CN113481189A

CN113481189A - A sucrose isomerase mutant and its application

Info

Publication number: CN113481189A
Application number: CN202110868362.2A
Authority: CN
Inventors: 刘洋; 郭川志; 周晶辉; 赵强; 赵士敏; 许岗
Original assignee: Hunan Flag Biotechnology Co ltd
Current assignee: Hunan Flag Biotechnology Co ltd
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2021-10-08
Anticipated expiration: 2041-07-30
Also published as: CN113481189B

Abstract

本发明属于酶工程技术领域，涉及一种蔗糖异构酶突变体及其应用。所述的突变体在SEQ ID NO.1所示氨基酸序列的野生型蔗糖异构酶中突变R434P与E273N。突变体较野生型蔗糖异构酶热稳定性和发酵活力均显著提高。利用突变体制备的固定化酶活力更高，稳定性显著增加，蔗糖转化率和异麦芽酮糖生成率有所提高，杂质含量更低，更适用于工业应用。The invention belongs to the technical field of enzyme engineering, and relates to a sucrose isomerase mutant and its application. Said mutants mutate R434P and E273N in the wild-type sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1. Compared with the wild-type sucrose isomerase, the mutant had significantly improved thermal stability and fermentation activity. The immobilized enzyme prepared by the mutant has higher activity, significantly increased stability, improved sucrose conversion rate and isomaltulose generation rate, and lower impurity content, which is more suitable for industrial application.

Description

Sucrose isomerase mutant and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and relates to a sucrose isomerase mutant and application thereof.

Background

Sucrose isomerase (EC 5.4.99.11) is an enzyme capable of converting sucrose into isomaltulose or trehalulose. Compared with sucrose, isomaltulose (Palatinose) not only has the physical and chemical properties and mouthfeel similar to those of sucrose, but also has the advantages of good acid stability, low hygroscopicity, high safety and the like, and has wide market application prospect in food. Meanwhile, isomaltulose is used as a novel sweetener, has the advantages of low sweetness (about half of that of cane sugar), no decayed tooth, low calorie and the like, and is particularly suitable for patients with diabetes and obesity. In addition, the functional sugar alcohol can be used as a precursor for producing novel functional edible sugar alcohol.

At present, the research on the production method of isomaltulose mainly comprises four methods, namely an enzyme catalysis method, a microbial transformation method, a synthetic chemistry method and a plant transformation method. The isomaltulose produced by the chemical synthesis method is difficult and easily pollutes the environment. Although the medium Pueraria achieves the conversion of sucrose in plants to obtain isomaltulose, the method also has the problems of immature technology, low repetition rate, high environmental requirement, long period, high cost and the like. Thus, the biological method remains a method for mainly producing isomaltulose. In 1984, Nippon Xin Mitsui company successfully developed a special enzyme technology to industrially produce isomaltulose in large quantities. The prior process for preparing isomaltulose still mainly adopts microbial fermentation production, but the problems that sucrose and other heterosugars are difficult to separate, the obtained isomaltulose has low purity, the preparation process is complex, the production cost is high, a large amount of labor resources are consumed and the like exist in the preparation process.

With the rapid development of genetic engineering technology, sucrose isomerases from different sources are heterologously expressed in escherichia coli, and the yield of the enzymes is remarkably improved. The growing maturity of heterologous expression technologies solves the problem of low enzyme yields. Currently, the conversion rate for the production of isomaltulose by enzymatic conversion is between 65 and 92%. Wherein, the Liu Tong and the like successfully express sucrose isomerase derived from pantoea dispersa in Escherichia coli BL21, and the conversion rate of free enzyme after optimizing the enzyme conversion condition is the highest level reported by domestic and foreign documents and reaches 92.0%. The isomaltulose has high purity, and downstream can obtain the high-purity isomaltulose by only a simple purification and crystallization process, thereby obviously reducing the generation difficulty and the cost.

However, neither wild-type sucrose isomerase nor the reported modified enzyme can achieve both good stability and high conversion rate. Poor stability will lead to difficulty in continuous industrial enzyme conversion of sucrose isomerase and storage and transportation of the enzyme, low conversion rate not only increases difficulty for development of downstream process, but also leads to waste of raw materials, both of which result in high cost of enzyme preparation of isomaltulose, and thus, the enzyme preparation cannot be applied in large scale. Therefore, the development of a sucrose isomerase mutant with both stability and conversion rate is urgently needed to be applied to the industrial production of isomaltulose in a large scale. Based on the purpose, the invention carries out mutation screening on the pantoea dispersata-source sucrose isomerase with high conversion rate, and further improves the activity and stability of the enzyme without reducing the conversion rate, so that the pantoea dispersata-source sucrose isomerase is more suitable for industrial application.

