NL2038057B1

NL2038057B1 - Arabinose isomerase mutant and construction method and application thereof

Info

Publication number: NL2038057B1
Application number: NL2038057A
Authority: NL
Inventors: Yan Jianguo; Zhang Xian; Du Xuexian; Wu Yingle; Lu Jian; Luo Jiaoyan; Miao Peiyu; Sun Yanfang; Zhou Xiping
Original assignee: Ningxia Saishang Dairy Co Ltd
Priority date: 2023-11-28
Filing date: 2024-06-25
Publication date: 2025-06-17
Also published as: CN117660429A; NL2038057A

Abstract

The present invention discloses an arabinose isomerase mutant and a construction method and application thereof, which belongs to the fields of genetic engineering and enzyme engineering. According to the present invention, on the basis of an arabinose isomerase derived from Escherichia coli, two mutant strains V230L and A313E with improved single-point enzyme activity and a combined mutant V230L/A313E are screened. The enzyme activity of the three mutants is significantly improved, and meanwhile, the thermal stability is performed well. These mutants in the present invention are more suitable for industrial production than a natural arabinose isomerase, and have very great application prospects and industrial values.

Description

ARABINOSE ISOMERASE MUTANT AND CONSTRUCTION METHOD AND APPLICATION

THEREOF

Technical Field

The present invention relates to an arabinose isomerase mutant with improved enzyme activity and a construction method and application thereof, which belongs to the technical fields of genetic engineering and enzyme engineering.

Background

Arabinose isomerase (EC 5.3.1.4) belongs to the arabinose isomerase family. The enzyme can transform L-arabinose into L-ribulose and can isomerize D-galactose into D-tagatose. It is an isomerase with a very high industrial application value. D-tagatose is a natural rare hexose, an epimer of D-fructose and an aldo-keto isomer of D-galactose. The sweetness of D-tagatose is most similar to that of sucrose, and its sweetness stimulation is faster than that of sucrose, but its calorific value is only 30% of that of sucrose, without aftertaste and other bad flavours. It is a recognized low-calorie sweetener and has broad application prospects in food, medicine and other industries. The arabinose isomerase is the most economical enzyme for industrial production of D-tagatose by a biological enzyme method at present.

At present, reported wild-type arabinose isomerases show limited enzyme activity in a biocatalysis process, such as arabinose isomerase derived from Escherichia coli K12, which is the most commonly used enzyme in biotransformation of D-tagatose. However, there are many defects in its enzymatic properties, such as an optimum catalytic temperature and pH, substrate affinity and enzyme activity, which still cannot meet actual production needs.

However, most researches only improve the yield of products by optimizing enzymatic synthesis conditions; and industrial application of the arabinose isomerase optimized by a production process is still not satisfactory. Therefore, the research on molecular modification of the arabinose isomerase to improve the activity will be the research focus of the present invention.

Summary isomerases cannot meet the requirements of industrialization, the present invention provides an arabinose isomerase mutant, which is obtained by mutating the amino acid at position 230 and/or position 313 of the amino acid sequence of arabinose isomerase shown in SEQ ID NO.1.

A specific technical solution provided by the present invention is as follows:

In a first aspect of the present invention, an arabinose isomerase mutant is provided, which is obtained by mutating the amino acid at position 230 and/or position 313 of the amino acid sequence of arabinose isomerase shown in SEQ ID NO. 1.

In one implementation of the present invention, the mutant is obtained by mutating valine at position 230 of the amino acid sequence of arabinose isomerase shown in SEQ ID NO.1 into leucine, and is named as V230L; and an amino acid sequence is shown in SEQ ID NO.3.

In one implementation of the present invention, the mutant is obtained by mutating alanine at position 313 of the amino acid sequence of arabinose isomerase shown in SEQ ID NO.1 into glutamic acid, and is named as A313E; and an amino acid sequence is shown in SEQ ID NO 4.

In one implementation of the present invention, the mutant is obtained by mutating valine at position 230 of the amino acid sequence of arabinose isomerase shown in SEQ ID NO.1 into leucine and simultaneously mutating alanine at position 313 into glutamic acid, and is named as

V230L/A313E; and an amino acid sequence is shown in SEQ ID NO.5.

In a second aspect of the present invention, a gene encoding the arabinose isomerase mutant is provided.

In a third aspect of the present invention, a recombinant vector carrying the gene is provided.

In one implementation of the present invention, the recombinant vector chooses any one of pXMJ19 vector, pMA5 vector, pHT43 vector, pET-20b(+} vector or pDXW-10 vector as an expression vector.

In a fourth aspect of the present invention, a recombinant cell including the recombinant vector is provided.

In one implementation of the present invention, the recombinant cell is from an expression host, chosen from any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or

Saccharomyces.

