CN117821433A

CN117821433A - Sucrose isomerase mutant and efficient synthesis of isomaltulose thereof

Info

Publication number: CN117821433A
Application number: CN202311803653.9A
Authority: CN
Inventors: 张荣珍; 黄润仪
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-04-05

Abstract

The invention discloses a sucrose isomerase mutant and an efficient synthesis method of isomaltulose, belonging to the technical fields of genetic engineering and enzyme engineering. The specific enzyme activity of the mutant M282S/M441F/A470E can reach 871.3U/mg at 30 ℃ and pH 6.0, the stability is improved by 1.33 times compared with the wild type, and the half life is about 120min at 45 ℃. The recombinant sucrose isomerase mutant is applied to isomaltulose production, the yield and the conversion rate are improved, the supernatant of the fermentation liquor is used as crude enzyme liquid to be added into a reaction system, when the concentration of sucrose is 650g/L, the yield is 98.7%, the yield is 641.6g, and isomaltulose can be efficiently synthesized and high yield can be obtained. The invention provides a high-quality sucrose isomerase mutant for the biocatalytic synthesis of isomaltulose, and the yield of the isomaltulose prepared by the method reaches the highest level of public report at home and abroad.

Description

Sucrose isomerase mutant and efficient synthesis of isomaltulose thereof

Technical Field

The invention relates to a sucrose isomerase mutant and an efficient synthesis method of isomaltulose, belonging to the technical fields of genetic engineering and enzyme engineering.

Background

Isomaltulose (isoallose), i.e. 6-O-glucopyranosyl-D-fructose, also known as palatinose, is an isomer of sucrose. Isomaltulose has only half the sweetness of sucrose, no odor, and stable sweetness. The isomaltulose can not be decomposed in the oral cavity and the stomach after being eaten, so that dental caries can be prevented, oral diseases can be effectively reduced, and the number of variable streptococcus in saliva is obviously reduced; after entering the small intestine, isomaltulose can be slowly hydrolyzed and absorbed by sucrase and isomaltase, and the insulin and blood sugar level in the body can not be rapidly changed, so that the anti-diabetic and anti-fatigue health care food can effectively prevent diabetes, refresh mind, resist fatigue, prevent diarrhea, accelerate fat metabolism and the like.

Natural isomaltulose is present in small amounts in foods such as beet and honey, but it is difficult to meet market demands due to low content and difficult extraction. The existing preparation method of isomaltulose has the problems of high cost, large pollution, difficult microbial conversion, separation and extraction, weak production strength and effective avoidance of the problems by an enzyme method, and the existing sucrose isomerase is the most effective enzyme for industrially producing isomaltulose by a biological enzyme method.

Sucrose isomerase (Sucrose isomerase, SIase, EC 5.4.99.11) is capable of isomerising sucrose to form isomaltulose and trehalulose, the isomerised product being affected by temperature and pH. Sucrose isomerase is mainly present in bacteria such as eubacterium rheum officinale (Erwinia rhapontici), serratia (Serratia plymuthica), gram Lei Bashi bacillus (Klebsiella sp.) and multi-source divergent bacteria (Pantoea dispersoa) and the like. However, sucrose isomerase from different sources has the problems of poor stability, low conversion rate, byproducts and the like.

The bacillus subtilis has a clearer genetic metabolism path, is non-pathogenic, has no codon preference, has a short fermentation period, is suitable for high-cell-density culture, has strong protein secretion capacity, can be directly secreted outside cells, and is a common food safety strain. The efficient expression of the recombinant sucrose isomerase outside the bacillus subtilis and the efficient synthesis of isomaltulose are realized, so that the method has important significance for the industrial production of the recombinant sucrose isomerase.

Disclosure of Invention

The invention aims to solve the technical problems of high efficiency and high concentration synthesis of isomaltulose due to the problems of poor enzyme stability, low conversion rate, byproducts and the like in the process of synthesizing isomaltulose by sucrose isomerase.

The invention provides a sucrose isomerase mutant, which is obtained by mutating at least one of 282 rd, 441 th and 470 th positions of sucrose isomerase; the sucrose isomerase is a sucrose isomerase with an amino acid sequence shown as SEQ ID NO.1 or a sucrose isomerase with at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the sequence shown as SEQ ID NO. 1.

