Method for producing isomaltulose by using sucrose isomerase with improved activity

Abstract

The invention discloses a method for producing isomaltulose by using sucrose isomerase with improved activity, the isomaltulose is catalytically produced by using sucrose isomerase, and the sucrose isomerase is as follows: (a) the amino acid sequence of the sucrose isomerase is as shown in SEQ ID NO.1; (b) on the basis of the amino acid sequence as shown in SEQ ID NO.1, only any one of the following mutations is generated: S87R, K254R and Q352A. According to the method, the activity of sucrose isomerase is improved and byproducts are reduced through a biotechnological means, so that the conversion rate, the production efficiency and the purity of isomaltulose are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of isomaltulose, and particularly relates to a method for producing isomaltulose by utilizing sucrose isomerase with enhanced activity. Background Art

[0002] Isomaltulose is a low-calorie sweetener with the advantages of being non-fermentable and non-cariogenic, and is widely used in the food industry. At present, the production method of isomaltulose is a biological method, which mainly produces sucrose isomerase through fermentation, and uses sucrose as a raw material to produce isomaltulose using sucrose isomerase catalysis. However, the traditional biological method has problems such as low enzyme activity and low conversion rate. Therefore, improving the activity of sucrose isomerase and then increasing the yield of isomaltulose is one of the most important means to reduce costs in industrialization. Summary of the invention

[0003] The object of the present invention is to provide a method for producing isomaltulose by using sucrose isomerase with enhanced activity, which method improves the activity of sucrose isomerase and reduces by-products by biotechnological means, thereby improving the conversion rate and purity of isomaltulose.

[0004] The above-mentioned object of the present invention can be achieved by the following technical scheme: a method for producing isomaltulose using sucrose isomerase with improved activity, wherein sucrose isomerase is used to catalyze the production of isomaltulose, and the sucrose isomerase is as follows:

[0005] (a) The amino acid sequence of the sucrose isomerase is shown in SEQ ID NO.1;

[0006] (b) Based on the amino acid sequence shown in SEQ ID NO.1, only one of the following mutations occurs: S87R, K254R, Q352A.

[0007] In the preliminary research of the present invention, it was found that the amino acid sequence shown in SEQ ID NO.1 has at least one or more mutations at the following sites: positions 87, 254, and 352, and at least one or more mutations at these sites: S87R, K254R, and Q352A. Therefore, in the amino acid sequence shown in SEQ ID NO.1, by replacing, deleting or adding one or more amino acids, a sucrose isomerase with higher catalytic activity than SEQ ID NO.1 can be obtained.

[0008] Furthermore, the method for producing isomaltulose using sucrose isomerase with enhanced activity provided by the present invention specifically comprises the following steps:

[0009] (1) fermenting the genetically engineered bacteria expressing the sucrose isomerase to obtain a fermentation broth, centrifuging the fermentation broth, collecting the bacterial cells, homogenizing the bacterial cells and then centrifuging them, taking the supernatant, and obtaining a crude enzyme solution containing sucrose isomerase with enhanced activity;

[0010] (2) adding a sucrose solution as a substrate to the crude enzyme solution described in step (1), reacting at a temperature of 20 to 40° C. for 2 to 12 hours, then inactivating the enzyme at high temperature to terminate the reaction, and obtaining a reaction solution containing isomaltulose;

[0011] (3) Decolorizing the isomaltulose-containing reaction solution obtained in step (2) with activated carbon, concentrating, cooling, centrifuging and drying to obtain isomaltulose crystals.

[0012] In the method for producing isomaltulose using sucrose isomerase with increased activity:

[0013] Preferably, in step (1), the process of fermenting the genetically engineered bacteria expressing the sucrose isomerase to obtain a fermentation broth is as follows: inoculating the genetically engineered bacteria expressing the sucrose isomerase into an LB medium, first culturing on a shaking table at a speed of 180 to 220 r / min for 7 to 9 hours, and then inoculating into a TB fermentation medium at an inoculum size of 0.8 to 1.2% (V / V) for fermentation culture, setting the ventilation volume to 0.8 to 1.2 VVM, setting the stirring speed to 300 to 400 rpm, fermenting at 37° C. for 3 to 5 hours, cooling to 25° C. and continuing fermentation for 16 to 20 hours to obtain a fermentation broth.