In a fifth aspect of the present invention, a method for improving enzyme activity of the arabinose isomerase is provided, which is to mutate the amino acid at position 230 and/or position 313 of the amino acid sequence of arabinose isomerase shown in SEQ ID NO.1.

In a sixth aspect of the present invention, an application of the recombinant vector or the recombinant cell in preparation of the arabinose isomerase is provided.

In a seventh aspect of the present invention, an application of the mutant or the gene or the recombinant vector or the recombinant cell in transforming D-galactose to produce D-tagatose is provided.

The present invention further provides a method for preparing the mutant, which includes the following specific steps: (1) performing digestion and ligation of the nucleotide sequence of arabinose isomerase araA with shown in SEQ ID NO.2 with a recombinant plasmid to obtain a recombinant plasmid containing the arabinose isomerase araA; using the recombinant plasmid containing the arabinose isomerase araA as a template; designing site-directed mutation primers; and performing PCR amplification to obtain a vector containing a mutant encoding gene; (2) transforming the vector containing the mutant encoding gene into a host cell to obtain a recombinant cell;

(3) screening and verifying the recombinant cell to obtain a positive clone; and then culturing and fermenting to produce an enzyme to obtain a crude enzyme solution containing an arabinose isomerase mutant.

Compared with the prior art, the present invention has the beneficial effects that: (1) On the basis of a natural arabinose isomerase, a molecular structure of the arabinose isomerase is modified by rational design in combination with the site-directed mutation biotechnology; effects of residues after mutation on enzyme activity are analysed; and two mutant strains V230L and A313E with improved single-point mutation stability and a combined mutant strain V230L/A313E are finally obtained. (2) The specific enzyme activity of the natural arabinose isomerase is 34.5U/mg; and the specific enzyme activity of the arabinose isomerase mutant V230L provided by the present invention is 84.2U/mg at 50°C, which is 2.44 times that of the natural arabinose isomerase; the specific enzyme activity of the arabinose isomerase mutant A313E is 89.5U/mg at 50°C, which is 2.59 times that of the natural arabinose isomerase; and the specific enzyme activity of

V230L/A313E at 50°C is 127.3U/mg, which is 3.69 times that of the natural arabinose isomerase. (3) The arabinose isomerase mutant provided by the present invention still has good thermal stability after the enzyme activity is obviously improved. Specifically, after thermal treatment at 45°C for 20 min when the enzyme catalytic activity is basically unchanged, the relative enzyme activities of the mutants V230L and A313E and the combined mutant V230L/A313E are remained by 51.7%, 48.5% and 95.3%, respectively, while the relative enzyme activity of the control group is only remained by 15.2%. (4) After significantly increasing the specific enzyme activity and improving the thermal stability, the arabinose isomerase mutant obtained by the present invention also improves a transformation rate of catalysing D-galactose to generate D-tagatose. The transformation rate of the natural arabinose isomerase is 30%. The transformation rate of the arabinose isomerase mutant V230L provided by the present invention is 35% at 50°C, which is 1.17 times that of the natural arabinose isomerase; the transformation rate of the arabinose isomerase mutant A313E is 38% at 50°C, which is 1.27 times that of the natural arabinose isomerase; and the transformation rate of V230L/A313E is 40% at 50°C, which is 1.33 times that of the natural arabinose isomerase. (5) Compared with the wild type, the arabinose isomerase mutant obtained by the present invention is more suitable for catalysing D-galactose to generate D-tagatose, which is more conducive to flexibility of a production process.

Description of Drawings

Fig. 1 shows specific enzyme activity of a mutant strain constructed after 20 min reaction at pH 7.0 and 37°C;

Fig. 2 shows half-lives of a wild-type arabinose isomerase and arabinose isomerase mutants

V230L, A313E and V230L/A313E at 45°C;

Fig. 3 shows effects of pH on enzyme activity of a wild-type arabinose isomerase and arabinose isomerase mutants V230L, A313E and V230L/A313E;

Fig. 4 shows effects of temperatures on enzyme activity of a wild-type arabinose isomerase and arabinose isomerase mutants V230L, A313E and V230L/A313E;

Fig. 5 shows transformation rates of a wild-type arabinose isomerase and arabinose isomerase mutants V230L, A313E and V230L/A313E of catalysing D-galactose to generate D- tagatose at 50°C.

Detailed Description

The present invention will be further clarified below by the detailed description of specific implementation. However, the specific implementation is not a limitation of the present invention, but only an illustrative example.

A pMAS vector involved in the following embodiments is preserved by a team laboratory of the present invention.

Media involved in the following embodiments are as follows:

LB liquid medium: 10g/l peptone, 5g/l yeast powder and 10g/I NaCl.

