Sucrose isomerase and preparation method thereof

Abstract

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to sucrose isomerase and a preparation method thereof. The sucrose isomerase is prepared through the steps of preparing the sucrose isomerase through prokaryotic expression, chemically modifying carboxyl-containing anhydride and the like. In the application, in combination with analysis of a spatial structure of sucrose isomerase, the inventor utilizes an anhydride chemical agent containing carboxyl to chemically modify the sucrose isomerase. Preliminary experiment results show that after chemical modification and transformation, the affinity of the transformed biological enzyme to a substrate is obviously increased, subsequent transformation application is facilitated, a good application prospect is shown, and meanwhile, a good scientific research reference is provided for chemical modification and transformation of other biological enzymes.

Description

Technical Field

[0001] The invention belongs to the technical field of bioenzyme engineering, and specifically relates to a sucrose isomerase and a preparation method thereof. Background Art

[0002] Isomaltulose, also known as palatinose, is an isomer of sucrose. It exists naturally in foods such as beets and honey, and is therefore also a natural sugar. Due to its characteristics of not causing tooth decay and having little effect on blood sugar levels, isomaltulose is in great demand in the fields of food and beverages. However, due to its very low content in natural products, isomaltulose in food industry production relies on artificial preparation through chemical synthesis, biotransformation and other methods.

[0003] In the prior art, the chemical synthesis method is used to prepare isomaltulose, but it is not suitable for industrial promotion and application due to its high preparation cost and serious environmental pollution problems. The microbial fermentation conversion method is used to prepare isomaltulose, although it has the advantage of low fermentation cost, but due to the complex composition of the fermentation product, the subsequent purification cost is high, so there is still a lot of room for technical improvement.

[0004] In the metabolism of organisms, sucrose isomerase is a biological enzyme that can isomerize sucrose to form isomaltulose and trehalulose. Therefore, the preparation of isomaltulose based on the biological enzyme conversion method has the advantages of high conversion efficiency and easy purification, and is also the most promising method for preparing isomaltulose for industrial application. However, since the preparation method of natural sucrose isomerase and the enzyme activity are the key factors restricting the preparation of isomaltulose using the biological enzyme conversion method, the preparation method of sucrose isomerase and the improvement of the enzyme activity are technical problems that need to be solved urgently. Summary of the invention

[0005] The main purpose of the present application is to provide a method for preparing sucrose isomerase, and to further improve the enzymatic activity of the prepared sucrose isomerase through chemical modification, thereby laying a certain technical foundation for the subsequent preparation of isomaltulose.

[0006] The specific technical solutions adopted in this application are as follows.

