[0028] Figure 3 The results are for Comparative Example 1 after 20 hours of reaction.

[0029] Figure 4 The results are from the test after 20 hours of reaction in Example 6. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, all instruments and reagents used in this embodiment are commercially available products.

[0032] The liquid phase detection conditions involved in the following examples are as follows:

[0033] Mobile phase: Acetonitrile: Water = 70:30

[0034] Detector: Differential detector

[0035] Flow rate: 1 mL / min

[0036] Column temperature: 40℃

[0037] Differential detector cell temperature: 40℃

[0038] Use a 250mm*4.6μm amino column.

[0039] Example 1: Obtaining the wild-type glucose isomerase gene sequence

[0040] High expression in *E. coli* was achieved by modifying the secondary structure and codon bias of the gene using whole-genome synthesis. Primers were designed using Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), ensuring the annealing temperature (Tm) difference was controlled within 3°C and the primer length within 60 bases. The primer sequences are shown in Table 2. The obtained primers were dissolved in double-distilled water and added to the reaction system to achieve a final primer concentration of 30 nM and a final concentration of 0.6 μM for the first and last primers.

[0041] Table 1

[0042] 10×Pfubuffer 5μl Pfu DNA polymerase (10 U / μl) 0.5μl <![CDATA[ddH2O]]> This brings the total volume of the reaction system to 50 μl.

[0043] The prepared PCR reaction system was placed in a Borgi XP cycler gene amplification instrument and amplified according to the following program: 98℃ for 30s, 55℃ for 45s, 72℃ for 120s, 35x. The DNA fragment obtained by PCR was purified by gel extraction and cloned into the NdeI / XhoI site of pET30a using homologous recombination. Single clones were picked for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 4, named CSGIwt, and its corresponding amino acid sequence is SEQ ID NO: 3.

[0044] Table 2

[0045]

[0046]

[0047] Example 2: Obtaining the gene sequence of the glucose isomerase mutant

[0048] The present invention provides a thermostable glucose isomerase mutant derived from the wild-type glucose isomerase of SEQ ID NO: 3. The glucose isomerase mutant and the polynucleotide encoding such mutant can be prepared using methods commonly employed by those skilled in the art. The mutant can be obtained through in vitro recombination of the enzyme, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, and directed evolution methods.

[0049] High expression in *E. coli* was achieved by modifying the secondary structure and codon bias of the gene using whole-genome synthesis. Primers were designed using Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), ensuring the annealing temperature (Tm) difference was controlled within 3°C and the primer length within 60 bases. The primer sequences are shown in Table 4. The obtained primers were dissolved in double-distilled water and added to the reaction system to achieve a final primer concentration of 30 nM and a final concentration of 0.6 μM for the first and last primers.

[0050] Table 3

[0051] 10×Pfubuffer 5μl Pfu DNA polymerase (10 U / μl) 0.5μl <![CDATA[ddH2O]]> This brings the total volume of the reaction system to 50 μl.

[0052] The prepared PCR reaction system was placed in a Borgi XP cycler gene amplification instrument and amplified according to the following program: 98℃ for 30s, 55℃ for 45s, 72℃ for 120s, 35x. The DNA fragment obtained by PCR was purified by gel extraction and cloned into the NdeI / XhoI site of pET30a using homologous recombination. Single clones were picked for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 2, named CSGI1, and its corresponding amino acid sequence is SEQ ID NO: 1. Compared with the CSGIwt sequence, it has three mutations: A44S, K380R, and K390R.

[0053] Table 4

[0054]

[0055]

[0056] Example 3: Shaking Bottle Expression Test

[0057] Single colonies of *E. coli* containing the expression vector were picked and inoculated into 10 ml of autoclaved medium containing: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.8 g / L glucose, and kanamycin to a final concentration of 50 mg / L. The medium was incubated overnight at 30°C and 250 rpm.

[0058] The next day, take a 1L Erlenmeyer flask and inoculate it into 100ml of autoclaved culture medium at a 1:100 ratio: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, potassium dihydrogen phosphate 3.4g / L, ammonium chloride 2.68g / L, sodium sulfate 0.71g / L, magnesium sulfate heptahydrate 0.493g / L, ferric chloride hexahydrate 0.027g / L, glycerol 5g / L, glucose 0.3g / L, and add kanamycin to a final concentration of 50mg / L. Incubate at 30℃ until the bacterial OD reaches 5-6, then immediately place the Erlenmeyer flask in a 25℃ shaker at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue incubating at 25℃ and 250rpm for 16 hours.