[0014] Preferably, the concentration of the sucrose solution in step (2) is 100-500 g / L, and the volume ratio of the sucrose solution to the crude enzyme solution in step (1) is 10-20:1.

[0015] Preferably, the high temperature inactivation of the enzyme in step (2) is carried out at 90° C. for 10 to 15 minutes.

[0016] Preferably, in step (3), the isomaltulose is concentrated at 40-60° C. to a concentration of 50-70% (w / v), then slowly cooled to 15-25° C., and finally centrifuged and dried to obtain isomaltulose crystals.

[0017] The present invention has the following advantages:

[0018] (1) The present invention can improve the activity of sucrose isomerase and reduce by-products by causing any one of the following mutations S87R, K254R, and Q352A on the basis of the amino acid sequence shown in SEQ ID NO.1, thereby improving the conversion rate, production efficiency and purity of isomaltulose;

[0019] (2) The method of the present invention can achieve a sucrose conversion rate of more than 88% and a purity of more than 99%, which can more effectively improve the utilization rate of raw materials and help reduce costs. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention, but the present invention is not limited thereby.

[0021] Example 1: Production of sucrose isomerase

[0022] Using restriction endonucleases NdeI and XhoI, the sucrose isomerase gene (sucrose isomerase, Pantoea dispersa, GenBank: AAP57083.1, designed by conventional methods, reverse PCR amplification, and then ligated with T4 ligase to obtain a single point mutation complete plasmid) was inserted into the expression vector pET-22b (+), and recombinant expression vectors pET-22b (+) -SI, pET-22b (+) -SI-S87R, pET-22b (+) -SI-K254R, pET-22b (+) -SI-Q352A were constructed. The recombinant vector was transferred into Escherichia coli BL21 (DE3) using conventional chemical transformation technology; the acquisition process of genetically engineered bacteria can also refer to but not be limited to the description in CN111518782A.

[0023] The amino acid sequence encoded by the sucrose isomerase gene is shown below: 1 MASPLTKPST PIAATNIQKS ADFPIWWKQA VFYQIYPRSF KDSNGDGIGD IPGIIEKLDY

[0024] 61 LKMLGVDAIW INPHYESPNT DNGYDISDYR KIMKEYGSMA DFDRLVAEMN KRGMRLMIDI

[0025] 121 VINHTSDRHR WFVQSRSGKD NPYRDYYFWR DGKQGQAPNN YPSFFGGSAW QLDKQTDQYY

[0026] 181 LHYFAPQQPD LNWDNPKVRA ELYDILRFWL DKGVSGLRFD TVATFSKIPG FPDLSKAQLK

[0027] 241 NFAEAYTEGP NIHKYIHEMN RQVLSKYNVA TAGEIFGVPV SAMPDYFDRR REELNIAFTF

[0028] 301 DLIRLDRYPD QRWRRKPWTL SQFRQVISQT DRAAGEFGWN AFFLDNHDNP RQVSHFGDDS

[0029] 361 PQWRERSAKA LATLLLTQRA TPFIFQGAEL GMTNYPFKNI EEFDDIEVKG FWNDYVASGK

[0030] 421 VNAAEFLQEV RMTSRDNSRT PMQWNDSVNA GFTQGKPWFH LNPNYKQINA AREVNKPDSV

[0031] 481 FSYYRQLINL RHQIPALTSG EYRDLDPQNN QVYAYTRILD NEKYLVVVNF KPEQLHYALP

[0032] 541 DNLTIASSLL ENVHQPSLQE NASTLTLAPW QAGIYKLN.