[0007] A sucrose isomerase is prepared by the following steps: (I) Prokaryotic expression of sucrose isomerase The sucrose isomerase was prepared by using the genetic engineering method and the Escherichia coli prokaryotic expression system; The sucrose isomerase (599AA), whose amino acid sequence is shown in SEQ ID No. 1, is as follows: When the sucrose isomerase is prepared by prokaryotic expression, according to the codon preference of Escherichia coli, the restriction enzyme sites of the plasmid vector (BamH I and Xho 1 restriction enzyme sites are introduced at the 5' end and 3' end, respectively), and the convenience of subsequent separation and purification (six His tags are inserted at the 3' end), the corresponding coding gene sequence (1830 bp) is shown in SEQ ID No. 2, as follows: Specific preparation: (1) The coding gene sequence shown in SEQ ID No. 2 was ligated and recombined with plasmid pET-28a(+) to prepare a recombinant plasmid expression vector named ROSI (commissioned to Shanghai Shenggong Biotechnology Co., Ltd. for preparation and provision, and the finished product provided was in the form of dry powder); (2) Add the above-mentioned recombinant plasmid expression vector ROSI to the E. coli BL21 DE(3) competent cells that had been thawed on ice in advance, flick to mix, and then let stand in the ice-water mixture for 10 minutes; after standing, heat shock at 42°C for 60-90 seconds, and immediately ice bath for 3 minutes; after the ice bath, add 600 μL of LB liquid culture medium, and resuscitate and culture at 37°C, 180 rpm on a shaking incubator for 45 minutes; Pipette 50-100 μL of the resuscitation product and spread it evenly on a resistance (containing Kana 50 μg / mL) LB plate. After culturing at 37°C for 12 hours, pick out the positive single clone and transfer it to resistance (containing Kana 50 μg / mL) LB liquid culture medium. Amplify and culture at 37°C and 180 rpm for 12 hours. The culture fluid after the above amplification culture is sampled, and the plasmid is extracted for double restriction digestion identification with BamH I and Xho I, and the correctly identified plasmid is further sequenced to ensure that the plasmid recombination is correct, and finally the strain containing the correct recombinant plasmid is preserved for future use; (3) The strain containing the correct recombinant plasmid was inoculated into resistant (Kana, 50 μg / mL) LB liquid culture medium, and cultured at 37°C and 180 rpm until OD600 = about 0.6-0.8, then IPTG was added at a final concentration of 0.5 mM, and cultured at 16-25°C for 18-20 h to induce protein expression; (4) After the induction expression is completed, the fermentation broth is centrifuged (8000 rpm for 10 min) to collect the bacterial cells; the bacterial cell pellet is washed 2 to 3 times with 50 mM disodium hydrogen phosphate citrate buffer (pH 5.5) (to remove the residual culture medium attached to the bacterial cell surface); After cleaning, add 10-15 mL of binding-washing buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.0) per gram of bacterial precipitation to resuspend the bacterial precipitation, place it in ice water (1:1 ratio) for precooling, and then ultrasonically disrupt the bacterial cells (ultrasonic power 350 W, ultrasonic 4 s, pause 5 s, treatment time 15 min); after the disruption is completed, the mixture after disruption is centrifuged (4 ° C, 12000 rpm for 10 min), and the supernatant is retained. The supernatant is the crude enzyme solution; Furthermore, the supernatant can be filtered using a 0.22 μm water filter membrane to reduce impurities and improve the efficiency of subsequent protein purification; (5) The supernatant obtained in step (4) is further purified using a Ni-NTA column. The specific operation is as follows: First, rinse with ultrapure water for 3-5 column volumes (to remove ethanol in the filler protection solution), and then add 3-5 column volumes of binding-washing buffer (500mM NaCl, 50mM Tris, 20mM imidazole, pH 7.0) to balance the column (Ni-NTA column); after the balance is completed, add the supernatant to the affinity chromatography column (Ni-NTA column) and let it stand on ice for no less than 30 minutes (shake every 10 minutes or so during this period to ensure that the target protein is fully in contact with and bound to the filler); After the rest period: First, use 3-5 column volumes of binding-washing buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.0) to elute the impurities that are weakly bound to the Ni-NTA filler and collect the flow-through; Then, the histidine-tagged protein on the column was eluted with 3-5 column volumes of elution buffer (500 mM NaCl, 50 mM Tris, 500 mM imidazole, pH 7.0) and the eluate was collected; Finally, the collected eluate was ultrafiltered and concentrated using an ultrafiltration tube (30 kDa molecular weight cutoff), and the obtained product was purified sucrose isomerase; (II) Chemical modification chemically modifying the sucrose isomerase obtained in step (1) using a chemical modifier; The chemical modifier is an acid anhydride containing a carboxyl group. During the chemical modification process, the chemical modifier is coupled with the ε-NH2 at the end of the Lys residue of the sucrose isomerase to generate an amide bond; The chemical modifier is specifically, for example, phthalic anhydride (PA), maleic anhydride (MA), succinic anhydride (SA), or citraconic anhydride (CA); The specific chemical modification operation process is as follows: First, 100 μL of 10 mM chemical modifier solution and 1 mL of 0.1 mg / mL pure sucrose isomerase prepared in step (i) were mixed evenly and reacted at 4° C. for 3 to 5 h (preferably, magnetic stirring was used during the reaction to facilitate the reaction); Subsequently, the reaction solution was dialyzed against 50 mmol / L phosphate buffer (pH=7.5) at 4°C overnight to obtain chemically modified sucrose isomerase.

[0008] Application of the sucrose isomerase in the preparation of isomaltulose.

[0009] In the prior art, in order to improve the stability and catalytic efficiency of biological enzyme molecules, the main modification strategy is to modify the enzyme molecules themselves and the enzyme reaction environment conditions. The modification of the enzyme molecules themselves mainly involves site-directed mutagenesis and chemical modification of amino groups at specific sites. The fundamental purpose of this modification is to adjust the spatial conformation of the enzyme molecules to better react with the reaction substrate.

