Expression strain of sucrose isomerase fused with tag as well as production method and application of expression strain

Abstract

The invention belongs to the field of bioengineering and genetic engineering, and designs an expression strain of sucrose isomerase fused with a tag as well as a production method and application of the expression strain. The method comprises the following steps: by taking a GFP coding gene and a CBM2a-PalI coding gene as templates, respectively carrying out amplification through an overlap PCR (Polymerase Chain Reaction) method to obtain a GFP-CBM2a-PalI coding gene, then converting the GFP-CBM2a-PalI coding gene into a competent cell of expression host bacteria E. coli Rosetta (DE3) through heat shock, and finally obtaining an expression strain of the sucrose isomerase containing the fusion tag. Compared with a traditional immobilization method, the conversion rate is remarkably improved, the visual immobilization strategy provides innovative help for industrial production application, and the monitoring intuition of the production process is improved.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and genetic engineering, and designs an expression strain of sucrose isomerase with fusion tag, its production method and application. Background Technology

[0002] Isomaltulose is a reducing disaccharide formed by the α-1,6 glycosidic bond between glucose and fructose. Its taste and physicochemical properties are similar to sucrose, but its properties are superior. Therefore, isomaltulose is widely used in the food, pharmaceutical, and health product industries. Currently, the main methods for producing isomaltulose are biocatalysis and sucrose isomerase catalysis. Biocatalysis involves using microorganisms or their enzymes to convert sucrose into isomaltulose; this method has limitations and is therefore unsuitable for modern factory production. Sucrose isomerase catalysis directly uses sucrose isomerase to convert sucrose into isomaltulose; this method is suitable for large-scale factory production and the reaction is relatively simple. Currently, isomaltulose production mainly utilizes sucrose isomerase derived from microorganisms to convert sucrose.

[0003] Currently, the expression of sucrose isomerases faces several significant challenges. For example, free enzymes require complex and costly purification methods; immobilized enzymes or materials are expensive; and immobilization reduces enzyme conversion rates. Common sucrose isomerase immobilization methods include cross-linking, encapsulation, and adsorption. Contesini et al. prepared immobilized sucrose isomerases from Erwinia sp. D12 using two methods: encapsulation with low-methoxy pectin and fat microcapsules, and adsorption with tetracalcium aluminoferrite. The isomaltulose conversion rates of the resulting immobilized enzymes were only 30% and 60%, respectively. Optimization of the adsorption immobilization conditions further increased the isomaltulose conversion rate to 63%. Therefore, finding an economical, efficient, and highly effective immobilization method is a pressing issue in sucrose isomerase research. Summary of the Invention

[0004] The purpose of this invention is to provide an expression strain for producing sucrose isomerase, its production method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A strain expressing a fusion-tagged sucrose isomerase was amplified using overlap PCR with the GFP-encoding gene and the CBM2a-PalI-encoding gene as templates to obtain the GFP-CBM2a-PalI-encoding gene, which was then transformed into a heat-shock transformation host strain. E. coli In Rosetta (DE3) competent cells, an expression strain containing a fusion tag sucrose isomerase was finally obtained.

[0006] To elaborate further, (1) Using pET28a-CBM17-GFP as a template and GF / R as primers, the GFP coding gene was amplified. The amplification product was recovered by gel extraction to obtain the GFP coding gene. (2) Using pET32a-CBM2a-PalI as a template, and CF and PR as primers respectively, the CBM2a-PalI coding gene was amplified. The amplification product was recovered by gel extraction to obtain the CBM2a-PalI coding gene. (3) Using the gel recovery products of (1) and (2) as templates, the GFP encoding gene and the CBM2a-PalI encoding gene were fused using the overlap PCR method to obtain the first round overlap PCR product (GFP-CBM2a-PalⅠ encoding gene). Using the first round overlap PCR product as templates, and GF and PR as primers, the first round product of the GFP-CBM2a-PalⅠ encoding gene RF-clone was amplified using the overlap PCR method. The amplified product was gel recovered to obtain the GFP-CBM2a-PalⅠ encoding gene. (4) Using the GFP-CBM2a-PalⅠ encoding gene of the gel recovery product in (3) as primers, and pET32a-PalI as template, the second round of RF clone product amplification was performed to construct the pET32a-GFP-CBM2a-PalI recombinant expression vector; (5) The above RF-clone product was digested with QuickCut™ DpnI at 37°C for 12 h and then electroporated into the cloning bacteria. E. coli DH10B competent cells were plated on LB agar plates containing 25 μg / mL chloramphenicol (CM) and 100 μg / mL ampicillin (Amp), respectively, and incubated at 37°C for 12–16 h until single colonies appeared. Plasmids were then extracted after sequencing. (6) The plasmids of correctly sequenced transformants were heat-shocked into competent cells of the expression host bacterium E. coli Rosetta (DE3) to finally obtain the expression strain (GC2aPalIE) containing the fusion tag sucrose isomerase.