LB solid medium: 2% agar is added based on an LB liquid medium.

Detection methods involved in the following embodiments are as follows:

A determination method of arabinose isomerase enzyme activity. enzyme activity determination reaction system (1 ml). 100 g/l substrate, 200 pl of pure enzyme, NazHPQO,-citric acid buffer (0.2 mol/l, pH 7.0), reaction at 37°C for 20 min, and boiling water bath for terminating the reaction.

Protein concentration determination adopts a Bradford method.

Definition of enzyme activity: under standard conditions (pH 7.0, 37°C), galactose is used as a substrate; and the amount of enzyme required to catalyze production of 1 umol of tagatose product per min is one enzyme activity unit.

Specific enzyme activity: defined as enzyme activity per unit protein, U/mg.

A determination method of an arabinose isomerase transformation rate: transformation reaction system (30 ml): 100 g/l galactose, 15 ml of crude enzyme solution, Na;HPO,-citric acid buffer (0.2 mol/l, pH 7.0), reaction at 50°C for 60h, and boiling water bath for terminating the reaction.

Embodiment 1

Construction of a recombinant plasmid containing an arabinose isomerase mutant in

Escherichia coli (1) Construction of a recombinant plasmid containing a wild-type arabinose isomerase araA

Suzhou GENEWIZ Life Sciences was entrusted to synthesize the nucleotide sequence of a wild-type arabinose isomerase araA with in SEQ ID NO.2. It was ligated with a pMA5 vector after digestion by Hind II enzyme and EcoR/ enzyme to prepare a recombinant vector pMA5-araA.

The amino acid sequence of arabinose isomerase araA is shown in SEQ ID NO. 1: 5 MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITA

ICRDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLN

QTAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALVDEYESCYTMTPATQIHG

KKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEGDW

KTAALLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQHLGI

GGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAQPDLPTA

SEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALR WNEVYYGFRR

The nucleotide sequence of arabinose isomerase araA:

ATGACGATTTTTGATAATTATGAAGTGTGGTTTGTCATTGGCAGCCAGCATCTGTATGG

CCCGGAAACCCTGCGTCAGGTCACCCAACATGCCGAGCACGTCGTTAATGCGCTGAATAC

GGAAGCGAAACTGCCCTGCAAACTGGTGTTGAAACCGCTGGGCACCACGCCGGATGAAAT

CACCGCTATTTGCCGCGACGCGAATTACGACGATCGTTGCGCTGGTCTGGTGGTGTGGCT

GCACACCTTCTCCCCGGCCAAAATGTGGATCAACGGCCTGACCATGCTCAACAAACCGTT

GCTGCAATTCCACACCCAGTTCAACGCGGCGCTGCCGTGGGACAGTATCGATATGGACTT

TATGAACCTGAACCAGACTGCACATGGCGGTCGCGAGTTCGGCTTCATTGGCGCGCGTAT

GCGTCAGCAACATGCCGTGGTTACCGGTCACTGGCAGGATAAACAAGCCCATGAGCGTAT

CGGCTCCTGGATGCGTCAGGCGGTCTCTAAACAGGATACCCGTCATCTGAAAGTCTGCCG

ATTTGGCGATAACATGCGTGAAGTGGCGGTCACCGATGGCGATAAAGTTGCCGCACAGAT

CAAGTTCGGTTTCTCCGTCAATACCTGGGCGGTTGGCGATCTGGTGCAGGTGGTGAACTC

CATCAGCGACGGCGATGTTAACGCGCTGGTCGATGAGTACGAAAGCTGCTACACCATGAC

GCCTGCCACACAAATCCACGGCAAAAAACGACAGAACGTGCTGGAAGCGGCGCGTATTGA

GCTGGGGATGAAGCGTTTCCTGGAACAAGGTGGCTTCCACGCGTTCACCACCACCTTTGA

AGATTTGCACGGTCTGAAACAGCTTCCTGGTCTGGCCGTACAGCGTCTGATGCAGCAGGG

TTACGGCTTTGCGGGCGAAGGCGACTGGAAAACTGCCGCCCTGCTTCGCATCATGAAGGT

GATGTCAACCGGTCTGCAGGGCGGCACCTCCTTTATGGAGGACTACACCTATCACTTCGA

GAAAGGTAATGACCTGGTGCTCGGCTCCCATATGCTGGAAGTCTGCCCGTCGATCGCCGC

AGAAGAGAAACCGATCCTCGACGTTCAGCATCTCGGTATTGGTGGTAAGGACGATCCTGC

CCGCCTGATCTTCAATACCCAAACCGGCCCAGCGATTGTCGCCAGCTTGATTGATCTCGG

CGATCGTTACCGTCTACTGGTTAACTGCATCGACACGGTGAAAACACCGCACTCCCTGCC

GAAACTGCCGGTGGCGAATGCGCTGTGGAAAGCGCAACCGGATCTGCCAACTGCTTCCGA

AGCGTGGATCCTCGCTGGTGGCGCGCACCATACCGTCTTCAGCCATGCACTGAACCTCAA

CGATATGCGCCAATTCGCCGAGATGCACGACATTGAAATCACGGTGATTGATAACGACACA

CGCCTGCCAGCGTTTAAAGACGCGCTGCGCTGGAACGAAGTGTATTACGGGTTTCGTCGC

TAA

(2) Obtaining of a recombinant vector containing the mutant:

A whole plasmid PCR technology was adopted; the recombinant vector pMA5-araA prepared instep (1) was used as a template to perform site-directed mutation of the amino acid at positions 99, 163, 190, 270, 286, 349, 382, 230, 313, 230 and 313 (here, it refers to simultaneous site- directed mutation at position 230 and position 313); and recombinant plasmids pMA5-W99L, pMA5-W163K, pMA5-R190P, pMA5-G270Q, pMA5-K286H, pMA5-S349W, pMA5-L382V, pMA5-

V230L, pMA5-A313E and pMA5-V230L/A313E containing mutant genes were obtained respectively.

Primer sequences designed respectively are as follows:

W99L-F: TTTTCCCCGGCCAAAATGCTTATCAACGGCCTGACCATGC TC, as shown in

SEQ ID NO:6;

W99L-R: TGTTGAGCATGGTCAGGCCGTTGATAAGCATTTTGGCCG, as shown in SEQ ID

NO:7;

W163K-F: AACAAGCACATGAGCGTATCGGCTCCAAGATGCGTCAG GCGGT, as shown in SEQ ID NO:8;

W183K-R:TTAGAGACCGCCTGACGCATCTTGGAGCCGATACGCTC, as shown in SEQ ID

NO:9;

R190P-F: CCGTCATCTGAAAGTCTGCCCTTTTGGCGATAACATGCGT GAAGTAGC, as shown in SEQ ID NO:10;

R190P-R: CGCATGTTATCGCCAAAAGGGCAGACTTTCAGATGACGG GTATCC, as shown in SEQ ID NO:11;

V230L-F: GGCGATGTTAACGCGCTGCTAGATGAGTACGAAAGCTGC, as shown in SEQ

ID NO:12;

V230L-R:TGGTGTAGCAGCTTTCGTACTCATCTAGCAGCGCGTTAACATCGCCATCGCT, as shown in SEQ ID NO:13;

G270Q-F: GATGAAGCGTTTCCTGGAACAAGGTCGATTCCACGCGT TC, as shown in

SEQ ID NO:14;

G270Q-R: TGGTGAACGCGTGGAATCGACCTTGTTCCAGGAAACGC TTCAT, as shown in

SEQ ID NO:15;

K286H-F: TTTGAAGATTTGCACGGTCTGCATCAGCTTCCTGGTCTG GCCGTA, as shown in SEQ ID NO: 16;

K286H-R: CGCTGTACGGCCAGACCAGGAAGCTGATGCAGACCGTGC AAATCT, as shown in SEQ ID NO:17;

A313E-F: GGGCGAAGGCGACTGGAAAACTGCCGAACTGCTTCGCA TC, as shown in

SEQ ID NO:18;

A313E-R: TTCATGATGCGAAGCAGGGTTCCAGTTTTCCAGTCGCCT, as shown in SEQ

ID NO:19;

S349W-F: AATGACCTGGTGCTCGGCTGGCATATGCTGGAAGTCTGT CCGT, as shown in SEQ ID NO:20;

S349W-R: GATCGACGGACAGACTTCCAGCATATGCCAGCCGAGCAC CAG, as shown in

SEQ ID NO:21;

L382V-F: GACGATCCTGCCCGCGTTATCTTCAACACTCAAACCGG, as shown in SEQ ID

NO:22;

L382V-R: CCGGACCGGTTTGAGTGTTGAAGATAACGCGGGCAGGA TC, as shown in

SEQ ID NO:23.

Specifically, a PCR amplification procedure is set as follows: firstly, pre-denaturation at 95°C for 5min; then 30 cycles; and denaturation at 95°C for 30s, annealing at 72°C for 30s, extension at 58°C for 3.5min, and heat preservation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.

A final amplified fragment was treated with a Dpn | enzyme in a water bath at 37°C for 1 h to remove the template; then the PCR mixture was chemically transformed into competent cells of

E. coli JM108; the transformation solution was coated on a solid medium containing ampicillin (200 ug/ml); and plasmids were extracted and sequenced. The sequencing work was completed by Suzhou GENEWIZ.