[0010] Although chemical modification is an effective method to improve and enhance the catalytic performance of enzymes, this modification method lacks certain specificity. Therefore, in practical applications, factors such as the original conformation of the enzyme molecule, the amino residue group, and the distribution characteristics of the side chain must be considered in a comprehensive manner. In this application, in combination with the spatial structure analysis of sucrose isomerase, the inventors used carboxyl-containing anhydride chemicals to chemically modify it. Preliminary experimental results show that after chemical modification, the modified enzyme has a significantly increased affinity for the substrate, which is beneficial to subsequent conversion applications, showing a good application prospect. At the same time, it also provides a good scientific research reference for the chemical modification of other enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The results of double enzyme electrophoresis for different recombinant plasmids (KMSI, PASI, PCSI and ROSI); in the figure: 1 is DL5000bp DNA marker; 2 is DL 2000bp DNA marker; 3 is recombinant plasmid KMSI; 4 is recombinant plasmid PASI; 5 is recombinant plasmid PCSI; 6 is recombinant plasmid ROSI; Figure 2 The SDS-PAGE test results of samples taken at different operation stages during the purification process of sucrose isomerase (Ro-SIase) from Raoultella ornithinolytica; in the figure: M is a protein marker, 1 is the supernatant after cell disruption, 2 is the result of the flow-through, 3, 4, 5 are the results after washing with 20mM imidazole, 6, 7, 8 are the results after elution with 500mM imidazole; Figure 3 The Michaelis-Menten diagram of sucrose isomerase Ro-SIase using sucrose as substrate; Figure 4 is the isomaltulose conversion rate under different reaction times of sucrose isomerase Ro-SIase; Figure 5 The ratio of different reaction products under different reaction times; Figure 6The three-dimensional structural analysis results of sucrose isomerase Ro-SIase; A is the model diagram of sucrose isomerase Ro-SIase docking with substrate sucrose; B is the ball-and-stick model of sucrose isomerase Ro-SIase, in which the ball represents the main chain of amino acids, the stick represents the side chain of amino acids, and Lys is shown in red; Figure 7 It is the relative ratio of enzyme activity after chemical modification with different concentrations and different chemical modifiers. DETAILED DESCRIPTION

[0012] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0013] Biomaterials: E. coli BL21 (DE3), plasmid pET-28a (+), etc. are all commonly used biological materials in genetic engineering experiments and can be obtained from public channels; the inventor, as a professional scientific researcher, has also preserved relevant biological materials for a long time; Experimental reagents LB liquid culture medium and LB solid culture medium can be prepared by referring to the conventional preparation and sterilization operation of existing biotechnology. The specific components can also be referred to as follows: LB liquid medium components: Peptone 10g / L, Yeast extract 5g / L, NaCl 10g / L, dissolved in deionized water; LB solid medium: LB liquid medium + 2% (w / v) agar; Phthalic anhydride (PA) (99.5% pure, product of Anage Chemical Company), maleic anhydride (MA) (>99% pure, product of Aladdin Company), succinic anhydride (SA) (97% pure, product of Xinhengyan Company) and citraconic anhydride (CA) (98% pure, product of Xinhengyan Company), etc., are all common and commonly used chemical reagents and can be purchased through public channels.

[0014] Example 1 Considering the difference in enzymatic activity of sucrose isomerase from different sources, the inventors took sucrose isomerase from different sources as an example and prepared them using prokaryotic expression systems. The specific experimental process is briefly described as follows.