In one embodiment of the invention, the mutant is obtained by mutating amino acids 282 and/or 441 and/or 470 of a sucrose isomerase having an amino acid sequence shown in SEQ ID NO. 1.

In one embodiment of the present invention, the mutant is obtained by mutating methionine at position 282 of sucrose isomerase having an amino acid sequence shown in SEQ ID NO.1 to serine.

In one embodiment of the present invention, the mutant is obtained by mutating methionine at position 441 of sucrose isomerase having an amino acid sequence shown in SEQ ID NO.1 to phenylalanine.

In one embodiment of the present invention, the mutant is obtained by mutating alanine at position 470 of sucrose isomerase having an amino acid sequence shown in SEQ ID NO.1 to glutamic acid.

In one embodiment of the present invention, the mutant is obtained by mutating methionine at 282 of sucrose isomerase having an amino acid sequence shown in SEQ ID NO.1 to serine, mutating methionine at 441 to phenylalanine, and mutating alanine at 470 to glutamic acid; named as：PdSlase ^{M282S/M441F/A470E} The amino acid sequence of the mutant is shown as SEQ ID NO. 2.

In one embodiment of the present invention, the nucleotide sequence encoding the sucrose isomerase parent enzyme is as shown in SEQ ID NO.3, encoding the PdSlase ^{M282S/M441F/A470E} The nucleotide sequence of the mutant is shown as SEQ ID NO. 4.

The invention also provides a gene for encoding the mutant.

The invention also provides a recombinant vector containing the mutant.

In one embodiment of the invention, the vector includes, but is not limited to, pMA0911.

The invention also provides a recombinant vector carrying the gene.

The present invention also provides vectors carrying the gene encoding the base sequence shown in SEQ ID NO.3 or SEQ ID NO.4, including but not limited to pMA0911.

The invention also provides a recombinant cell for expressing the sucrose isomerase mutant, or carrying the gene or the recombinant vector.

In one embodiment of the invention, the recombinant cell is a fungal or bacterial expression host.

In one embodiment of the invention, the recombinant cells include, but are not limited to, bacillus subtilis 168.

The invention also provides an enzyme preparation, which contains the sucrose isomerase mutant or the mixture of the sucrose isomerase mutant enzyme and an enzyme protective agent.

In one embodiment of the invention, the enzyme preparation is a lyophilized powder of the sucrose isomerase, comprising the sucrose isomerase and a protecting agent thereof.

The invention also provides a method for improving the enzyme activity and/or stability of the sucrose isomerase, which comprises the following steps of mutating methionine at 282 rd position of the sucrose isomerase with an amino acid sequence shown as SEQ ID NO.1 into serine;

or the 441 st methionine of sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1 is mutated into phenylalanine;

or mutating alanine at 470 position of sucrose isomerase with amino acid sequence shown in SEQ ID NO.1 into glutamic acid;

or the 282 th methionine of sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1 is mutated into serine, the 441 th methionine is mutated into phenylalanine, and the 470 th alanine is mutated into glutamic acid.

The invention also provides a recombinant bacillus subtilis which uses pMA0911 as a vector and expresses sucrose isomerase shown in SEQ ID NO.1 or SEQ ID NO.2 in bacillus subtilis 168.

The invention also provides a method for constructing the recombinant bacterium, which comprises the following steps: the sucrose isomerase gene is connected with a vector pMA0911, and the obtained recombinant expression vector is transformed into bacillus subtilis 168 to obtain recombinant bacteria.

The invention also provides a method for soluble expression of sucrose isomerase, which is prepared by inoculating the recombinant cells into a culture medium for fermentation.

In one embodiment of the present invention, the recombinant bacillus subtilis described above is inoculated into a medium and cultured at 30-35 ℃ for 24-48 hours.

In one embodiment of the invention, the medium contains tryptone, yeast extract, glycerol, monobasic potassium phosphate, dibasic potassium phosphate.

In one embodiment of the invention, the incubation temperature is 30℃and the incubation time is 48 hours.