Disclosure of Invention

The invention aims to provide a sucrose isomerase mutant so as to enable the mutant to have higher activity and stability than wild sucrose isomerase, and increase continuous reaction batches of sucrose isomerase immobilized enzymes to meet industrial use requirements.

To achieve this object, in a basic embodiment, the present invention provides a sucrose isomerase mutant having a mutation in the amino acid sequence shown in SEQ ID NO.1 at an amino acid position including at least one of R434P and E273N.

Furthermore, the amino acid sequences of the two-point mutant of R434P and E273N are shown as SEQ ID NO. 2.

The second objective of the invention is to provide a polynucleotide encoding the sucrose isomerase mutant, so that the encoded sucrose isomerase mutant has higher activity and stability than the wild sucrose isomerase, and the continuous reaction batches of the sucrose isomerase immobilized enzyme are increased, thereby meeting the industrial use requirements.

To achieve this object, in a basic embodiment, the present invention provides a polynucleotide encoding the aforementioned sucrose isomerase mutant.

The third purpose of the invention is to provide the application of the sucrose isomerase mutant: used for catalyzing sucrose reaction to better prepare isomaltulose.

To achieve the object, in a basic embodiment, the present invention provides a use of a sucrose isomerase mutant for catalyzing a sucrose reaction with the sucrose isomerase mutant as described above in a reaction system to produce isomaltulose.

Further, in the reaction system, the enzyme activity of the sucrose isomerase mutant is 60-150U/g substrate sucrose.

Furthermore, in the reaction system, the concentration of sucrose is 30-70%.

Further, the reaction temperature is 25-40 ℃, and the reaction time is 120-180 min.

Further, the reaction pH was 5.5 to 7.0.

Further, the reaction stirring speed is 150-.

Further, the sucrose isomerase mutant is an immobilized sucrose isomerase mutant.

The sucrose isomerase mutant has the beneficial effects that the sucrose isomerase mutant has higher stability and fermentation activity than wild sucrose isomerase. Meanwhile, the sucrose conversion rate and the isomaltulose generation rate are improved, the impurity content is lower, and the prepared immobilized enzyme can continuously react in multiple batches, thereby meeting the requirements of industrial application.

The sucrose isomerase SIM from the disperse Pantoea is selected as a starting point, and compared with the SIMs from other sources, the sucrose isomerase SIM has the following advantages: the product inhibition is small, the catalytic efficiency is high, and the final product concentration is high. On the basis, the SIM is mutated by means of genetic engineering and enzyme engineering technology, and the obtained SIM-1 mutant has the advantages of improving enzyme fermentation activity by 3 times, improving thermal stability by 4 times, obviously improving immobilized enzyme activity and stability and the like compared with the SIM, and is more suitable for synthesis of isomaltulose because the sucrose conversion rate and the isomaltulose generation rate are improved and the impurity content is lower.

Drawings

FIG. 1 is a schematic diagram of isomaltulose synthesis.

Detailed Description

The present invention is further illustrated by, but is not limited to, the following examples.

The enzyme activity of the SIM and the mutant thereof is determined as follows.

Sucrose with a mass concentration of 15% was prepared as a substrate solution using 0.05mol/L of a citric acid-disodium hydrogen phosphate buffer solution with pH 6.5.

And (3) measuring the enzyme activity of the crude enzyme solution: adding 1ml of crude enzyme solution into 10ml of substrate solution at 30 ℃ and pH6.5, reacting for 10min, and calculating the enzyme activity according to the amount of isomaltulose produced.

Enzyme activity determination of fermentation thalli: taking 50mL of zymocyte liquid, centrifuging at 12000r/min, removing supernatant, collecting thalli, re-suspending the thalli by using 50mL of normal saline, ultrasonically crushing (the ultrasonic time is 3s each time, the interval time is 2s, the ultrasonic frequency is 99 times, the ultrasonic power is 500W), taking 1mL of crushed liquid after completion, adding 10mL of reaction substrate, controlling the temperature at 30 ℃, stirring at the rotating speed of 150r/min, reacting for 10min, sampling 1mL after completion, and performing enzyme activity determination by using HPLC.