[0015] (I) Selection of sucrose isomerase gene sequence and preparation of recombinant plasmid expression vector Based on the amino acid sequence of sucrose isomerase derived from Klebsiella sp. LX3 strain, which has been widely studied in the prior art, and with the amino acid sequence similarity of >50% and structural analysis indicating that it has sucrose isomerase function as the standard, combined with the amino acid sequences in the prior art, four biological enzymes from different sources were initially screened and selected as research objects: (1)Bioenzyme (607 AA) from Klebsiella michiganensis: MSFIMLRTGVAVALSSLMISLACPTVSAAPSVNQDINIQKESEYPAWWKEAVFYQIYPRSFKDTNDDGIGDIRGIIEKLDYLKSLGIDAIWINPHYDSPNTDNGYDISNYRQIMKEYGTMEDFDNLVAEMKKRNMRLMIDVVINHTSDQHPWFIQSKSDKNSPYRNYYFWRDGKDNQPPNNYPSFFGGSAWQKDVKSGQYYLHYFARQQPDLNWDNPKVREDLYAMLRFWLDKGVSGMRFDTVATYSKIPGFPNLTPEQQKNFAQQYTMGPNIHRYIQEMNREVLSRYDVATAGEIFGVPLDRSSQFFDRRRHELNMAFMFDLIRLDRDSNERWRHKPWSLSQFRQIISKMDVTVGKYGWNTFFLDNHDNPRAVSHFGDDRPQWREASAKALATITLTQRATPFIYQGSELGMTNYPFRQLNEFDDIEVKGFWQDYVQSGKVTATEFLDNVRLTSRDNSRTPFQWDDSLNAGFTHGKPWFRINPNYVEINAEREEAREDSVLNYYKKMIQLRHHIPALVYGAYQDLNPQDNTVYAYTRTLGNERYLVVVNFKEYPVRYTLPANDAIGEVVIETQHQATAPQGTSLSLSPWQAGVYKLRGKHDQTQGQ; (2)Bioenzyme (599 AA) from Raoultella ornithinolytica: MSFIKRRTAAALALSSLMMTWACPSLSATPSFADDIDVHKENTFPAWWKEAVFYQVYPRSFKDTNGDGIGDIRGIIEKLDYLKSLGIDAIWINPHYDSPNTDNGYDISNYQQIMKEYGTMADFDTLIAEMKKRNMRLMIDVVINHTSDRHPWFIQSKSGKNNPYRDYYFWRDGKDNQPPNNYPSFFGGSAWQKDAKSGQYYLHYFARQQPDLNWDNPKVREDLYAMLRFWLDKGVSGMRFDTVATYSKIPGFPDLTPEQQKNFAQQYTMGPNIHQYIQEMNREVLSHYDVATAGEIFGVPLDRSSQFFDRRRHELNMAFMFDLIRLDRDSNERWRHRPWTLSQFRQIVSKMDTTVGEYGWNTFFLDNHDNPRAVSHFGDDSPQWREASAKALATVTLTQRATPFIYQGSELGMTNYPFKKLSEFDDIEVKGFWHDYVESGKVTAAEFLDNVRLTSRDNSRTPFQWDDSQNAGFTSGKPWFHVNPNYVQINAARELTENKSVLNYYKQMIHLRHELPALVYGAYQDLNPQDNSVYAYTRTLDNERYLVVVNFKEQPVRYALPDNDAIEQVMIETNQQSDTAKESTAIALSPWQAGVYKLR; (3) Enzyme derived from Pectobacterium carotovorum (591 AA): MLAAGLSFSAVNYAATNHNEQDTKTVIAVNDGVSAHPVWWKEAVFYQVYPRSFKDSNGDGIGDLKGLTEKLDYLKTLGINAIWINPHYDSPNTDNGYDIRDYRKIMKEYGTMDDFDNLIAEMKKRDMRLMIDVVVNHTSNEHKWFVESKKSKDNPYRDYYIWRDGKDGTPPNNYPSFFGGSAWQKDNVTQQYYLHYFGVQQPDLNWDNPKVREEVYDMLRFWIDKGVSGLRMDTVATFSKNPAFPDLTPEQLKNFAYTYTQGPNLHRYIQEMHQKVLAKYDVVSAGEIFGVPLEEAAPFIDQRRKELDMAFSFDLIRLDRAVEERWRRNDWTLSQFRQINNRLVDMAGQYGWNTFFLSNHDNPRAVSHFGDDRPEWRIRSAKALATLALTQRATPFIYQGDELGMTNYPFTSLSEFDDIEVKGFWQDFVETGKVKPDVFLENVKQTSRDNSRTPFQWSNAEQAGFTTGTPWFRINPNYKNINAEDQTQNPDSIFHFYRQLIALRHATPAFTYGAYQDLDPNNNEVLAYTRELNQQRYLVVVNFKEKPVHYALPKTLSIKQTLLESGQKDKVAPNATSLELQPWQSGIYQLN; (4)Biological enzyme (591AA) derived from Pectobacterium atrosepticum: .

[0016] Based on the above-screened and determined enzyme amino acid sequences, combined with the codon preference of E. coli during subsequent prokaryotic expression, the coding gene of the corresponding enzyme was optimized (when synthesizing the coding gene sequence, combined with the expression plasmid, BamH I and Xho 1 restriction enzyme sites were introduced at the 5' and 3' ends, respectively, and for subsequent separation and purification, 6 His tags were inserted at the 3' end); the optimized coding gene sequence is as follows: For "(1) biological enzyme derived from Klebsiella michiganensis", the coding gene sequence is as follows (1854 bp): For "(4) biological enzyme from Pectobacterium atrosepticum", the coding gene sequence is as follows (1806 bp):

[0017] Based on the above coding gene sequence, Shanghai Sangon Biotechnology Co., Ltd. was commissioned to synthesize the corresponding sequence and to connect the corresponding gene sequence with plasmid pET-28a(+) to recombinantly construct the relevant recombinant plasmid expression vectors, and the corresponding plasmid expression vectors were named: KMSI (for "(1) biological enzyme derived from Klebsiella michiganensis"), PASI (for "(4) biological enzyme derived from Pectobacterium atrosepticum"), PCSI (for "(3) biological enzyme derived from Pectobacterium carotovorum") and ROSI (for "(2) biological enzyme derived from Raoultella ornithinolytica").