In one embodiment of the invention, the culture medium formula is peptone 12g/L, yeast powder 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.3g/L, dipotassium hydrogen phosphate 12.5g/L.

The invention also provides a method for preparing isomaltulose, which comprises the steps of adopting the mutant, the recombinant cell, the enzyme preparation or the recombinant enzyme prepared by fermenting the recombinant cell, and reacting sucrose as a substrate to prepare isomaltulose.

In one embodiment of the invention, the reaction conditions are: 400-800g/L sucrose was added to 50mM pH 6.0 citrate-disodium hydrogen phosphate buffer, and the fermentation broth of recombinant cells was reacted at 200rpm in a shaker at 30℃for 12 hours.

The invention also provides the application of the recombinant cell, or the sucrose isomerase, or the enzyme preparation in the field of isomaltulose production.

In one embodiment of the invention, the method is used for efficient synthesis of isomaltulose and high yields are obtained.

The invention also provides application of the mutant, the gene, the vector, the recombinant cell, the enzyme preparation or the method in preparing isomaltulose or food, medicine, cosmetic or health care product containing isomaltulose.

Advantageous effects

(1) The invention uses Pantoea disperso sucrose isomerase and mutant PdSlase thereof ^M282S ^/M441F/A470E The sucrose isomerase mutant which is expressed in the food safety level strain bacillus subtilis 168 and is high-efficiency in expression and simultaneously improves the enzyme activity, stability, conversion rate and yield is obtained;

(2) Positive mutant PdSlase ^M282S 、PdSlase ^M441F 、PdSlase ^A470E The specific enzyme activity is respectively improved to 808.5U/mg, 772.9U/mg and 694.1U/mg;

(3) The obtained mutant PdSlase ^{M282S/M441F/A470E} The expression quantity in the recombinant bacteria is not lower than that of the wild type, the stability is improved, the specific enzyme activity is improved to 871.3U/mg, the yield of isomaltulose is improved to 641.6g by 1.33 times compared with that of the wild type, the conversion rate is 98.7%, and the cost is reduced for preparing the recombinant sucrose isomerase by downstream separation.

(4) The results showed that, at 45 ℃, mutant PdSlase ^{M282S/M441F/A470E} The half-life of (2) is about 120 minutes. The sucrose isomerase mutant has good stability. The recombinant sugarcaneThe sugar isomerase is applied to isomaltulose production.

Drawings

FIG. 1 is an amino acid sequence alignment of PdSlase with other sucrose isomerase.

FIG. 2 is a SDS-PAGE analysis of PdSlase and its mutants.

FIG. 3 shows specific enzyme activities of PdSlase and mutants thereof.

FIG. 4 shows the PdSlase and its mutant PdSlase ^{M282S/M441F/A470E} Optimum pH.

FIG. 5 shows the PdSlase and its mutant PdSlase ^{M282S/M441F/A470E} Optimum temperature.

FIG. 6 shows the PdSlase and its mutant PdSlase ^{M282S/M441F/A470E} pH stability.

FIG. 7 shows the PdSlase and its mutant PdSlase ^{M282S/M441F/A470E} Temperature stability.

FIG. 8 is a PdSlase ^{M282S/M441F/A470E} Yield and yield of isomaltulose synthesized under high concentration sucrose.

Detailed Description

The following examples relate to the following media:

LB liquid medium: 10g/L of sodium chloride, 10g/L of peptone and 5g/L of yeast powder.

TB liquid medium: 12g/L peptone, 24g/L yeast powder, 5g/L glycerol, 2.3g/L potassium dihydrogen phosphate and 12.5g/L dipotassium hydrogen phosphate.

The detection method involved in the following examples is as follows:

the method for measuring the enzyme activity of sucrose isomerase comprises the following steps:

to 1mL of 50mM pH 6.0 citric acid-disodium hydrogen phosphate buffer containing 200g/L sucrose was added 0.5uM sucrose isomerase; the reaction was stopped at 30℃for 10min, followed by 10min in a boiling water bath at 100℃and the supernatant was centrifuged and the isomaltulose content of the reaction solution was checked by HPLC.