[0007] The above plasmids pET28a-CBM17-GFP (see this article for details: Li Sipeng, Lü Yifei, Li Xianzheng, et al. Immobilization of CBM-mediated sucrose isomerase [J]. Journal of Microbiology, 2024, 44(03):12-23). ​​pET32a-CBM2a-PalI and pET32a-PalI vector plasmids (preliminary laboratory construction) were derived from the National-Local Joint Engineering Laboratory for Biocatalysis Technology of Dalian University of Technology; PalI was derived from Klebsiella Klebsiella pneumoniae sp. LX3 (GenBank: KJ452353.1), see this paper for details, by Wang Bingbing. (Source: [Insert source here]) Klebsiella Site-directed mutagenesis of sucrose isomerase PalⅠ of sp. LX3 and fermentation transformation analysis of Bacillus subtilis expression product [D]. Dalian University of Technology, 2019. DOI:10.26992 / d.cnki.gdlqc.2019.000141.

[0008] The electroporation conversion method is as follows: Take a clean electroporation cup and place it at -20°C. o C's refrigerator was pre-chilled. (Take out) E. coli DH10B electroporated competent cells were thawed in an ice-water mixture. After thawing, the competent cells were aspirated. Dpn 3 μL of the digestion product was carefully added to the competent cells. The mixture was then gently pipetted and placed in an ice-water bath for 10 min. After the ice bath, the pre-chilled electroporation cuvette was removed, and the mixture was transferred to the cuvette. The cuvette was capped and inserted into the electroporator to begin electroporation at 25,000 V for 5 ms. Immediately after electroporation, 1 mL of sterile LB broth was added to the cuvette. The culture was gently mixed with the competent cells using a pipette, and the entire culture was then transferred from the cuvette to a 2.5 mL centrifuge tube and incubated at 37°C. o Incubate at 200 rpm for 1 h. After 1 h, take out 200 μL of the cultured bacterial solution, spread it on a plate, and incubate at 37℃ for 12-16 h.

[0009] The thermal shock conversion method is as follows: Take E. coli Rosetta (DE3) heat-stress competent cells were thawed in an ice-water mixture. After thawing, 1 μL of plasmid was carefully added to the competent cells, followed by gentle pipetting of the mixture. The mixture was then placed in an ice-water mixture for an ice bath for 30 min. After the ice bath, the competent cells were placed at 42°C. o Incubate in a C2 water bath for 1 min, then immediately remove and incubate on ice for 5 min. Add 1 mL of sterilized LB liquid medium to the competent cells, gently pipette to mix the medium with the competent cells, and then incubate at 37°C. o Incubate at 200 rpm for 45 min at C. Take 200 μL of the cultured bacterial solution and spread it onto LB agar plates of the corresponding antibiotic resistance. Invert the plates and incubate at 37°C. oIncubate statically at C for 12-16 hours until single colonies appear. The cells can then be cultured in liquid form and stored at -80°C to prepare a bacterial culture. o C.

[0010] The LB medium consists of the following components: NaCl 10 g / L, tryptone 5 g / L, yeast extract 5 g / L, and pH adjusted to 7 in water. The LB solid culture medium consists of the following components in water: NaCl 10 g / L, tryptone 5 g / L, yeast extract 5 g / L, agar powder 1.5%-2%, and pH adjusted to 7. The TB liquid culture medium consists of the following components in water: 3 mL / L glycerol, 7.2 g / L tryptone, 14.4 g / L yeast extract, 1.4 g / L potassium dihydrogen phosphate, 9.86 g / L dipotassium hydrogen phosphate, and pH adjusted to 7. All of the above culture media were at 121 o Sterilize at C for 20 min; The method for preparing the 1.0 mmol / L IPTG is as follows: Weigh 0.238g of IPTG, add ddH2O to dissolve it completely, and then bring the volume to 1L.

[0011] Application of an expression strain of the fusion-tagged sucrose isomerase, and application of the expression strain in the preparation of a visualized immobilized sucrose isomerase. A method for preparing a visualized immobilized sucrose isomerase involves fermenting the strain (GC2aPalIE) and then immobilizing it with microcrystalline cellulose to obtain the immobilized sucrose isomerase GC2aPalI-A. Further, the strain is inoculated into LB liquid medium containing the corresponding antibiotic and cultured at 37°C and 200 r / min with shaking until the OD600 nm reaches 2.0 to obtain a seed culture. The seed culture is then inoculated into TB liquid medium at a volume ratio of 1-5% for expansion culture. When the OD600 nm reaches 0.6, IPTG at a final concentration of 1.0 mmol / L is added, and the culture is carried out at 16°C and 200 r / min for 16-20 h to induce sucrose isomerase expression. After induction, the precipitate is collected by centrifugation, washed, and then resuspended in citrate-disodium hydrogen phosphate buffer. The precipitate is then sonicated, centrifuged, and collected again to obtain the crude sucrose isomerase solution. The obtained crude sucrose isomerase solution is then incubated with microcrystalline cellulose at 30°C and 60°C. Immobilization of GC2aPalI on the surface was carried out under immobilization conditions of min to prepare immobilized sucrose isomerase GC2aPalI-A.