[0033] The above-mentioned E. coli was inoculated into LB medium, cultured on a shaking table at a speed of 200 r / min for 8 hours, and then transferred to TB medium at an inoculum of 1% (V / V) for fermentation culture, the ventilation volume was set to 1VVM, the stirring speed was 350rpm, and after fermentation at 37°C for 4 hours, the temperature was lowered to 25°C, and fermentation was continued for 18 hours to obtain a fermentation liquid. The fermentation liquid was centrifuged to collect the bacterial cells, and the bacterial cells were subjected to high-pressure homogenization and then centrifuged to obtain the supernatant to obtain a crude enzyme liquid containing sucrose isomerase.

[0034] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.0.

[0035] TB medium: tryptone 15 g / L, yeast extract 20 g / L, sodium chloride 10 g / L, glucose 2 g / L, lactose 2 g / L, pH 7.0.

[0036] Example 2 Enzyme activity determination

[0037] Prepare a 40% concentration (400 g / L, i.e. 40%, w / v) sucrose solution, take 10 mL of the sucrose solution, add 500 μL of crude sucrose isomerase solution, react at 30° C. for 15 min, heat at 90° C. for 10 min to terminate the reaction, and take a sample for HPLC detection (Agilent zorbax amino column, mobile phase acetonitrile: water = 7:3, differential refractometer, flow rate 1.0 ml / min).

[0038] The sucrose isomerase activity is expressed as sucrose isomerase activity units, which is defined as the amount of enzyme required to convert 1 μmol of isomaltulose per minute, that is, 1 enzyme activity unit, expressed as U / mL. The sucrose isomerase activities of different mutants are shown in Table 1.

[0039] Table 1 Activities of different mutants

[0040]

[0041] Example 3 Conversion rate determination

[0042] Prepare 40% (400 g / L, i.e. 40%, w / v) sucrose solution, take 10 mL of sucrose solution, add 500 μL of sucrose isomerase crude enzyme solution, react at 30°C, take samples every 15 minutes, and the reaction end point is when sucrose is completely converted. The isomaltulose conversion rates of different mutants are shown in Table 2.

[0043] Table 2 Activities of different mutants

[0044] Mutants wild type S87R K254R Q352A Isomaltulose(%) 87.5 90.8 88.5 89.9 Fructose + glucose (%) 6 3.5 5.9 5.2 Trehalulose(%) 6.5 5.7 5.6 4.9

[0045] Example 4 Production of Isomaltulose by Sucrose Isomerase Prepared from pET-22b(+)-SI-S87R Mutant

[0046] Prepare a 40% concentration (400 g / L, i.e. 40%, w / v) sucrose solution, take 1000 mL of sucrose solution, add 50 mL of crude sucrose isomerase obtained by fermentation of pET-22b(+)-SI-S87R mutant, react at 30°C for 2 h, heat at 90°C for 10 min to terminate the reaction, and measure the isomaltulose conversion rate to be 90.8% (isomaltulose content / sucrose addition amount HPLC detection (Agilent zorbax amino column, mobile phase acetonitrile: water = 7:3, differential refractometer, flow rate 1.0 mL / min), and use the external standard method for quantification, refer to GB1886.182-2016). After adding 1 g of activated carbon to the reaction solution for decolorization, the filtrate was filtered and concentrated to a concentration of 55% (w / v) at 50° C., slowly cooled to 20° C. while stirring, centrifuged and dried to obtain 232 g of isomaltulose product with an isomaltulose purity of 99.6% (i.e., the isomaltulose content in GB1886.182-2016).

[0047] Example 5 Production of isomaltulose by sucrose isomerase prepared from pET-22b(+)-SI-K254R mutant

[0048] Prepare 40% (400g / L, i.e. 40%, w / v) sucrose solution, take 1000mL sucrose solution, add 50mL crude sucrose isomerase solution obtained by fermentation of pET-22b(+)-SI-K254R mutant, react at 30℃ for 2h, heat at 90℃ for 10min to terminate the reaction, and the isomaltulose conversion rate is measured to be 88.5%. After adding 1g activated carbon to decolorize the reaction solution, filter it by suction, concentrate the filtrate at 50℃ to a concentration of 55% (w / v), slowly cool it to 20℃ while stirring, centrifuge it, and dry it to obtain 205g of isomaltulose product with an isomaltulose purity of 99.4%.