[0018] (II) Transformation and screening The recombinant plasmid obtained in step (I) was transformed into E. coli BL21 DE (3) competent cells, and the correct transformed strain was obtained by screening. The specific operation process is as follows: Take 3 μL (10 ng / μL) of the recombinant plasmid obtained in step (a) and add it to the E. coli BL21DE (3) competent cells thawed on ice, flick the centrifuge tube to mix, and place it in an ice-water mixture for 10 min; After the standing period, heat shock was performed at 42°C (metal bath) for 60 seconds, and then immediately placed in an ice bath and allowed to stand for 3 minutes; Subsequently, 600 μL of LB medium was added and the cells were resuscitated and cultured at 37°C and 180 rpm (shaking incubator) for 45 min. After the recovery culture, 50 μL of the transformation product was evenly spread on a resistance (Kana, 50 μg / mL) LB plate, cultured at 37°C for 12 h, and then a single clone was picked and transferred to a resistance (Kana, 50 μg / mL) LB liquid culture medium, cultured at 37°C and 180 rpm for 12 h; After the culture was completed, samples were taken to extract plasmids (refer to the instructions of the plasmid extraction kit (Shanghai Shenggong product) for operation) and double restriction enzyme digestion with BamH I and Xho I was performed for identification (identification results were as follows Figure 1 As shown, the results are in line with expectations, indicating that the plasmid transformation is correct); the plasmid that is correct after restriction digestion verification is further sequenced and identified (Shanghai Shenggong is entrusted to conduct the identification) to ensure that the target gene SIase (sucrose isomerase gene) is correctly recombined. Finally, the strain containing the correct recombinant plasmid is preserved for future use (50% sterilized glycerol is added to the bacterial solution at a ratio of 1:1 and stored at -80°C) (III) Inducible expression The recombinant bacteria screened and identified correctly in step (ii) were inoculated into LB liquid culture medium containing Kana (50 μg / mL) resistance, and cultured at 37°C and 180 rpm until OD600 = about 0.8, then IPTG was added at a final concentration of 0.5 mM, and cultured at 16°C for 20 h to induce protein expression.

[0019] (IV) Preparation of crude enzyme solution After the induction expression in step (iii) is completed, the fermentation liquid is taken and centrifuged at 8000 rpm for 10 min to collect the bacteria; the bacterial precipitate is washed three times with 50 mM disodium hydrogen phosphate citrate buffer (pH = 5.5) (to remove the residual culture medium attached to the surface of the bacteria); After cleaning, add 15 mL of binding-washing buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH = 7.0) per gram of bacterial precipitation to resuspend the bacterial precipitation, place it in ice water (1:1 ratio) for precooling, and then ultrasonically disrupt the bacterial cells (ultrasonic power 350 W, ultrasonic 4 s, pause 5 s, treatment time 15 min); after the disruption is completed, the mixture after disruption is centrifuged (4 ° C, 12000 rpm centrifugation for 10 min), and the supernatant is retained. The supernatant is the crude enzyme solution; Furthermore, the supernatant was filtered using a 0.22 μm water filter membrane to reduce impurities and improve the efficiency of subsequent protein purification.

[0020] (V) Testing the Isomaltulose Conversion Rate of the Prepared Sucrose Isomerase In order to further determine the application effect of sucrose isomerase from different sources, the inventors conducted an experimental test on the isomaltulose conversion rate of the sucrose isomerase prepared in step (iv). The specific test method and results are briefly described as follows.

[0021] Detection method: Pipette 450 μL of sucrose solution (20% (w / v), prepared with 50 mM disodium hydrogen phosphate citrate buffer, pH = 5.5) into a centrifuge tube, add 50 μL of the crude enzyme solution prepared in step (iv) respectively, and keep the reaction at 45°C (in a water bath) for 3 h; After the reaction was completed, the reaction was terminated by boiling water bath for 10 min; the supernatant was then centrifuged and diluted 10 times with ultrapure water, filtered with a 0.22 μm aqueous filter membrane, and the content of isomaltulose in the reaction system was determined by high performance liquid chromatography; When performing high performance liquid chromatography testing, the test parameters are as follows: Chromatographic column: Agilent ZORBAX Carbohydrate Analysis Colum (4.6mm×250mm, 5μm); Mobile phase: acetonitrile / ultrapure water = 75 / 25 (V / V); Flow rate: 1.0 mL / min; Column temperature: 30°C; Injection volume: 10L; Running time: 18 minutes; Detector: Refractive Index Detector (RID).

[0022] According to the liquid chromatography test results, the conversion rate of isomaltulose was calculated according to the following formula: Conversion rate of isomaltulose (%) = (mass of isomaltulose in the reaction system / mass of sucrose in the reaction system) × 100.