Enzyme activity determination of immobilized enzyme: accurately weighing 0.1g of immobilized enzyme, adding the immobilized enzyme into 10mL of reaction substrate for reaction, controlling the temperature at 30 ℃, stirring at the rotating speed of 150r/min, reacting for 10min, sampling 1mL of immobilized enzyme after the reaction is finished, and performing enzyme activity determination by using HPLC.

The HPLC measurement conditions were as follows:

liquid phase conditions: mobile phase: 75% acetonitrile in water

A chromatographic column: amide-805 um, 4.6 x 250mm

A detector: RID

Column temperature: 75 deg.C

Flow rate: 1.0ml/min

Sample introduction amount: 10 μ l

Peak time of each substance: sucrose: 13.844min

Isomaltulose: 14.929min

Glucose: 3.317min

Unit of enzyme activity: the amount of enzyme required to produce 1. mu. mol of isomaltulose per minute at a temperature of 30 ℃ and a pH of 6.5 is one unit (1U).

Example 1: construction of Pantoea dispersa-derived sucrose isomerase zymogen nuclear expression strain

Downloading an amino acid sequence (SEQ ID NO.1, corresponding to GenBank accession number: AAP57083.1) of Sucrose Isomerase (SIM) from Pantoea dispersa in GenBank, removing 1-21 signal peptide (MFLNGFKTVIALTMASSFYLA), and providing the signal peptide for Beijing Ongchoideae biotechnology Limited company to perform whole gene synthesis of coding nucleic acid (adopting preferred codon of Escherichia coli). The C-end of the synthetic gene is provided with a His label (the pET30a (+) vector is provided with the His label), the synthetic gene is constructed into a prokaryotic expression vector pET30a (+), and the restriction enzyme site of the prokaryotic expression vector is as follows: nde I at the 5 'end, Xho I at the 3' end. Passing the constructed plasmid pET30a (+) -SIM through CaCl₂The heat shock transformation method is used for transforming the strain into an escherichia coli expression strain BL21(DE3), the strain is coated on an LB solid medium plate containing 50 mu g/ml Kanamycin, the plate is cultured overnight at 37 ℃, and a colony growing on the plate is the sucrose isomerase zymogen nuclear expression recombinant strain E.coli BL21(DE3)/pET30a (+) -SIM.

Carefully picking out a single colony of the sucrose isomerase zymogen nuclear expression recombinant strain in the LB solid culture medium plate by using a sterilized gun head, inoculating the single colony into a triangular flask containing 20mL of LB liquid culture medium, culturing at 37 ℃ at 200r/min, and shaking overnight. Inoculating the shake flask bacterial liquid into a triangular flask containing 100mL of TB liquid culture medium according to the inoculation amount of 1% the next day, carrying out shake culture at 37 ℃ at 220r/min, measuring the OD value of the culture solution every 1h, supplementing lactose with the final concentration of 1% (m/v) when the OD value of the culture solution is 1.5, continuously culturing at 25 ℃ at 220rpm for 4-6 h, and stopping culturing.

Example 2: purification and immobilization of Pantoea dispersa-derived sucrose isomerase

The fermentation broth obtained in example 1 was purified for protein using activated IDA Resin (His. bind Resin, Ni-charged, available from Annuan Biotech Ltd., Beijing) using His tag carried in the SIM recombinant protein, in the following manner: centrifuging the fermentation liquid for 10min at 4 ℃ and 10000r/min, discarding the supernatant, collecting the thallus, repeatedly washing the thallus twice with phosphate buffer solution (pH 7.5 and 0.1mol/L), centrifuging, and then concentrating the thallus by 5 times and suspending in 20ml of phosphate buffer solution (pH 7.5 and 0.1 mol/L). And (3) placing the treated bacterial liquid in ice water for ultrasonic crushing until the bacterial liquid is clarified, wherein the ultrasonic crushing conditions are as follows: work 2s, interval 5s, ultrasonic power 500W. And (3) placing the crushed lysate into a low-temperature high-speed centrifuge for centrifugation (12000rpm, 4 ℃ and 20min), and collecting supernatant to obtain crude protein. Loading the crude protein onto the activated IDA resin, performing gradient elution by using imidazole solution (10mM-300mM), performing real-time monitoring by using a protein chromatography system (Bio-Rad), and collecting the stable protein peak, namely the SIM recombinant protein purified protein, for preparing the immobilized enzyme.