[0012] The beneficial effects of this invention are as follows: (1) The constructed PalI fusion of CBM2a and GFP tags was successfully immobilized on the surface of microcrystalline cellulose, achieving one-step purification and visualization immobilization of sucrose isomerase. The optimal temperature and pH for immobilized sucrose isomerase were 45℃ and 6.5, respectively, and its temperature and pH stability were not significantly different from those of the free enzyme. The highest enzyme activity was 7.89±0.22 U / mg microcrystalline cellulose (Avicel PH101). The affinity of the immobilized enzyme for the substrate sucrose was enhanced, with a Km of 83.03±3.93 mmol / L. The storage stability of the enzyme was also improved. After 133 days of storage at 4℃, the enzyme activity was still above 60%, and (2) Compared with the free enzyme, the temperature stability and pH stability of the visualized immobilized enzyme are not significantly different. Moreover, the visualized immobilized enzyme has good operational stability and storage stability. After 12 consecutive reactions, the enzyme activity still remains at about 70% of the initial enzyme activity, which improves its reusability and increases the total sugar content of isomaltulose in the enzymatic hydrolysis product. The visualized immobilization of sucrose isomerase increases the proportion of isomaltulose in the total sugar content of sucrose enzymatic hydrolysis product to 72.51%±1.3%, which is 1.28 times higher than that of the free enzyme, and the conversion rate is significantly improved compared with the traditional immobilization method.

[0013] The visualization and fixation strategy of this invention provides innovative assistance for industrial production applications and increases the intuitiveness of production process monitoring. Attached Figure Description

[0014] Figure 1 Visualization of fluorescence detection of immobilized sucrose isomerase.

[0015] Figure 2 This is the optimal temperature for free enzymes.

[0016] Figure 3 To visualize the optimal temperature for immobilized enzymes.

[0017] Figure 4 This is to ensure the temperature stability of the free enzyme.

[0018] Figure 5 To visualize the temperature stability of immobilized enzymes.

[0019] Figure 6 The optimal pH for free enzymes.

[0020] Figure 7 To visualize the optimal pH for immobilized enzymes.

[0021] Figure 8 This refers to the pH stability of the free enzyme.

[0022] Figure 9 To visualize the pH stability of immobilized enzymes.

[0023] Figure 10 To visualize the operational stability of immobilized enzymes.

[0024] Figure 11 To visualize the storage stability of immobilized enzymes.

[0025] Figure 12 Thin-layer chromatography to visualize the sucrose conversion products of immobilized and free enzymes.

[0026] Figure 13 High-performance liquid chromatography (HPLC) results for visualizing the conversion of sucrose products by immobilized and free enzymes. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] This invention utilizes overlap extension PCR technology to synthesize the carbohydrate domain CBM2a, the green fluorescent protein gene GFP, and a protein derived from... Klebsiella sp. LX3's sucrose isomerase PalI (where PalI is derived from...) Klebsiella Klebsiella pneumoniae sp. LX3 (GenBank: KJ452353.1) was fused to construct the fusion gene GFP-CBM2a-PalI. Next, the sucrose isomerase visualization and immobilization recombinant vector pET32a-GFP-CBM2a-PalI containing the fusion gene GFP-CBM2a-PalI was constructed using RF cloning technology. Then, the recombinant vector was transformed into the expression strain. E. coli Rosetta (DE3) was used to induce the expression of sucrose isomerase using IPTG. Subsequently, the induced cells were disrupted by sonication to separate the intracellular soluble components, which were then immobilized on a cellulose carrier. Finally, the immobilization efficiency was determined using fluorescence detection. This method successfully achieved visualized immobilization of sucrose isomerase. This invention provides a green and efficient method for the immobilization of sucrose isomerase, offering a reference for further promoting the industrial application of sucrose isomerase.

[0029] (a) Sources of biological materials: Cloned strains E. coli DH10B, expression strain E. coliRosetta(DE3); the plasmids used in the experiment were pHT43-PalI (see this article for details: Jiang Chen, Wu Xintong, Chen Huiling, et al. Expression and fermentation optimization of recombinant sucrose isomerase in Bacillus subtilis [J]. Journal of Biology, 2024, 41(06):39-46.), pET32a-CBM2a-PalⅠ, pET28a-CBM17-GFP (see this article for details: Li Sipeng, Lü Yifei, Li Xianzheng, et al. Immobilization study of CBM-mediated sucrose isomerase [J]. Journal of Microbiology, 2024, 44(03):12-23.) and pET32a-PalⅠ (where PalI is derived from Klebsiella Klebsiella pneumoniae sp. LX3 (GenBank: KJ452353.1), see this paper for details, by Wang Bingbing. (Source: [Insert source here]) Klebsiella Site-directed mutagenesis of sucrose isomerase PalⅠ of sp. LX3 and fermentation transformation analysis of Bacillus subtilis expression product [D]. Dalian University of Technology, 2019. DOI:10.26992 / d.cnki.gdlqc.2019.000141. All images are from the National-Local Joint Engineering Laboratory for Biocatalysis Technology, Dalian University of Technology (and are publicly available).

[0030] (ii) Purchasing reagents: Tryptone, sodium chloride, sucrose, yeast extract, and Goldview I nucleic acid dye were purchased from Beijing Dingguo Changsheng Co., Ltd.; QuickCut™ DpnI rTaq DNA Polymerase, Prime STAR HS DNA Polymerase, Premixed Protein Marker (High), Buffer, DNA Marker, TEMED, Tris, SDS, Acrylamide, APS, EDTA, Glycine, Bovine Serum Protein, DNA Fragment Purification Kit, Plasmid Extraction Kit, and Gel Recovery Kit were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; Avicel PH101 was purchased from Fluka; Citric acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Disodium hydrogen phosphate, sodium dihydrogen phosphate, and dipotassium hydrogen phosphate were purchased from Tianjin Kemeo Chemical Reagent Co., Ltd.; Chloramphenicol (CM) and Ampicillin (Amp) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Isopropyl-β-D-thiogalactoside (IPTG) was purchased from Beijing Jintai Hongda Biotechnology Co., Ltd.