[0023] The calculation results of the isomaltulose conversion rate detection of the four kinds of sucrose isomerase crude enzyme solutions are shown in Table 1.

[0024] Table 1. Isomaltulose conversion results of different sucrose isomerases source Isomaltulose conversion rate / % Klebsiella michiganensis 40.29 Pectobacterium atrosepticum 35.81 Pectobacterium carotovorum 22.87 Raoultella ornithinolytica 66.31 From the results in the above table, it can be seen that the sucrose isomerase from Raoultella ornithinolytica has the highest isomaltulose conversion rate. Therefore, the sucrose isomerase from Raoultella ornithinolytica can be used as a basis to further improve the isomaltulose conversion rate effect in its application process.

[0025] Example 2 Based on the crude enzyme solution (Raoultella ornithinolytica derived sucrose isomerase Ro-SIase) prepared in Example 1, the inventors further purified it (using Ni-NTA column) to further measure the enzyme activity, kinetic performance parameters and other characteristics of the prepared sucrose isomerase. The specific experimental results are briefly described as follows.

[0026] (I) Purification of sucrose isomerase After the Ni-NTA filler is evenly filled, wash with ultrapure water for 3-5 column volumes to remove the ethanol in the filler protection solution; then equilibrate the column with 3 column volumes of binding-washing buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.0); After the balance is completed, the crude enzyme solution (supernatant) is added to the affinity chromatography column (Ni-NTA column) and placed on ice for 30 minutes (shake every 10 minutes or so during this period to ensure that the target protein is fully in contact with and combined with the filler); After the rest period: First, use 3 column volumes of binding-washing buffer (500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH 7.0) to elute the impurities that are weakly bound to the Ni-NTA filler and collect the flow-through; The histidine-tagged protein on the column was then eluted with 3 column volumes of elution buffer (500 mM NaCl, 50 mM Tris, 500 mM imidazole, pH 7.0) and the eluate was collected; Finally, the collected eluate is ultrafiltered and concentrated using an ultrafiltration tube (30 kDa molecular weight cutoff), and the resulting product is purified sucrose isomerase.

[0027] During the above purification process, samples were taken at different experimental stages for SDS-PAGE detection. Figure 2 The analysis showed that the molecular weight of the purified sucrose isomerase was about 70 kDa, which was consistent with the theoretical molecular weight, and the band was single, indicating that the purity was high after purification.

[0028] (II) Enzyme activity detection The enzyme activity of the sucrose isomerase purified and prepared in the above step (i) was determined by the DNS method. The specific operation process is as follows.

[0029] Pipette 450 μL of sucrose solution (20% (w / v), prepared with 50 mM disodium hydrogen phosphate citrate buffer, pH = 5.5) into a centrifuge tube, warm it in a 45°C water bath for 10 min, then add 50 μL of diluted enzyme solution and react for 15 min (during this period, use the same ratio of inactivated enzyme solution and substrate for water bath reaction as a control); After the reaction was completed, 1 mL of DNS solution was immediately added to terminate the reaction, and then the mixture was boiled in boiling water for 5 min and then placed on ice water to return to room temperature. After diluting the sample to an appropriate multiple, 200 μL of the sample was pipetted into an ELISA plate, OD540 was measured, and the generated isomaltulose content was calculated using the standard curve.

[0030] Enzyme activity definition: Under the above reaction conditions, the amount of enzyme required to convert sucrose into 1 μmol of isomaltulose within 1 min is defined as one activity unit.

[0031] The results showed that the activity of the purified sucrose isomerase Ro-SIase was 441 U / mg.

[0032] (III) Kinetic parameters of Ro-SIase sucrose isomerase When specifically measuring the kinetic performance parameters of sucrose isomerase Ro-SIase, the measurement process (refer to the above description for relevant specific operations) is as follows.

[0033] Using different concentrations of sucrose (25, 50, 75, 100, 150, 200, 250 mmol / L) as substrate, the enzyme concentration of sucrose isomerase Ro-SIase was 0.05 mg / ml, and the enzyme activity was measured and calculated after 15 min of reaction; According to the measurement results, with substrate concentration (mM) as the horizontal axis and initial velocity (U / mg) as the vertical axis, the data of v and [S] were linearly fitted according to the Michaelis-Menten equation, and the reaction kinetic parameters corresponding to the enzyme were calculated based on the chart.

[0034] The results are as follows Figure 3 The results showed that the maximum reaction rate Vmax was 814.2U / mg, the Michaelis constant Km was 41.3mmol / L, and Kcat was 947.73s -1 .