The purified SIM recombinant protein is used for preparing immobilized enzyme, and the specific method comprises the following steps:

(1) activating an immobilized carrier: 30ml of 60% (m/v) glutaraldehyde and dipotassium hydrogen phosphate (K) are accurately measured₂HPO₄·3H₂O)4.76g is added into 600ml deionized water, after dissolution, the volume is adjusted to 1000ml by deionized water, and the pH value is adjusted to 8.0 by phosphoric acid solution. An epoxy-based carrier ECEP (Resindions S.r.l. Italy) of 250g was put into the above solution and activated at 25 ℃ with low stirring for 2 hours, and the carrier was collected by filtration, washed 2 to 3 times with sterile deionized water and vacuum-filtered to dryness for use.

(2) Immobilization of SIM recombinant protein: diluting a certain amount of the purified SIM recombinant protein with a phosphate buffer solution (pH 6.5 and 0.5mol/L), adding 50g of the activated carrier, immobilizing for 16h at 25 ℃ and 120rpm, washing the obtained immobilized enzyme with the phosphate buffer solution (pH 6.5 and 0.02mol/L) for 3-5 times, and vacuum filtering to obtain the final immobilized enzyme product.

Example 3: construction of error-prone mutation library of SIM prokaryotic expression strain E.coli BL21(DE3)/pET30a (+) -SIM

pET30a (+) -SIM recombinant plasmid is used as PCR template, conventional T7F/R is used as universal primer (primer sequence: T7F: 5'-TAATACGACTCACTATAGGG-3' T7R: GCTAGTTATTGCTCAGCGG is shown in SEQ ID NO.3 and 4) to carry out error-prone PCR amplification on SIM gene, and Mg in a PCR amplification reaction system is adjusted²⁺、Mn²⁺dCTP and dTTP oligonucleotide concentration, base-mismatching of the mutant libraryThe rate is only two thousandth, that is, only 1 to 2 amino acids of one mutant are mutated.

Error-prone PCR reaction system:

error-prone PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 1min, and extension at 72 ℃ for 1.5min for 25 cycles; finally, extension is carried out for 10min at 72 ℃.

And sampling 2 mu L of the error-prone PCR product, detecting the error-prone PCR product through agarose gel electrophoresis, and purifying the error-prone PCR product by using a PCR product purification kit after the error-prone PCR product is detected. The PCR purified product and the prokaryotic expression vector pET30a (+) were subjected to double digestion with Nde I and Xho I restriction enzymes at 37 ℃ respectively, and the digestion product was subjected to gel cutting and recovery (wherein the size of the recovered PCR purified product fragment is about 1700bp, and the size of the recovered vector pET30a (+) fragment is about 5400bp) followed by error-prone PCR: the prokaryotic expression vector pET30a (+) is 3: 1, and T4DNA ligase was added thereto and ligated overnight at 16 ℃. The next day, the ligation product is transferred into escherichia coli BL21(DE3) by an electric shock transformation method to construct engineering bacteria, and a random mutant library with large library capacity can be obtained.

Example 4: screening of error-prone mutation library of SIM prokaryotic expression strain E.coli BL21(DE3)/pET30a (+) -SIM

The screening principle is as follows:

based on the principle that a fibhlin reagent can react with reducing sugar to generate brick red precipitate, sucrose as a substrate is non-reducing sugar, and the product isomaltulose (reducing sugar) can be identified by using the fibhlin reagent.