Embodiment 2

Construction of a recombinant Bacillus subtilis engineering strain producing an arabinose isomerase mutant and separation and purification of an arabinose isomerase (1) The recombinant plasmids pMA5-araA, pMA5-W99L, pMA5-W163K, pMA5-R190P, pMA5-G270Q, pMA5-K286H, pMA5-S349W, pMA5-L382V, pMA5-V230L, pMA5-A313E and pMA5-V230L/A313E obtained by embodiment 1 were chemically transformed into B.subtilis 168 competent cells. Gene engineering strains were prepared respectively: B.subtilis13032/pMA5- araA, B.subtilis13032/pMA5-W99L, B.subtilis13032/pMA5- W163K, B.subtilis13032/pMA5-

R190P, B.subtilis13032/pMA5-G270Q, B.subtilis13032/pMA5-K286H, B.subtilis13032/pMA5-

S349W, B.subtilis13032/pMA5-L382V, B.subtilis13032/pMA5-V230L, B.subtilis13032/pMA5-

A313E and B.subtilis13032/pMA5-V230L/A313E. (2) The gene engineering strains prepared in step (1) were inoculated into 10 ml of LB liquid culture medium containing 100 pg/ml kanamycin sulphate respectively; and culturing overnight was performed at 37°C and 200 rpm to prepare a seed solution.

The prepared seed liquid was transferred to 100 ml of LB liquid culture medium containing 100 pg/ml kanamycin sulphate according to an inoculation amount of 2% (v/v}, and cultured at 37°C and 200 rpm for 24 h to obtain a fermentation liquid. The prepared fermentation liquid was centrifuged at 8000 x g and 4°C for 5 min to obtain cell thalli. After washing for three times, the cells were resuspended with 10 ml of 50 mM sodium citrate and sodium dihydrogen phosphate buffer (pH 8.0).

After adding 20 pl of 100 mg/l lysozyme to the cell suspension, the resuspended cells were treated for 30 min with an ultrasonic crusher under conditions of an ice bath and centrifuged for 30 min (8000 x g, 4°C). The obtained supernatant was a crude enzyme solution.

The supernatant was filtered through a 0.22-um filter, and then further loaded on a 1 ml Ni affinity column, which was pre-balanced with a 50 mM washing buffer (20 mM Tris and 500 mM

NaCl, pH 7.4). Then the unbound protein and arabinose isomerase were eluted with a linear gradient by an elution buffer (20 mM Tris, 500 mM NaCl and 500 mM imidazole, pH 7.4). Pure enzyme solutions containing wild-type araA4, WO9L, W163K, R190P, G270Q, K286H, S349W,

L382V, V230L, A313E and V230L/A313E were prepared respectively. (3) Determination of specific enzyme activity of the pure enzyme solutions prepared in step (2)

Specific enzyme activities of the pure enzyme solution containing the wild-type araA, W9S9L,

W163K, R190P, G270Q, K286H, S349W, L382V, V230L, A313E and V230L/A313E prepared in step (2) were detected respectively. Results are shown in Table 1 and Fig. 1.

Table 1 Specific Enzyme Activities of Different Arabinose Isomerases

Enzyme | SPeciicenzyme activity (U-mg")

OO Wildtype 345

V230L 84.2

A313E 89.5

V230L/A313E 127.3

Note: Table 1 only lists the specific enzyme activities of three arabinose isomerases with the largest specific enzyme activities.

Embodiment 3

Enzymatic properties of an arabinose isomerase mutant 1. Thermal stability

In order to test effects of single-point mutation on thermal stability, the three pure enzymes with the largest specific enzyme activities prepared in embodiment 2 were incubated at 50°C for 20 min for an enzyme activity determination test; and the remaining enzyme activity was obtained.

The relative enzyme activity was determined with the initial pure enzyme without incubation as 100%.

The results showed that the mutants V230L and A313E and the combined mutant

V230L/A313E remained 51.7%, 48.5% and 95.3% relative enzyme activity respectively, while the control group (pure enzyme containing the wild-type araA) only remained 15.2% relative enzyme activity. The relative enzyme activities of other mutants were all below 20%. Therefore, the mutants V230L and A313E and the combined mutant V230L/A313E of the present invention have good stability on the basis of significantly improved enzyme activity. 2. Half-life

The pure enzyme solutions containing the wild-type araA, V230L, A313E and V230L/A313E prepared in step (2) of embodiment 2 were taken respectively and placed in a constant temperature water bath at 50°C; sampling was conducted at time intervals; the remaining enzyme activities thereof were determined according to the arabinose isomerase enzyme activity determination method; and their thermal stability was compared. Half-life results of the wild-type araA and its mutants were obtained, as shown in Table 2 and Fig. 2.