[0035] (IV) Isomaltulose conversion rate of purified sucrose isomerase Ro-SIase Referring to the relevant operating instructions of Example 1, the concentration of the purified sucrose isomerase Ro-SIase was adjusted to 0.05 mg / mL, 900 μL of sucrose solution (20% (w / v)) and 100 μL of enzyme solution were added to a 1 mL EP tube, and the reaction was carried out at 45° C. (water bath) for 4 h; during this period, samples were taken every 30 min to detect the conversion rate of isomaltulose.

[0036] The results are as follows Figure 4 , Figure 5 As shown. It can be seen that in the process of sucrose isomerase catalyzing the production of isomaltulose from sucrose, certain by-products (glucose, fructose, trehalulose, etc.) will be produced, and as the reaction time increases, the by-product ratio changes to a certain extent. When the reaction time is 2.5h, the maximum conversion rate of isomaltulose can reach more than 87%, and even if the reaction time is 0.5h, the minimum conversion rate of isomaltulose is more than 70%, showing a good application prospect.

[0037] (V) Structural analysis of sucrose isomerase Ro-SIase Based on the amino acid sequence of the aforementioned sucrose isomerase Ro-SIase, the inventors used AlphaFold 3 to predict and analyze its protein structure (the ligand substrate sucrose small molecule used for molecular docking was drawn by ChemDraw 20.0, the molecular docking calculation was performed using AutoDock Vina software, and the docking results were visualized by PyMol). The results are as follows Figure 6 As shown. The analysis shows that: Sucrose isomerase Ro-SIase contains 29 lysines, all of which are on the surface of the enzyme molecule, including 14 Lys on the α-helix, 2 Lys on the β-fold, and 13 Lys on the Loop ring, all of which are on the surface of the enzyme molecule.

[0038] Example 3 Based on the structural analysis of sucrose isomerase Ro-SIase in Example 2, combined with the inventor's previous research on the chemical modification of biological enzymes, considering that only the 13 free Lys on the Loop ring in sucrose isomerase Ro-SIase can be chemically modified (neither the α-helix nor the β-folded Lys can be modified), the inventor tried to use anhydride to chemically modify the surface of the enzyme molecule (the use of anhydride for chemical modification depends on the number of free lysines on the surface of the enzyme molecule), in order to further regulate and enhance the enzyme activity. This embodiment briefly introduces the relevant experimental process as follows.

[0039] Chemical modification process: First, the purified sucrose isomerase prepared in Example 2 was diluted with 50 mM disodium hydrogen phosphate citrate buffer at pH = 5.5 to a 0.1 mg / mL enzyme solution; Subsequently, considering the differences in modification effects caused by different chemical modifiers and reagent concentrations, phthalic anhydride (PA), maleic anhydride (MA), succinic anhydride (SA), and citraconic anhydride (CA) were prepared into 10-200 mM solutions using DMSO; Then, 100 μL of the 10 mM chemical modifier solution and 1 mL of the 0.1 mg / mL pure sucrose isomerase solution were mixed evenly and reacted at 4° C. for 4 h (magnetic stirring was used during the reaction to facilitate the reaction). Finally, the reaction solution was placed in a dialysis bag (molecular weight cutoff 30 kD), and dialyzed in 50 mmol / L phosphate buffer at 4° C. for 12 h to obtain chemically modified sucrose isomerase.

[0040] Referring to the operation of Example 2, the enzyme activity of chemically modified sucrose isomerase obtained in different ways was measured, and the enzyme activity with a modifier concentration of 0 was used as a control to calculate the relative enzyme activity with different modifier concentrations. The specific measurement results of the relevant samples are as follows.

[0041] Based on the above results, the plot is as follows Figure 7 As shown. It can be seen that: The enzyme activity of sucrose isomerase Ro-SIase before chemical modification is about 440U / mg; after modification with different concentrations and types of chemical modifiers (CA, MA, PA, SA), the enzyme activity is improved, among which the optimal concentration of chemical modifier is 25mM~50mM; among which: When the CA concentration was 25 mM, the relative enzyme activity increased by 17.03%; When the MA concentration was 25 mM, the relative enzyme activity increased by 11.74%; When the PA concentration was 25 mM, the relative enzyme activity increased by 11.30%; When the SA concentration was 25 mM, the relative enzyme activity increased by 6.74%.