The specific method comprises the following steps:

using the sterilized toothpicks, single colonies of the mutant library (1 single colony per toothpick) were carefully picked and inoculated into different wells of a 96-well cell culture plate (LB liquid medium containing 50. mu.g/ml kanamycin had been added to each well). The 96-well cell culture plates were incubated for 6 hours at 37 ℃ and 700rpm in a constant temperature shaker, then lactose was added to each well using an 8-channel pipette to a final concentration of 1% (m/v), and incubation was induced for 8 hours at 25 ℃ and 250 rpm. After induction culture is finished, a 96-hole cell culture plate is placed into an ultralow temperature refrigerator with the temperature of minus 86 ℃ for freezing for 2 hours, the cell culture plate is taken out and placed at the room temperature for half an hour and then 4000r/min, the cell culture plate is centrifuged at the temperature of 4 ℃ for 20 minutes, and 100 mu L of supernatant is taken out of each hole and is placed at the temperature of 47 ℃ for standing for 30 minutes. After warming up, 25. mu.L of the supernatant from each well was transferred to a new 96-well plate, and 50. mu.L of 50% sucrose (prepared with 0.05mol/L phosphate buffer, pH 6.5) was added thereto and reacted at 30 ℃ for 120 min. Then 50 μ l of film formazan was added: the mixed solution of ethyl-5: 1 was developed at 60 ℃ for 30 min. The color change was observed.

Through repeated mass screening verification (about 140000 clones), sequencing analysis and enzyme activity determination, an SIM mutant strain with 4-fold improved thermal stability and 3-fold improved zymolase activity, namely an SIM-1 expression strain, is obtained, and is subjected to two-point mutation of R434P and E273N.

Example 5: comparison of immobilized enzyme-catalyzed isomaltulose Synthesis reactions of the mutant strains and the control strains

The immobilized enzymes were prepared from SIM-1 and SIM respectively (same method as in example 2) to catalyze the isomaltulose synthesis reaction.

Reaction conditions are as follows: accurately weighing 50.0g of sucrose, dissolving with deionized water, fixing the volume to 100ml (the concentration is 50%), adding 0.2g of sucrose isomerase immobilized enzyme into each gram of sucrose, reacting at 30 ℃ and pH6.5 at 180r/min, sampling, detecting a liquid phase, performing suction filtration to obtain a reaction solution after the reaction is finished, top washing the immobilized enzyme with 100ml of 2mmol/L phosphate buffer solution with pH6.5, mixing the top washing solution into the reaction solution for the same volume, detecting the concentration, calculating the yield, and adding the top-washed enzyme into the next batch for reaction. The batch reaction of SIM-1 and SIM is shown in tables 1 and 2 below.

TABLE 1SIM-1 batch reaction

TABLE 2SIM batch reactions

The table results show that the activity of the mutant SIM-1 immobilized enzyme is improved by about 54 percent compared with the wild type SIM immobilized enzyme, the activity of the immobilized enzyme is integrally kept stable after 33 batches of reactions, the reaction time is stabilized at 2-2.5h, the activity of the SIM immobilized enzyme is obviously reduced after each batch of reactions, and the reaction time is also obviously prolonged. Obviously, compared with wild immobilized enzyme, the activity and stability of the mutant are obviously improved. In addition, the mutant SIM-1 immobilized enzyme can completely convert the sucrose, and the isomaltulose production rate reaches 96.98% at most. And the wild type SIM immobilized enzyme is converted with a small amount of sucrose residues, and the highest isomaltulose production rate is 90.26%, which shows that the mutant is improved in substrate conversion rate and product production rate compared with the wild type immobilized enzyme.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.

Sequence listing

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Claims

1. A sucrose isomerase mutant, characterized in that: the amino acid sequence shown in SEQ ID NO.1 is mutated, and the mutated amino acid site comprises at least one of R434P and E273N.

2. The mutant according to claim 1, characterized in that: the amino acid sequences of the two-point mutants of R434P and E273N are shown in SEQ ID NO. 2.

3. A nucleotide encoding the sucrose isomerase mutant of claim 1 or 2.

4. Use of a sucrose isomerase mutant as claimed in claim 1 or 2 wherein: used for catalyzing sucrose reaction to prepare isomaltulose.

5. Use according to claim 4, characterized in that: in the reaction system, the enzyme activity of the sucrose isomerase mutant is 60-150U/g substrate sucrose.

6. Use according to claim 4, characterized in that: in the reaction system, the concentration of the sucrose is 30-70%.

7. Use according to claim 4, characterized in that: the reaction temperature is 25-40 ℃, and the reaction time is 120-180 min.

8. Use according to claim 4, characterized in that: the reaction pH is 5.5-7.0.

9. Use according to claim 4, characterized in that: the reaction stirring speed is 150-200 r/min.

10. Use according to claim 4, characterized in that: the sucrose isomerase mutant is an immobilized sucrose isomerase mutant.