[0049] Example 6 Production of Isomaltulose by Sucrose Isomerase Prepared from pET-22b(+)-SI-Q352A Mutant

[0050] Prepare 40% (400g / L, i.e. 40%, w / v) sucrose solution, take 1000mL sucrose solution, add 50mL crude sucrose isomerase solution obtained by fermentation of pET-22b(+)-SI-Q352A mutant, react at 30℃ for 2h, heat at 90℃ for 10min to terminate the reaction, and the isomaltulose conversion rate is measured to be 88.5%. After adding 1g activated carbon to decolorize the reaction solution, filter it by suction, concentrate the filtrate at 50℃ to a concentration of 55% (w / v), slowly cool it to 20℃ while stirring, centrifuge it, and dry it to obtain 218g of isomaltulose product with an isomaltulose purity of 99.6%.

[0051] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A method for producing isomaltulose using sucrose isomerase with enhanced activity, characterized in that: Sucrose isomerase is used to catalyze the production of isomaltulose, and the sucrose isomerase is as follows: (a) The amino acid sequence of the sucrose isomerase is shown in SEQ ID NO.1; (b) Based on the amino acid sequence shown in SEQ ID NO.1, only one of the following mutations occurs: S87R, K254R, Q352A.

2. The method for producing isomaltulose using sucrose isomerase with enhanced activity according to claim 1, characterized in that: The specific steps include: (1) fermenting the genetically engineered bacteria expressing the sucrose isomerase according to claim 1 to obtain a fermentation broth, centrifuging the fermentation broth, collecting the bacterial cells, homogenizing the bacterial cells and then centrifuging them, taking the supernatant, and obtaining a crude enzyme solution containing sucrose isomerase with enhanced activity; (2) adding a sucrose solution as a substrate to the crude enzyme solution described in step (1), reacting at a temperature of 20 to 40° C. for 2 to 12 hours, then inactivating the enzyme at high temperature to terminate the reaction, and obtaining a reaction solution containing isomaltulose; (3) Decolorizing the isomaltulose-containing reaction solution obtained in step (2) with activated carbon, concentrating, cooling, centrifuging and drying to obtain isomaltulose crystals.

3. The method for producing isomaltulose using sucrose isomerase with enhanced activity according to claim 2, characterized in that: The process of fermenting the genetically engineered bacteria expressing the sucrose isomerase to obtain a fermentation broth in step (1) is as follows: the genetically engineered bacteria expressing the sucrose isomerase is inoculated into an LB medium, first cultured on a shaking table at a speed of 180 to 220 r / min for 7 to 9 hours, then inoculated into a TB fermentation medium at an inoculum amount of 0.8 to 1.2%, V / V for fermentation culture, the ventilation volume is set to 0.8 to 1.2 VVM, the stirring speed is set to 300 to 400 rpm, and after fermenting at 37° C. for 3 to 5 hours, the temperature is lowered to 25° C. and the fermentation is continued for 16 to 20 hours to obtain a fermentation broth.

4. The method for producing isomaltulose using sucrose isomerase with enhanced activity according to claim 2, characterized in that: The concentration of the sucrose solution in step (2) is 100-500 g / L, and the volume ratio of the sucrose solution to the crude enzyme solution in step (1) is 10-20:

1.

5. The method for producing isomaltulose using sucrose isomerase with enhanced activity according to claim 2, characterized in that: In step (2), the enzyme is inactivated at high temperature at 90° C. for 10 to 15 minutes.

6. The method for producing isomaltulose using sucrose isomerase with enhanced activity according to claim 2, characterized in that: In step (3), the isomaltulose is concentrated at 40-60° C. to a concentration of 50-70%, w / v, then slowly cooled to 15-25° C., and finally centrifuged and dried to obtain isomaltulose crystals.

Images