Detection of isomaltulose content: isomaltulose was measured using an Agilent 1260 liquid chromatograph and a Thermo amino column with acetonitrile as mobile phase, water (80:20), 10 μl sample injection, flow rate of 0.8mL/min, column temperature of 30deg.C, and differential refractive detector (RID).

Sucrose isomerase enzyme activity is defined as the amount of enzyme required to catalyze the production of l. Mu. Mol isomaltulose per minute from sucrose and is one enzyme activity unit U. Specific enzyme activity is defined as the enzyme activity U/mg of a unit protein.

Example 1: construction of recombinant Bacillus subtilis 168/pMA0911-PdSlase

The method comprises the following specific steps:

(1) The amino acid sequence of the sucrose isomerase encoding PdSlase from Pantoea disc was subjected to 1-21 amino acid sequences of the front end signal peptide (SEQ ID NO. 1) and sent to the division of biological engineering (Shanghai) for codon optimization, a 6 XHis tag was inserted into the C-terminal of the gene, and the gene was cloned into a vector pMA0911 with BamH I/EcoRI cloning site to obtain a recombinant plasmid pMA0911-PdSlase for preservation at-20 ℃.

SEQ ID NO.1:

ASPLTKPSTPIAATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINP

HYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNP

YRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFW

LDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPV

SAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDN

PRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASG

KVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQ

LINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSL

QENASTLTLAPWQAGIYKLN

(2) 10. Mu.L of plasmid was added to 500. Mu.L of B.subtilis 168 competent cells, gently mixed, and then placed in a metal bath at 45℃for 90s, and immediately after heat-shock, placed on ice for 1min. The culture was carried out at 37℃and 200rpm with shaking for 2.5 hours. The cells were collected by centrifugation at 6,000rpm for 5min, 50. Mu.L of the supernatant was resuspended and then spread on LB solid medium plates containing 50. Mu.g/mL kanamycin sulfate, and the plates were inverted and cultured overnight in a constant temperature incubator at 37 ℃.

Preparing recombinant bacillus subtilis: the subulis 168/pMA0911-PdSlase.

Example 2: mutant design and construction of sucrose isomerase

Certain amino acid sites are rationally designed according to the amino acid sequence alignment of FIG. 1 to carry out site-directed mutagenesis. Primers shown in Table 1 were designed, and the sucrose isomerase mutant was obtained by site-directed mutagenesis using the pMA0911-PdSlase expression vector prepared in example 1 as a template by whole plasmid PCR, and then the obtained positive mutant was subjected to combinatorial mutation.

Table 1: primers for mutation design of sucrose isomerase

The PCR amplification reaction system is as follows: 2 XPimeSTAR 25. Mu. L, pMA0911-PdSlase plasmid 1. Mu. L, ddH ₂ O10. Mu.L, 2. Mu.L each of the primers. PCR procedure: 98 ℃ for 5min; 30 cycles were performed at 98℃for 30s,55℃for 30s, and 72℃for 30 s; 72℃for 10min and 16℃for 10min.

After the amplified fragments after the PCR are purified by using the kit, 100 mu L of E.coli BL21 (DE 3) competent cells are added, the mixture is subjected to ice bath for 30min, then is subjected to metal bath heat shock at 42 ℃ for 90s, and is rapidly placed on ice for 5min after the heat shock, 700 mu L of LB liquid medium is added into a centrifuge tube, and the mixture is subjected to shaking culture at 37 ℃ and 200rpm for 1h. The cells were collected by centrifugation at 5,000Xg for 5min, and 50. Mu.L of the supernatant was used to resuspend the cells and then spread on LB solid medium plates containing 100. Mu.g/mL ampicillin. The cells were placed in a constant temperature incubator at 37℃overnight (about 10 hours). Single colonies were picked up and cultured in 5mL LB liquid medium tubes containing 100. Mu.g/mL ampicillin at 37℃and 200rpm with shaking for about 12 hours, and the extracted plasmids were sent to the company, inc. of biological engineering (Shanghai) for sequencing, and verification of correctness.