[0031] (III) Determination of sucrose isomerase activity The enzyme activity of sucrose isomerase was determined using the 3,5-dinitrosalicylic acid colorimetric saccharification method (DNS method).

[0032] First, a standard curve for sucrose isomerase needs to be plotted. The method is as follows: Accurately weigh 0.034 g of isomaltulose, dissolve it completely in ddH2O, and then add ddH2O to bring the volume to 10 mL to prepare an isomaltulose standard solution with a concentration of 0.1 μmol / mL. Then, dilute this solution with ddH2O to prepare isomaltulose solutions with concentrations of 0, 0.02, 0.04, 0.06, 0.08, and 0.1 μmol / mL, respectively. Take a new centrifuge tube, add 150 μL of isomaltulose solution and 200 μL of DNS reagent, mix thoroughly, and then place it in a boiling water bath for 10 min. After the boiling water bath, mix 900 μL of ddH2O with the reaction solution. The absorbance of the mixture needs to be measured at 540 nm, using 0 μmol / mL as a blank group for zeroing. The x-axis represents the concentration of isomaltulose, and the y-axis represents OD. 540 The absorbance values ​​were used to plot the standard curve of isomaltulose (R0) on the x and y axes. 2 It must be greater than 0.999).

[0033] The specific method for determining enzyme activity is as follows: Take 100 μL of sucrose isomerase solution, add 400 μL of 4% sucrose prepared with citrate-disodium hydrogen phosphate (pH 6.0), react at 40℃ for 15 min, then quickly place in a water bath and boil for 15 min to inactivate and terminate the enzyme reaction. Take 150 μL of the reaction solution and add 200 μL of DNS, boil in a water bath for 5 min, and after the reaction is complete, quickly remove it, add 900 μL of ddH2O, repeatedly invert and mix, then take 200 μL of the reaction liquid and measure its absorbance at 540 nm. Use inactivated sucrose isomerase as a negative control, and set up 3 parallel samples for each reaction. Calculate the reducing sugar produced based on the plotted isomaltulose standard curve, and calculate the enzyme activity of sucrose isomerase according to the definition and formula of enzyme activity.

[0034] Enzyme activity definition and calculation: The amount of enzyme required to release 1 μmol of reducing sugar per minute in an enzyme reaction system is 1 U. formula: In the formula, R: concentration of reducing sugar released in the test group (μmol); N: concentration of reducing sugar released in the control group (μmol); S: volume of the reaction system (mL); V: volume of enzyme solution (mL); t: reaction time (min); C: concentration of enzyme (mg / mL).

[0035] The LB medium consists of the following components: NaCl 10 g / L, tryptone 5 g / L, yeast extract 5 g / L, and pH adjusted to 7 in water. The LB solid culture medium consists of the following components in water: NaCl 10 g / L, tryptone 5 g / L, yeast extract 5 g / L, agar powder 1.5%-2%, and pH adjusted to 7. The TB liquid culture medium consists of the following components in water: 3 mL / L glycerol, 7.2 g / L tryptone, 14.4 g / L yeast extract, 1.4 g / L potassium dihydrogen phosphate, 9.86 g / L dipotassium hydrogen phosphate, and pH adjusted to 7. All of the above culture media were at 121 o Sterilize at C for 20 min; The method for preparing the 1.0 mmol / L IPTG is as follows: Weigh 0.238g of IPTG, add ddH2O to dissolve it completely, and then bring the volume to 1L.

[0036] Example 1: Construction of plasmids pET32a-CBM2a-PalⅠ and pET32a-PalⅠ (1) Using the vector pHT43-PalI containing the sucrose isomerase encoding gene preserved in the laboratory as a template, and using PF / R and P2-F / R as primers, the PalI encoding gene was amplified. The amplification product was recovered by gel extraction to obtain the PalI encoding gene. (2) Using the PalI encoding gene of the first-round purified product (1) as primers, and pET32a-CBM2a and pET32a vector as templates, PalI was inserted into pET32a-CBM2a and pET32a vector respectively to construct the target vectors pET32a-CBM2a-PalⅠ and pET32a-PalⅠ; (3) The above RF-clone product was digested with QuickCut™ DpnI at 37°C for 12 h and then electroporated into the cloning bacteria. E. coli DH10B competent cells were plated on LB agar plates containing 25 μg / mL chloramphenicol (CM) and 100 μg / mL ampicillin (Amp) respectively, and incubated at 37°C for 14 h until single colonies appeared. (4) Use the universal E. coli primer T7-F / R to perform colony PCR verification on single colonies appearing on the plate; perform LB liquid culture on the correctly identified transformants, extract plasmids, and send them to BGI Genomics for sequencing.

[0037] Details of the primers in Example 1 are shown in Table 1-1. Details of the PCR reaction system and amplification conditions for PalI amplification in Example 1 are shown in Tables 1-2 and 1-3. Details of the RF-clone II reaction system and amplification conditions are shown in Tables 1-4 and 1-5.