Table 2 Half-Lives of Different Arabinose Isomerases

Enzyme tua(min) (50°C) © Wildtype 98

V230L 26.9

A313E 38.2

V230L/A313E 100 3. Optimum pH

The pure enzyme solutions containing the wild-type araA, V230L, A313E and V230L/A313E prepared in step (2) of embodiment 2 were placed in a 50 mM buffer containing citric acid/sodium phosphate (pH 6.0-8.0), Tris/hydrochloric acid (pH 8.0-9.0), and glycine/sodium hydroxide (pH 9.0-10.0); and the enzyme activity was determined with the initial enzyme activity without incubation as 100%. Results are shown in Fig. 3.

The results showed that the optimum pH of the mutant was 9.0, which was similar to that of the wild type. 4. Optimum temperature

The pure enzyme solutions containing the wild-type araA, V230L, A313E and V230L/A313E prepared in step (2) of embodiment 2 were placed in a 50 mM buffer containing citric acid/sodium phosphate (pH8.0); the reaction temperature was set at 30-70°C; and the enzyme activity was determined with the initial enzyme activity without incubation as 100%. Results are shown in Fig. 4.

The results showed that the optimum temperature of the mutant was 50°C, which was similar to that of the wild type. 5. Kinetic parameters of arabinose isomerase

Using galactose as a substrate, kinetic parameters of the pure enzyme solutions containing the wild-type araA, V230L, A313E and V230L/A313E prepared in step (2) of embodiment 2 were determined under standard determination conditions. Specifically, the substrate concentration of

D-galactose was 13.8, 25.2, 53.1, 109, 156, 234, 301 and 805mM, respectively. The addition amount of pure enzyme solution was 10 pg. The reaction was carried out at 40°C for 10 min. After the reaction, experimental data was treated by regression analysis by using GraphPad Prism 8.0 to determine Vmax and Km values. The results are shown in Table 3.

The results showed that the mutant showed similar activity compared with the wild type. The kinetic parameters such as Km and Kcat/Km of these mutants were changed little, which indicates that mutation had little effect on catalytic properties of the enzyme.

Table 3 Kinetic Parameters of Different Arabinose Isomerases

Enzyme Kin(mM) Keat(S™) Keat/Kon(S mM) ~ Widtype ~~ 587+14 699+58 119

V230L 5711.1 69814 4 12.2

A313E 59.2+2.5 705+4.8 11.9

V230L/A313E 52.811.7 710+5.2 13.4 6. Transformation rate of catalysing D-galactose to generate D-tagatose by an arabinose isomerase

Using galactose as a substrate, transformation rates of the crude enzyme solutions containing the wild-type araA, V230L, A313E and V230L/A313E prepared in step (2) of embodiment 2 were determined under transformation conditions (pH 7.0, 50°C). Specifically, the substrate concentration of D-galactose was 100 g/l. The addition amount of the crude enzyme solution was 15 ml. The reaction was carried out at 50°C for 60h. After the reaction, the transformation rate was calculated. The results are shown in Table 4 and Fig. 5.

Table 4 Transformation Rates of Different Arabinose Isomerases

Enzyme Transformation rate (%)

OO Wildtype 30

V230L 35

A313E 38

V230L/A313E 40 -

The results showed that compared with the wild type, the three mutants constructed by the present invention showed higher transformation rates.

The amino acid sequence of the arabinose isomerase mutant V230L constructed by the present invention is shown in SEQ ID NO. 3:

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITA

ICRDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLN

QTAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALLDEYESCYTMTPATQIHG

KKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEGDW

KTAALLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQHLGI

GGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAGQPDLPTA

SEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALR WNEVYYGFRR

The amino acid sequence of the arabinose isomerase mutant A313E constructed by the present invention is shown in SEQ ID NO.4:

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITA

ICRDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLN

QTAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALVDEYESCYTMTPATQIHG

KKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEGDW

KTAELLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQHLGI

GGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAGQPDLPTA

SEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALR WNEVYYGFRR

The amino acid sequence of the arabinose isomerase mutant V230L/A313E constructed by the present invention is shown in SEQ ID NO.5:

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITA

ICRDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLN

QTAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALLDEYESCYTMTPATQIHG

KKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEGDW

KTAELLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQHLGI

GGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAGQPDLPTA

SEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALR WNEVYYGFRR

The above embodiments only describe the preferred modes of the present invention, instead of limiting the scope of the present invention. On the premise of not deviating from the design spirit of the present invention, various transformations and improvements made to the technical solution of the present invention by those ordinary skilled in the art shall fall within the protection scope determined by the claims of the present invention.