Respectively preparing plasmids pMA0911-PdSlase ^N109K 、pMA0911-PdSlase ^G112N 、pMA0911-PdSlase ^R127Q 、pMA0911-PdSlase ^L171K 、pMA0911-PdSlase ^P185K 、pMA0911-PdSlase ^N267D 、pMA0911-PdSlase ^M282S 、pMA0911-PdSlase ^Y307D 、pMA0911-PdSlase ^S359R 、pMA0911-PdSlase ^L374T 、pMA0911-PdSlase ^F384Y 、pMA0911-PdSlase ^A387S 、pMA0911-PdSlase ^E428N 、pMA0911-PdSlase ^M441F 、pMA0911-PdSlase ^Q466E 、pMA0911-PdSlase ^A470E 、pMA0911-PdSlase ^Q554D 、pMA0911-PdSlase ^{M282S/M441F/A470E} 。

Example 3: expression and purification of recombinant sucrose isomerase and mutants thereof

The method comprises the following specific steps:

(1) Preparation of crude enzyme solution

10. Mu.L of the plasmid prepared in example 2 was added to 500. Mu.L of B.subtilis 168 competent cells, mixed gently, and then placed in a metal bath at 45℃for 90s, and immediately after heat-shock, placed on ice for 1min. The culture was carried out at 37℃and 200rpm with shaking for 2.5 hours. The cells were collected by centrifugation at 6,000Xg for 5min, 50. Mu.L of the supernatant was resuspended and then spread on LB solid medium plates containing 50. Mu.g/mL kanamycin sulfate, and the plates were inverted and cultured overnight in a constant temperature incubator at 37 ℃.

Recombinant bacillus subtilis containing different mutants is prepared respectively.

Recombinant bacillus subtilis single colonies on solid medium plates were picked separately and cultured in 5mL LB liquid medium tubes containing 50 μg/mL kanamycin sulfate at 37 ℃ at 200rpm with shaking for about 8h. The seed solution grown in the test tube was inoculated into a TB liquid medium shake flask containing 50. Mu.g/mL kanamycin sulfate at 37℃and 200rpm in accordance with an inoculum size of 1%, and cultured with shaking for 2 hours. Transferring the shake flask to 30 ℃, and performing induction culture at 200rpm for 48 hours to obtain a fermentation broth. Centrifuging the fermentation liquor for 10min under the condition of 10,000Xg, separating the supernatant of the fermentation liquor and thalli, wherein the quantity of wet thalli exceeds 10g/L, and the supernatant of the fermentation liquor is crude enzyme liquid.

(2) Preparation of pure enzyme solution

The supernatant of the fermentation broth was filtered through a 0.22 μm aqueous filter head, and the filtrate was purified by a nickel column. The HisTrap HP column (GE Healthcare, chicago, usa) was equilibrated for 5 column volumes with protein purification buffer a (20mM Tris,150mM NaCl,pH 7.0), after loading, the hybrid protein was washed with 3% protein purification buffer B (20mM Tris,150mM NaCl,1M imidazole, pH 7.0), then the protein of interest was eluted with 30% protein purification buffer B and collected.

The target protease solution collected after the purification of the nickel column is desalted by a Sephadex G-25 desalting column. The pure enzyme solution of the sucrose isomerase and the pure enzyme solution of the sucrose isomerase mutant are obtained by respectively flushing and balancing 3 column volumes with ultrapure water and buffer C (50 mM pH 6.0 citric acid-disodium hydrogen phosphate buffer), loading the sample and eluting protein with the buffer C. The concentration of the pure enzyme obtained after desalination is more than 0.4mg/mL, the total amount of the pure enzyme is more than 50mL, and the total expression amount of sucrose isomerase is more than 20mg/L. After the pure enzyme solution was concentrated to a concentration of 1mg/mL, SDS-PAGE was performed, and the result of the bands was observed to determine whether a single purified protein of interest was obtained.