[0038] Table 1-1 Primers used in Example 1

[0039] Table 1-2 PCR reaction system for PalI amplification

[0040] Table 1-3 PCR reaction conditions for PalI amplification

[0041] Table 1-4 RF-clone II reaction system

[0042] Table 1-5 RF-clone II Reaction Conditions

[0043] Note: This indicates a temperature drop of 1°C per cycle.

[0044] The above-mentioned electroporation conversion method is as follows: Take a clean electroporation cup and place it at -20°C. o C's refrigerator was pre-chilled. (Take out) E. coli DH10B electroporated competent cells were thawed in an ice-water mixture. After thawing, the competent cells were aspirated. Dpn 3 μL of the digestion product was carefully added to the competent cells. The mixture was then gently pipetted and placed in an ice-water bath for 10 min. After the ice bath, the pre-chilled electroporation cuvette was removed, and the mixture was transferred to the cuvette. The cuvette was capped and inserted into the electroporator to begin electroporation at 25,000 V for 5 ms. Immediately after electroporation, 1 mL of sterile LB broth was added to the cuvette. The culture was gently mixed with the competent cells using a pipette, and the entire culture was then transferred from the cuvette to a 2.5 mL centrifuge tube and incubated at 37°C. o Incubate at 200 rpm for 1 h. After 1 h, take out 200 μL of the cultured bacterial solution, spread it on a plate, and incubate at 37℃ for 12-16 h.

[0045] Example 2: Construction of GC2aPalIE (1) Using pET28a-CBM17-GFP as a template and GF / R of the primers shown in Table 1-1 as primers, the GFP coding gene was amplified. The amplification product was recovered by gel extraction to obtain the GFP coding gene. (2) Using pET32a-CBM2a-PalI as a template, and using CF and PR primers as shown in Table 1-1 respectively, the CBM2a-PalI coding gene was amplified. The amplification product was recovered by gel extraction to obtain the CBM2a-PalI coding gene. (3) Using the gel recovery products of (1) and (2) as templates, the GFP encoding gene and the CBM2a-PalI encoding gene were fused using the overlap PCR method to obtain the first round overlap PCR product (GFP-CBM2a-PalⅠ encoding gene). Using the first round overlap PCR product as templates, and GF and PR as primers, the first round product of the GFP-CBM2a-PalⅠ encoding gene RF-clone was amplified using the overlap PCR method. The amplified product was gel recovered to obtain the GFP-CBM2a-PalⅠ encoding gene. (4) Using the GFP-CBM2a-PalⅠ encoding gene from the gel recovery product in (3) as primers, and pET32a-PalI as a template, the second round of RF clone product amplification was performed to construct the pET32a-GFP-CBM2a-PalI recombinant expression vector; (5) The above RF-clone product was digested with QuickCut™ DpnI at 37℃ for 12 h and electroporated into the cloning bacteria. E. coli DH10B competent cells were plated on LB agar plates containing 25 μg / mL chloramphenicol (CM) and 100 μg / mL ampicillin (Amp) respectively, and incubated at 37°C for 14 h until single colonies appeared. (6) Use the universal E. coli primer T7-F / R to perform colony PCR verification on single colonies appearing on the plate; perform LB liquid culture on correctly identified transformants, extract plasmids, and send them to BGI Genomics for sequencing. (7) Heat shock transformation of plasmids of correctly sequenced transformants into expression host bacteria. E. coli In Rosetta (DE3) competent cells, the strain expressing sucrose isomerase with fusion tag was finally obtained and named GC2aPalIE; Details of the primers used in Example 2 are shown in Table 1-1. Details of the PCR reaction system and amplification conditions for GFP amplification in Example 2 are shown in Tables 2-1 and 2-2. Details of the PCR reaction system and amplification conditions for CBM2a-PalI amplification are shown in Tables 2-3 and 2-4. Details of the first round of overlap PCR reaction system and amplification conditions are shown in Tables 2-5 and 2-6. Details of the second round of overlap PCR reaction system and amplification conditions are shown in Tables 2-7 and 2-8. Details of the RF-clone II reaction system and amplification conditions are shown in Tables 2-9 and 2-10.

[0046] Table 2-1 PCR reaction system for GFP amplification

[0047] Table 2-2 PCR reaction conditions for GFP amplification

[0048] Table 2-3 PCR reaction system for CBM2a-PalI amplification

[0049] Table 2-4 PCR reaction conditions for CBM2a-PalI amplification

[0050] Note: This indicates a temperature drop of 1°C per cycle. Table 2-5 First-round reaction system for overlap PCR

[0051] Table 2-6 Conditions for the first round of overlap PCR

[0052] Table 2-7 Second-round reaction system for overlap PCR

[0053] Table 2-8 Conditions for the second round of overlap PCR

[0054] Table 2-9 RF-clone II reaction system

[0055] Table 2-10 RF-clone II Reaction Conditions

[0056] The above-mentioned thermal shock conversion method is as follows: Take E. coli Rosetta (DE3) heat-stress competent cells were thawed in an ice-water mixture. After thawing, 1 μL of plasmid was carefully added to the competent cells, followed by gentle pipetting of the mixture. The mixture was then placed in an ice-water mixture for an ice bath for 30 min. After the ice bath, the competent cells were placed at 42°C. o Incubate in a C2 water bath for 1 min, then immediately remove and incubate on ice for 5 min. Add 1 mL of sterilized LB liquid medium to the competent cells, gently pipette to mix the medium with the competent cells, and then incubate at 37°C. o Incubate at 200 rpm for 45 min at C. Take 200 μL of the cultured bacterial solution and spread it onto LB agar plates of the corresponding antibiotic resistance. Invert the plates and incubate at 37°C. oIncubate statically at C for 12-16 hours until single colonies appear. The cells can then be cultured in liquid form and stored at -80°C to prepare a bacterial culture. o C.