Claims

1. A sucrose isomerase, characterized in that The sucrose isomerase is prepared by the following steps: (I) Prokaryotic expression of sucrose isomerase The sucrose isomerase was prepared by using the genetic engineering method and the Escherichia coli prokaryotic expression system; The sucrose isomerase has an amino acid sequence as shown in SEQ ID No. 1; The coding gene sequence corresponding to the sucrose isomerase is shown in SEQ ID No.2; Specific preparation: (1) The coding gene sequence shown in SEQ ID No. 2 was ligated and recombined with plasmid pET-28a to prepare a recombinant plasmid expression vector named ROSI; (2) Transforming the above-mentioned recombinant plasmid expression vector ROSI into E. coli BL21 DE competent cells, and screening to obtain strains containing the correct recombinant plasmid; (3) The strain containing the correct recombinant plasmid was inoculated into LB liquid medium and cultured until OD600 = 0.6-0.8, and IPTG was added to induce protein expression; (4) After the induction of expression is completed, the fermentation liquid is centrifuged to collect the bacteria; after the bacterial precipitate is cleaned, 10-15 mL of binding-washing buffer is added per gram of bacterial precipitate to resuspend the bacterial precipitate, and the bacterial cells are broken after resuspension; The binding-washing buffer comprises: 500 mM NaCl, 50 mM Tris, 20 mM imidazole, pH = 7.0; After the crushing is completed, the crushed mixture is centrifuged and the supernatant is retained. The supernatant is the crude enzyme solution; (5) further purifying the supernatant obtained in step (4) using a Ni-NTA column; (II) Chemical modification chemically modifying the sucrose isomerase obtained in step (1) using a chemical modifier; The chemical modifier is an acid anhydride containing a carboxyl group; The specific chemical modification operation process is as follows: First, the chemical modifier solution and the pure sucrose isomerase prepared in step (i) are mixed evenly and reacted at 2-10° C. for 3-5 hours; Subsequently, the reaction solution was dialyzed against a phosphate buffer solution at pH 7.5 at 4° C. overnight to obtain chemically modified sucrose isomerase.

2. The sucrose isomerase according to claim 1, characterized in that In step "(2)" of step (a), when the recombinant plasmid expression vector ROSI is transformed into E.coli BL21 DE competent cells, the recombinant plasmid expression vector ROSI is added to the E.coli BL21 DE competent cells that have been thawed on ice in advance, mixed, and allowed to stand in the ice-water mixture for 10 minutes; after the standing period, heat shock is performed at 42°C for 60 to 90 seconds, and then ice bath is immediately performed for 3 minutes; after the ice bath, 600 μL of LB liquid culture medium is added, and the cells are resuscitated and cultured on a shaking platform at 37°C and 180 rpm for 45 minutes; Pipette 50-100 μL of the resuscitation product and spread it evenly on the resistant LB plate containing 50 μg / mL Kana. After culturing at 37°C for 12 hours, pick the positive single clone and transfer it to the resistant LB liquid culture medium containing 50 μg / mL Kana. Amplify and culture at 37°C and 180 rpm for 12 hours. The culture fluid after the above amplification culture was sampled, and the plasmid was extracted for double restriction digestion identification with BamH I and Xho I. The correctly identified plasmid was further sequenced to ensure that the plasmid recombination was correct.

3. The sucrose isomerase according to claim 1, characterized in that In step "(3)" of step (a), the final concentration of IPTG is 0.5 mM, and the culture is performed at 16-25°C for 18-20 h to induce protein expression.

4. The sucrose isomerase according to claim 1, characterized in that In step "(4)" of step (a), ultrasonic disruption is used to disrupt bacterial cells.

5. The sucrose isomerase according to claim 1, characterized in that In step "(5)" of step (a), when the Ni-NTA column is used for purification, the specific operation is as follows: First, wash with ultrapure water for 3-5 column volumes, then add 3-5 column volumes of binding-washing binding buffer to balance the column; after the balance is completed, add the supernatant to the affinity chromatography column and let it stand on ice for no less than 30 minutes; After the rest period: First, use 3-5 column volumes of bind-wash buffer to elute the impurities that are weakly bound to the Ni-NTA filler and collect the flow-through; Then, elute the histidine-tagged protein on the column with 3-5 column volumes of elution buffer and collect the eluate; The elution buffer comprises: 500 mM NaCl, 50 mM Tris, 500 mM imidazole, pH = 7.0; Finally, the collected eluate is ultrafiltered and concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to obtain purified sucrose isomerase.

6. The sucrose isomerase according to claim 1, characterized in that In step (ii), the chemical modifier is: phthalic anhydride PA, maleic anhydride MA, succinic anhydride SA, or citraconic anhydride CA.

7. The sucrose isomerase according to claim 1, characterized in that In step (ii), during chemical modification, the material dosage ratio is: 100 μL, 10 mM chemical modifier solution and 1 mL, 0.1 mg / mL sucrose isomerase pure enzyme.

8. Use of the sucrose isomerase according to any one of claims 1 to 7 in the preparation of isomaltulose.

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