The results showed that the protein band was consistent with the theoretical 68kDa (FIG. 2), that the recombinant isomerase and its mutant were successfully expressed and that pure enzymes were obtained, respectively: pdSlase ^WT 、PdSlase ^N109K 、PdSlase ^G112N 、PdSlase ^R127Q 、PdSlase ^L171K 、PdSlase ^P185K 、PdSlase ^N267D 、PdSlase ^M282S 、PdSlase ^Y307D 、PdSlase ^S359R 、PdSlase ^L374T 、PdSlase ^F384Y 、PdSlase ^A387S 、PdSlase ^E428N 、PdSlase ^M441F 、PdSlase ^Q466E 、PdSlase ^A470E 、PdSlase ^Q554D 、PdSlase ^{M282S/M441F/A470E} 。

Example 4: sucrose isomerase and mutant enzyme activity determination thereof

Specific enzyme activities of the pure enzymes prepared in example 3 were detected, specific enzyme activities of PdSlase and mutants thereof were analyzed, and the results are shown in table 2 and fig. 3:

table 2: wild sucrose isomerase and specific enzyme activity of different mutant pure enzymes thereof

The results show that: the specific enzyme activity of the wild type PdSlase is 657.3U/mg, wherein the mutant PdSlase is positive ^M282S 、PdSlase ^M441F 、PdSlase ^A470E The specific enzyme activities are respectively increased to 808.5, 772.9 and 694.1U/mg, and the three mutants PdSlase are obtained after the combined mutation of the positive mutants ^{M282S/M441F/A470E} The specific enzyme activity of (C) is 871.3U/mg (FIG. 3), which is improved by 1.33 times compared with the wild type.

Example 5: sucrose isomerase and mutant enzyme activity optimum pH

The method comprises the following specific steps:

the specific enzyme activities of the recombinant sucrose isomerase and its mutants were determined according to the enzyme activity assay method of example 4 under 50mM citrate-disodium hydrogen phosphate buffer conditions with different pH. The results are shown in Table 3 and FIG. 4:

table 3: pdSlase and mutant PdSlase thereof ^{M282S/M441F/A470E} Specific enzyme activity at different pH values

The results showed that wild-type and mutant pdslases ^{M282S/M441F/A470E} All have the highest specific enzyme activity at pH 6.0 (FIG. 4), and sucrose isomerase belongs to acid protease, and is more suitable for the reaction for synthesizing isomaltulose under the acid condition.

Example 6: sucrose isomerase and mutant enzyme activity optimum temperature

The method comprises the following specific steps:

the specific enzyme activities of the recombinant sucrose isomerase and its mutants were determined according to the enzyme activity assay method of example 4 under different temperature conditions. The results are shown in Table 4 and FIG. 5:

table 4: pdSlase and mutant PdSlase thereof ^{M282S/M441F/A470E} Specific enzyme activity at different temperatures

The results showed that wild-type and mutant pdslases ^{M282S/M441F/A470E} The specific enzyme activity is highest at 30 ℃.

Example 7: sucrose isomerase and mutant enzyme activity pH stability

The method comprises the following specific steps:

after incubating the recombinant sucrose isomerase for 1d at 4℃under 50mM citric acid-disodium hydrogen phosphate buffer at different pH, the specific enzyme activities of the recombinant sucrose isomerase and its mutants were determined at different pH according to the enzyme activity assay method of example 4.

The specific enzyme activity measured under the reaction condition of pH 6.0 and 30 ℃ is taken as 100%. The results are shown in Table 5 and FIG. 6:

table 5: pdSlase and mutant PdSlase thereof ^{M282S/M441F/A470E} Residual relative enzyme activities after incubation at different pH values

The results showed that wild-type and mutant pdslases ^{M282S/M441F/A470E} All are most stable at pH 6.0, and the stability of the mutant is improved compared with the wild type.

Example 8: sucrose isomerase and mutant enzyme activity temperature stability

The method comprises the following specific steps:

after incubation of the recombinant sucrose isomerase for 2h at different temperatures, the specific enzyme activities of the recombinant sucrose isomerase and its mutants were determined at different temperatures according to the enzyme activity determination method of example 4. The specific enzyme activity measured under the reaction condition of pH 6.0 and 30 ℃ is taken as 100%. The results are shown in Table 6 and FIG. 7:

table 6: pdSlase and mutant PdSlase thereof ^{M282S/M441F/A470E} Residual relative enzyme activities after incubation at different temperatures

The results showed that wild-type and mutant pdslases ^{M282S/M441F/A470E} All are most stable at 30 ℃, and the stability of the mutant is improved compared with that of the wild type, and the mutant PdSlase is at 45 DEG C ^{M282S/M441F/A470E} The half-life of (2) is about 120 minutes.