[0057] Example 3: Visualized Immobilized Sucrose Isomerase Process (1) Preparation of sucrose isomerase: GC2aPalIE from Example 2 was streaked onto LB agar plates containing 25 μg / mL CM and 100 μg / mL Amp. The plates were incubated at 37°C for 12–16 h until single colonies appeared. A single colony was picked and transferred to 10 mL of LB liquid medium containing the corresponding antibiotic, and cultured at 37°C with shaking at 200 r / min until OD500 was reached. 600 nm To achieve an OD of approximately 2.0, transfer 4 mL of seed culture to 200 mL of TB liquid medium for expansion culture, and culture with shaking until the OD reaches 2.0. 600 nm When the concentration was 0.6, IPTG was added to a final concentration of 1.0 mmol / L, and the cells were cultured at 16℃ and 200 r / min for 16–20 h with shaking to induce sucrose isomerase expression. The cells were collected by centrifugation at 8000 g for 5 min at 4℃. The cells were washed with deionized water and 0.1 mol / L citrate-disodium hydrogen phosphate buffer (pH 6.0), then resuspended in citrate-disodium hydrogen phosphate buffer and sonicated at 550 W (5 s working time, 2 s interval) for 20 min. The cells were then centrifuged at 10000 g for 20 min at 4℃, and the supernatant was separated to obtain the crude sucrose isomerase solution.

[0058] (2) Visualized immobilized sucrose isomerase process: GC2aPalI crude enzyme solution was immobilized on the surface of microcrystalline cellulose at 30℃ for 60 min to prepare visualized immobilized sucrose isomerase GC2aPalI-A. The immobilized enzyme was irradiated under ultraviolet light according to the method described above to preliminarily determine the immobilization effect (see above). Figure 1 Figure a shows microcrystalline cellulose with the fusion tag-immobilized sucrose isomerase; Figure b shows microcrystalline cellulose without the enzyme immobilized. The microcrystalline cellulose immobilized with the sucrose isomerase GC2aPalI-A exhibits significant green fluorescence.

[0059] Example 4: Optimal temperature and temperature stability of free enzymes and visualized immobilized enzymes (1) 100 μL of free sucrose isomerase enzyme solution (i.e., the crude sucrose isomerase enzyme solution obtained in Example 3 (1)) and the visualized immobilized enzyme solution GC2aPalI-A obtained in Example 3 (2) were resuspended in 400 μL of citrate-disodium hydrogen phosphate buffer (0.1 mol / L, pH 6.0) containing 4% sucrose by mass. The enzymes were reacted with the substrate at different temperature conditions (20, 30, 40, 50, 60 °C) to determine their enzyme activity (except for the temperature conditions, all other conditions were the same as the enzyme activity determination method above) to determine their optimal reaction temperature (see Figure 2 and 3 The optimal temperature for both the free enzyme and the visualized immobilized enzyme was 45℃, with no significant difference.

[0060] (2) The residual enzyme activity of the free and visualized immobilized enzyme solutions was measured after incubation at different temperatures for 30 min to determine the thermostability of sucrose isomerase (see [reference]). Figure 4 and 5 Immobilized enzymes exhibited better temperature stability; the residual enzyme activity of free enzymes remained above 80% between 20 and 45°C, while the residual enzyme activity of visualized immobilized enzymes remained above 90% between 20 and 45°C.

[0061] Example 5: Optimal pH and pH stability of free enzymes and visualized immobilized enzymes (1) 0.1 mg of sucrose isomerase (the free sucrose isomerase solution obtained in Example 3 (1) and the visualized immobilized enzyme solution GC2aPalI-A obtained in Example 3 (2)) was placed in 1 mL of buffer solution with pH 3.0~11 (where each pH value is a gradient of 0.5, pH 3.0~6.0 is 0.1 mol / L citrate-disodium hydrogen phosphate buffer, pH 6.0~8.0 is 0.1 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0~9.0 is 0.1 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and pH 9.0~11.0 is 0.1 mol / L sodium carbonate-sodium bicarbonate buffer). The sucrose isomerase activity was measured under the optimal temperature conditions (45 °C for both the free enzyme and the visualized immobilized enzyme measured in Example 3) to determine its optimal reaction pH (see Figure 6 and 7 The free and visualized immobilized sucrose isomerases have the same optimal pH, with the highest relative enzyme activity at pH 6.5.

[0062] (2) To determine the pH stability of sucrose isomerase, 0.1 mg of sucrose isomerase was incubated at room temperature for 24 h in 1 mL of different buffer solutions with pH values ​​ranging from 3.0 to 11 (where each pH value is a gradient of 0.5, pH 3.0–6.0 is 0.1 mol / L citrate-disodium hydrogen phosphate buffer, pH 6.0–8.0 is 0.1 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0–9.0 is 0.1 mol / L sodium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, and pH 9.0–11.0 is 0.1 mol / L sodium carbonate-sodium bicarbonate buffer). The remaining enzyme activity was then measured (see [reference]). Figure 8 and 9 Under the same treatment conditions, the pH stability range of both free and immobilized enzymes was 4.0–10.0, and the residual enzyme activity was above 60%.