NL Arabinose Isomerase NL Ningxia Saishang Dairy Industry Co. Ltd. SUN Yanfang

An arabinose isomerase mutant and its construction method, application 23 500 AA

PAT source 1..500 mol_type protein organism Escherichia coli

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITAIC

RDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLNQ

TAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALVDEYESCYTMTPATQI

HGKKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEG

DWKTAALLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQ

HLGIGGKDDPARLIFNTQTGPATVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAQP

DLPTASEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALRWNEVYY

GFRR 1503 DNA PAT source 1..1503 mol_type genomic DNA organism Escherichia coli atgacgatttttgataattatgaagtgtggtttgtcattggcagccagcatctgtatggcccggaaaccctgcgtcaggt cacccaacatgccgagcacgtcgttaatgcgctgaatacggaagcgaaactgccctgcaaactggtgttgaaaccgc tgggcaccacgccggatgaaatcaccgctatttgccgcgacgcgaattacgacgatcgttgcgctggtctggtggtgt ggctgcacaccttctccccggccaaaatgtggatcaacggcctgaccatgctcaacaaaccgttgctgcaattccacac ccagttcaacgcggcgctgccgtgggacagtatcgatatggactttatgaacctgaaccagactgcacatggcggtcg cgagttcggcttcattggcgcgcgtatgcgtcagcaacatgccgtggttaccggtcactggcaggataaacaagccca tgagcgtatcggctcctggatgcgtcaggcggtctctaaacaggatacccgtcatctgaaagtctgccgatttggcgat aacatgcgtgaagtggcggtcaccgatggcgataaagttgccgcacagatcaagttcggtttctccgtcaatacctgg gcggttggcgatctggtgcaggtggtgaactccatcagcgacggcgatgttaacgcgctggtcgatgagtacgaaag ctgctacaccatgacgcctgccacacaaatccacggcaaaaaacgacagaacgtgctggaagcggcgcgtattgag ctggggatgaagcgtttcctggaacaaggtggcttccacgcgttcaccaccacctttgaagatttgcacggtctgaaac agcttcctggtctggccgtacagcgtctgatgcagcagggttacggctttgcgggcgaaggcgactggaaaactgcc gccctgcttcgcatcatgaaggtgatgtcaaccggtctgcagggcggcacctcctttatggaggactacacctatcactt cgagaaaggtaatgacctggtgctcggctcccatatgctggaagtctgcccgtcgatcgccgcagaagagaaaccga tcctcgacgttcagcatctcggtattggtggtaaggacgatcctgcccgcctgatcttcaatacccaaaccggcccagc gattgtcgccagcttgattgatctcggcgatcgttaccgtctactggttaactgcatcgacacggtgaaaacaccgcact ccctgccgaaactgccggtggcgaatgcgctgtggaaagcgcaaccggatctgccaactgcttccgaagcgtggatc ctcgctggtggcgcgcaccataccgtcttcagccatgcactgaacctcaacgatatgcgccaattcgccgagatgcac gacattgaaatcacggtgattgataacgacacacgcctgccagcgtttaaagacgcgctgcgctggaacgaagtgta ttacgggtttcgtcgctaa 500 AA PAT source 1..500 mol_type protein organism