Example 9: application of sucrose isomerase and mutant crude enzyme liquid in high-yield isomaltulose

The method comprises the following specific steps:

100mL of the reaction system was carried out in 500mL shake flasks, 50mM citric acid-disodium hydrogen phosphate buffer pH 6.0, and the enzyme addition was one tenth of the total system, i.e., the mutant PdSlase as in example 3 ^{M282S/M441F/A470E} 10mL of fermentation broth supernatant obtained by the crude enzyme preparation method is added with sucrose with final concentration of 400 g/L, 450 g/L, 500 g/L, 550 g/L, 600g/L, 650g/L, 700 g/L, and 800g/L, and reacted for 12h at 200rpm in a shaking table at 30 ℃ to prepare a reaction solution, and the content of isomaltulose in the reaction solution is detected by HPLC.

The results are shown in Table 7 and FIG. 8:

table 7: yield and conversion of isomaltulose from wild-type enzyme and mutants thereof at different sucrose concentrations

The results show thatThe wild-type enzyme catalyzes, when the concentration of the substrate sucrose is lower than 600g/L, the conversion rate is more than 90%, when the concentration of the sucrose is 600g/L, the conversion rate is 91.7%, the yield is 550.2g, when the concentration of the sucrose is higher than 600g/L, the yield is slightly improved, but the conversion rate is reduced, when the concentration of the sucrose is 650g/L, the conversion rate is 85.4%, and the yield is 581.1g/L; with mutant PdSlase ^{M282S/M441F/A470E} When the concentration of the substrate sucrose is lower than 650g/L, the yield is more than 98%, when the concentration of the sucrose is 650g/L, the conversion rate is 98.7%, the yield is 641.6g, and when the concentration of the sucrose is higher than 650g/L, the yield is slightly improved, but the conversion rate is reduced.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A sucrose isomerase mutant, which is characterized in that the mutant is obtained by mutating methionine at 282 rd position of sucrose isomerase with an amino acid sequence shown as SEQ ID NO.1 into serine;

or the amino acid sequence is shown as SEQ ID NO.1, namely, the 441 st methionine of the sucrose isomerase is mutated into phenylalanine;

or mutation of methionine at 282 to serine, mutation of methionine at 441 to phenylalanine, and mutation of alanine at 470 to glutamic acid of sucrose isomerase having the amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the mutant of claim 1.

3. A vector carrying the gene of claim 2.

4. A recombinant cell expressing the sucrose isomerase mutant of claim 1, or carrying the gene of claim 2, or containing the vector of claim 3; preferably, the recombinant cell is a fungal or bacterial host.

5. An enzyme preparation comprising the sucrose isomerase mutant according to claim 1, or a mixture of the sucrose isomerase mutant enzyme and an enzyme protecting agent.

6. A method for improving the enzyme activity and/or stability of sucrose isomerase, which is characterized in that methionine at 282 rd position of sucrose isomerase with an amino acid sequence shown as SEQ ID NO.1 is mutated into serine;

7. A method for soluble expression of sucrose isomerase, which is characterized in that the recombinant cell is inoculated into a culture medium for fermentation to prepare the sucrose isomerase.

8. A method for preparing isomaltulose is characterized in that the method adopts the mutant according to claim 1, the recombinant cell according to claim 4, the enzyme preparation according to claim 5, or the recombinant enzyme prepared by fermenting the recombinant cell according to claim 4, and sucrose is used as a substrate to react to prepare isomaltulose.

9. The method of claim 8, wherein the reaction conditions are: sucrose was added to 50mM pH 6.0 citrate-disodium hydrogen phosphate buffer at 400-800g/L and reacted at 200rpm in a shaker at 30℃for 12h.

10. Use of a mutant according to claim 1, or a gene according to claim 2, or a vector according to claim 3, or a recombinant cell according to claim 4, or an enzyme preparation according to claim 5, or a method according to any one of claims 6 to 9, for the preparation of isomaltulose or a food, pharmaceutical, cosmetic or health product containing isomaltulose.