[0059] After culturing, the culture medium was centrifuged at 12000g for 20 minutes at 4℃ to collect the wet bacterial cells. The bacterial precipitate was then washed twice with distilled water, and the cells were collected and stored at -70℃. A small amount of the bacterial cells was also taken for SDS-PAGE analysis.

[0060] Example 4: Batch Feeding Fermentation

[0061] Feed-in fermentation was carried out in a computer-controlled bioreactor (Shanghai Guoqiang) with a capacity of 15L and a working volume of 8L. The culture medium used consisted of 24g / L yeast extract, 12g / L peptone, 0.4% glucose, 2.31g / L dihydrogen phosphate enzyme, and 12.54g / L dipotassium hydrogen phosphate, at a pH of 7.0.

[0062] A single colony of *E. coli* containing the expression vector was prepared in a 200 ml seed shake flask. When the culture in the seed shake flask reached an OD of 2.0, it was inoculated into the bioreactor. Throughout the fermentation process, the temperature was maintained at 37°C. The dissolved oxygen concentration was automatically controlled at 30% by a cascade of stirring speed (rpm) and aeration, while the pH of the culture medium was maintained at 7.0 by 50% (v / v) orthophosphate and 30% (v / v) ammonia. During fermentation, when a significant increase in dissolved oxygen occurred, fed-batch feeding was initiated. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When the OD600 was approximately 50.0 (wet weight approximately 100 g / L), the temperature was controlled at 25°C, and expression was induced with 0.1 mM IPTG for 16 hours. The cells were then collected by centrifugation and stored at -25°C. For use, 2 kg of purified water was added per kg of wet cells.

[0063] Example 5: Heat Treatment and Residual Enzyme Activity Detection

[0064] Each enzyme was incubated at 85°C in 10ml tubes (2ml system with 0.5ml crude enzyme solution) for 0, 0.5, 1, and 2 hours. Then, 500μl of the heat-treated enzyme extract and 500μl of substrate buffer (100mM PB buffer, pH 6.8, 20mM MgSO4, 1mM NCl2, 360g / L D-glucose, pH 6.8) were mixed and incubated at 55°C for 4 hours to evaluate residual enzyme activity. Finally, high-performance liquid chromatography (HPLC) was used to detect substrate residue and product formation, with the 0-hour CSGIwt sample as 100% standard. At 4 hours, the fructose concentration was 97.51g / L, and the glucose concentration was 91.13g / L, with a conversion rate of 54.17%. The results of a 2-hour heat-treated followed by a 4-hour reaction are shown below.

[0065]

[0066] It is evident that the mutants of this invention retain a significant amount of enzyme activity after high-temperature treatment, showing a substantial improvement over the wild-type protein.

[0067] Comparative Example 1: High-Temperature Reaction Control Example

[0068] Prepare a 2ml system in a 10ml reaction tube containing 50mM PB buffer (pH 6.8), 10mM MgSO4, 0.5mM MnCl2, 400g / L D-glucose, and 50μl / ml CSGIwt crude enzyme solution. React in an 80℃ water bath for 4 hours and overnight, then take samples. Spot the samples onto a plate, dilute 5-fold, and analyze by high-performance liquid chromatography (HPLC). Figure 3 As shown, the results indicate that the fructose concentration was 100.56 g / L after 20 hours, and the enzyme activity was significantly reduced during the high-temperature reaction.

[0069] Example 6: High-Temperature Reaction Example

[0070] Prepare a 2ml system in a 10ml reaction tube containing 50mM PB buffer (pH 6.8), 10mM MgSO4, 0.5mM MnCl2, 400g / L D-glucose, and 50μl / ml CSGI1 crude enzyme solution. React at 80℃ for 4 hours and overnight, then take samples. Spot the samples onto a plate, dilute 5-fold, and analyze by high-performance liquid chromatography (HPLC). Figure 4 As shown, the fructose concentration was 230.77 g / L after 20 hours.

[0071] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A glucose isomerase with enhanced heat resistance, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of the thermostable glucose isomerase of claim 1 in catalyzing the production of D-glucose from D-glucose.

3. The application of the thermostable glucose isomerase according to claim 2 in catalyzing the production of D-glucose from D-glucose, characterized in that: Take the D-glucose mother solution, add PB buffer and pure water, add the crude enzyme solution of glucose isomerase as described in claim 1, and react at 80°C.

4. The application of the thermostable glucose isomerase according to claim 3 in catalyzing the production of D-glucose from D-glucose, characterized in that, The method for preparing the crude enzyme solution includes the following steps: (1) The corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO:1 was assembled by primer splicing and cloned into a prokaryotic expression vector to achieve high expression in Escherichia coli; (2) Shake flask fermentation or batch feeding fermentation is adopted.

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