[0063] Example 6: Visualization of operational stability analysis of immobilized enzymes To evaluate the operational stability of the visualized immobilized enzyme, the activity of the immobilized enzyme was continuously measured under optimal reaction conditions (reaction temperature of 45°C, and other procedures as described in Specific Implementation Method (III) above) for 24 cycles. The efficiency of reusability was measured by the changes in the initial and final enzyme activity (see [link to relevant documentation]). Figure 10 The final results showed that the immobilized enzyme activity remained above 60% for the first 12 cycles, but dropped below 50% after 20 cycles.

[0064] Example 7: Visualization of storage stability analysis of immobilized enzymes To assess the storage stability of the visualized immobilized enzyme, the immobilized enzyme was stored at 4°C, and its enzyme activity was observed under optimal reaction conditions (reaction temperature 45°C, the rest of the process is described in the specific implementation method (III) above) (see Enzyme Activity Assay Method). Figure 11 The final results showed that the enzyme activity remained above 60% after 133 days of storage at 4°C.

[0065] Example 8: Qualitative Analysis of Visualized Immobilized Enzyme Digestion Products Qualitative analysis of the enzymatic hydrolysis products of the visualized immobilized enzyme GC2aPalI-A was performed using TLC.

[0066] 100 μL of free sucrose isomerase solution (i.e., the crude sucrose isomerase solution obtained in Example 4 (1)) and the visualized immobilized enzyme solution GC2aPalI-A obtained in Example 4 (2) were resuspended in 400 μL of citrate-disodium hydrogen phosphate buffer (0.1 mol / L, pH 6.0) containing 4% sucrose. The reaction was carried out at 45°C for 16 h, inactivated by boiling in a water bath for 5 min, and the supernatant was collected by centrifugation. The prepared reactants were spotted on a TLC plate (thin-layer chromatography plate) with a sample spacing of 0.5 cm, an upper line 0.5 cm from the top, and a lower line 1 cm from the bottom. The sample volume was 0.5 μL, and the sample was loaded multiple times and then dried with an air blower. 1% glucose, sucrose, fructose, and isomaltulose standards were prepared as controls, and the loading methods (process and conditions) were the same as for the sucrose sample. The results of the control group and the experimental group were on the same TLC plate. Next, place the TLC plate in the developing solvent and stop developing when it reaches the upper limit. Remove and dry with a hairdryer (develop twice). Then, immerse the TLC plate in the staining solution for no more than 5 seconds (4 seconds in this case) and dry in an oven at 105°C. The developing solvent for the above experiment is prepared as follows: mix 50 mL n-butanol, 30 mL ethanol, and 20 mL water thoroughly, seal to prevent evaporation, and store in a cool, dark place. The staining solution for the above experiment is prepared as follows: dissolve 0.4 g diphenylamine in 20 mL acetone, add 400 μL aniline, and finally add 2 mL phosphoric acid. Wait for the precipitate to completely dissolve before use, seal to prevent evaporation, and store in a cool, dark place (prepare fresh before use).

[0067] See results Figure 12 In the figures, lanes 1, 2, 3, and 4 in diagrams A and B represent 1% sucrose, 1% glucose, 1% fructose, and 1% isomaltulose, respectively. In diagram A, lane 5 represents the enzymatic hydrolysis product of GC2aPalI; lane 6 is the negative control for GC2aPalI. In diagram B, lane 5 represents the enzymatic hydrolysis product of GC2aPalI-A; lane 6 is the negative control for GC2aPalI-A. As shown in the figures, the enzymatic hydrolysis products of both GC2aPalI and GC2aPalI-A are isomaltulose, trehalose, fructose, and glucose, with isomaltulose being the most abundant.

[0068] Example 9: Quantitative Analysis of Visualized Immobilized Enzyme Digestion Products HPLC was used to quantitatively analyze the enzymatic hydrolysis products of the visualized immobilized enzyme GC2aPalI-A.

[0069] 1. Determine the elution times of each component in the sample based on the elution times of the standards (mass concentrations: 1% fructose, 1% glucose, 1% sucrose, 1% isomaltulose, 1% trehalose). Prepare different concentrations of the above standards (0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, 3%, 4%, 5%) and perform HPLC analysis, plotting standard curves. The x-axis represents the standard concentration, and the y-axis represents the peak area. The HPLC conditions are: mobile phase acetonitrile:water = 80:20 (v / v), column temperature 35℃, injection volume 10 μL, flow rate 1 mL / min, elution time 20 min, and prepare three replicates for each sample.

[0070] 2. 100 μL of free sucrose isomerase enzyme solution (i.e., the crude sucrose isomerase enzyme solution obtained in Example 2 (1)) and the visualized immobilized enzyme solution GC2aPalI-A obtained in Example 2 (2) were resuspended in 400 μL of citrate-disodium hydrogen phosphate buffer (0.1 mol / L, pH 6.0) containing 4% sucrose by mass. The reaction was carried out at 45°C for 16 h, and after inactivation by boiling water bath for 5 min, the supernatant of the reaction solution was collected by centrifugation and used for HPLC product analysis.