Escherichia coli

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITAIC

RDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLNQ

TAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALLDEYESCYTMTPATQIH

GKKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEGD

WKTAALLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQHL

GIGGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAQPDL

PTASEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALRWNEVYYGF

RR 500 AA PAT source 1..500 mol_type protein organism Escherichia coli

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITAIC

RDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLNQ

TAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALVDEYESCYTMTPATQI

HGKKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEG

DWKTAELLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQ

HLGIGGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAQP

DLPTASEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALRWNEVYY

GFRR 500 AA PAT source 1..500 mol_type protein organism Escherichia coli

MTIFDNYEVWFVIGSQHLYGPETLRQVTQHAEHVVNALNTEAKLPCKLVLKPLGTTPDEITAIC

RDANYDDRCAGLVVWLHTFSPAKMWINGLTMLNKPLLQFHTQFNAALPWDSIDMDFMNLNQ

TAHGGREFGFIGARMRQQHAVVTGHWQDKQAHERIGSWMRQAVSKQDTRHLKVCRFGDN

MREVAVTDGDKVAAQIKFGFSVNTWAVGDLVQVVNSISDGDVNALLDEYESCYTMTPATQIH

GKKRQNVLEAARIELGMKRFLEQGGFHAFTTTFEDLHGLKQLPGLAVQRLMQQGYGFAGEGD

WKTAELLRIMKVMSTGLQGGTSFMEDYTYHFEKGNDLVLGSHMLEVCPSIAAEEKPILDVQHL

GIGGKDDPARLIFNTQTGPAIVASLIDLGDRYRLLVNCIDTVKTPHSLPKLPVANALWKAQPDL

PTASEAWILAGGAHHTVFSHALNLNDMRQFAEMHDIEITVIDNDTRLPAFKDALRWNEVYYGF

RR 42 DNA PAT source 1..42 mol_type other DNA organism synthetic construct ttttccccggccaaaatgcttatcaacggcctgaccatgctc 39 DNA PAT source 1..39 mol_type other DNA organism synthetic construct tgttgagcatggtcaggccgttgataagcattttggccg 43 DNA PAT source 1..43 mol_type other DNA organism synthetic construct aacaagcacatgagcgtatcggctccaagatgcgtcaggcggt 38 DNA PAT source 1..38 mol_type other DNA organism synthetic construct ttagagaccgcctgacgcatcttggagccgatacgctc 48 DNA PAT source 1..48 mol_type other DNA organism synthetic construct ccgtcatctgaaagtctgcccttttggcgataacatgcgtgaagtagc 45 DNA PAT source 1..45 mol_type other DNA organism synthetic construct cgcatgttatcgccaaaagggcagactttcagatgacgggtatcc 39 DNA PAT source 1..39 mol_type other DNA organism synthetic construct ggcgatgttaacgcgctgctagatgagtacgaaagctgc 52 DNA PAT source 1..52 mol_type other DNA organism synthetic construct tggtgtagcagctttcgtactcatctagcagcgcgttaacatcgccatcgct 40 DNA PAT source 1..40 mol_type other DNA organism synthetic construct gatgaagcgtttcctggaacaaggtcgattccacgcgttc 43 DNA PAT source 1..43 mol_type other DNA organism synthetic construct tggtgaacgcgtggaatcgaccttgttccaggaaacgcttcat 45 DNA PAT source 1..45 mol_type other DNA organism synthetic construct tttgaagatttgcacggtctgcatcagcttcctggtctggccgta 45 DNA PAT source 1..45 mol_type other DNA organism synthetic construct cgctgtacggccagaccaggaagctgatgcagaccgtgcaaatct 40 DNA PAT source 1..40 mol_type other DNA organism synthetic construct gggcgaaggcgactggaaaactgccgaactgcttcgcatc 39 DNA PAT source 1..39 mol_type other DNA organism synthetic construct ttcatgatgcgaagcagggttccagttttccagtcgcct 43

DNA PAT source 1..43 mol_type other DNA organism synthetic construct aatgacctggtgctcggctggcatatgctggaagtctgtccgt 42 DNA PAT source 1..42 mol_type other DNA organism synthetic construct gatcgacggacagacttccagcatatgccagccgagcaccag 38 DNA PAT source 1..38 mol_type other DNA organism synthetic construct gacgatcctgcccgcgttatcttcaacactcaaaccgg 40

DNA PAT source 1..40 mol_type other DNA organism synthetic construct ccggaccggtttgagtgttgaagataacgcgggcaggatc

Claims

CONCLUSIONS

1. An arabinose isomerase mutant obtained by mutating the amino acid at position 230 and/or position 313 of the amino acid sequence of arabinose isomerase as shown in SEQ ID NO.1.

2. The arabinose isomerase mutant according to claim 1, wherein — valine at position 230 of the amino acid sequence of arabinose isomerase as shown in SEQ ID NO.1 is mutated to leucine; or — alanine at position 313 of the amino acid sequence of arabinose isomerase as shown in SEQ ID NO.1 is mutated to glutamic acid; or — valine at position 230 of the amino acid sequence of arabinose isomerase as shown in SEQ ID NO.1 is mutated to leucine and simultaneously alanine at position 313 is mutated to glutamic acid.

3. A gene encoding the arabinose isomerase mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. The recombinant vector according to claim 4, wherein one of the vectors pXMJ19, pMAS5, pHT43, pET-20b(+) or pDXW-10 is selected as the expression vector.

6. A recombinant cell containing the recombinant vector of claim 4.

7. The recombinant cell of claim 6, wherein the recombinant cell is derived from an expression host selected from: Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Saccharomyces.

8. A method for improving the enzyme activity of arabinose isomerase, comprising mutating the amino acid at position 230 and/or position 313 of the amino acid sequence of arabinose isomerase as shown in SEQ ID NO.1.

9. Use of the recombinant vector according to claim 4 or the recombinant cell according to claim 8 in the preparation of the arabinose isomerase.

10. Use of the mutant of claim 1, the gene of claim 3, the recombinant vector of claim 4 or the recombinant cell of claim 6 in converting D-galactose to form D-tagatose.