[0071] The high-performance liquid chromatography (HPLC) conditions were as follows: mobile phase acetonitrile:water = 80:20 (v / v), column temperature 35℃, injection volume 10 μL, flow rate 1 mL / min, elution time 20 min, and three replicates were prepared for each sample. The peak areas of different components in the enzymatic hydrolysate were used to calculate the specific amount of each component produced and its proportion in the total product.

[0072] See results Figure 13 Figures ①-⑤ show fructose, glucose, sucrose, isomaltulose, and trehalose, respectively. The results indicate that both the free enzyme and the visualized immobilized enzyme can convert sucrose, with isomaltulose being the main product. The content of each component in the product was calculated using a standard curve, and their proportion in the total product was also calculated. Isomaltulose was the main component. In the free enzyme, isomaltulose accounted for 56.71% ± 0.94% of the total sugar content, and the isomaltulose content in the product was 51.24 ± 2 mg / mL. In the visualized immobilized enzyme, isomaltulose accounted for 72.51% ± 1.3% of the total sugar content, and the isomaltulose content in the product was 94.25 ± 5.96 mg / mL. The conversion rate and yield of the visualized immobilized enzyme were significantly higher than those of the free enzyme. The isomaltulose conversion rate of visualized immobilization was 1.21-2.42 times higher than that of existing immobilization methods.

Claims

1. An expression strain of sucrose isomerase containing a fusion tag, characterized in that, Using the GFP-encoding gene and the CBM2a-PalI-encoding gene as templates, the GFP-CBM2a-PalI-encoding gene was amplified by overlap PCR to obtain the gene, which was then transformed into heat-shock-transformed host bacteria. E. coli In Rosetta (DE3) competent cells, an expression strain containing a fusion tag sucrose isomerase was finally obtained.

2. The expression strain of sucrose isomerase containing a fusion tag according to claim 1, characterized in that, (1) Using pET28a-CBM17-GFP as a template and GF / R as primers, the GFP coding gene was amplified. The amplification product was recovered by gel extraction to obtain the GFP coding gene. (2) Using pET32a-CBM2a-PalI as a template, and CF and PR as primers respectively, the CBM2a-PalI coding gene was amplified. The amplification product was recovered by gel extraction to obtain the CBM2a-PalI coding gene. (3) Using the gel recovery products of (1) and (2) as templates, the GFP encoding gene and the CBM2a-PalI encoding gene were fused using the overlap PCR method to obtain the first round overlap PCR product (GFP-CBM2a-PalⅠ encoding gene). Using the first round overlap PCR product as templates, and GF and PR as primers, the first round product of the GFP-CBM2a-PalⅠ encoding gene RF-clone was amplified using the overlap PCR method. The amplified product was gel recovered to obtain the GFP-CBM2a-PalⅠ encoding gene. (4) Using the GFP-CBM2a-PalⅠ encoding gene of the gel recovery product in (3) as primers, and pET32a-PalI as template, the second round of RF clone product amplification was performed to construct the pET32a-GFP-CBM2a-PalI recombinant expression vector; (5) The above RF-clone product was digested with QuickCut™ DpnI at 37°C for 12 h and then electroporated into the cloning bacteria. E. coli DH10B competent cells were plated on LB agar plates containing 25 μg / mL chloramphenicol (CM) and 100 μg / mL ampicillin (Amp), respectively, and incubated at 37°C for 12–16 h until single colonies appeared. Plasmids were then extracted after sequencing. (6) Heat shock transformation of plasmids from correctly sequenced transformants into expression host bacteria. E. coli In Rosetta (DE3) competent cells, the expression strain (GC2aPalIE) containing the fusion tag sucrose isomerase was finally obtained.

3. The application of the expression strain of sucrose isomerase containing a fusion tag as described in claim 1, characterized in that, Application of the expressed strain in the preparation of visualized immobilized sucrose isomerase.

4. A method for preparing a visualized immobilized sucrose isomerase, characterized in that: The strain (GC2aPalIE) described in claim 1 was fermented and then immobilized with microcrystalline cellulose to obtain the immobilized sucrose isomerase GC2aPalI-A.

5. The method for preparing a visualized immobilized sucrose isomerase according to claim 4, characterized in that: The strain was inoculated into LB liquid medium containing the corresponding antibiotic and cultured at 37°C and 200 r / min with shaking until the OD600 nm reached 2.0 to obtain the seed culture. The seed culture was then inoculated into TB liquid medium at a volume ratio of 1-5% for expansion culture. When the OD600 nm reached 0.6 with shaking, IPTG was added to a final concentration of 1.0 mmol / L and cultured at 16°C and 200 r / min for 16-20 h to induce the expression of sucrose isomerase. After induction, the precipitate was collected by centrifugation. The precipitate was washed and then resuspended in citrate-disodium hydrogen phosphate buffer. The precipitate was then sonicated, centrifuged, and collected to obtain the crude sucrose isomerase enzyme solution. The crude sucrose isomerase solution was mixed with microcrystalline cellulose at 30°C for 60 min to immobilize GC2aPalI on the surface, thus preparing immobilized sucrose isomerase GC2aPalI-A.

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