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High-heat-resistance D-psicose-3-epimerase mutant and application thereof

A technology of epimerase and psicose, applied in the field of genetic engineering, can solve the problems of poor thermal stability, limited industrial application of D-psicose, low catalytic activity of D-fructose, etc., and achieve thermal stability. performance and catalytic activity improvement, important industrial application value, excellent thermal stability and catalytic activity

Pending Publication Date: 2022-06-07
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, at least 20 DAEases have been screened and identified, but most of them have low catalytic activity to D-fructose and poor thermal stability. Production requires a higher temperature to react, which greatly limits the industrial application of D-psicose produced by biological enzymes
[0004] Patent 202010790290X discloses a DAEase mutant V105I whose enzymatic activity is 150% of that of the original parent, but the half-life of the mutant at 70°C is t 1 / 2 The value is only 0.5h. Compared with the general temperature requirement of 60-70°C for industrial catalysis, its thermal stability is still lacking, resulting in a sharp decline in its activity and stability during high-temperature catalysis.

Method used

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  • High-heat-resistance D-psicose-3-epimerase mutant and application thereof
  • High-heat-resistance D-psicose-3-epimerase mutant and application thereof
  • High-heat-resistance D-psicose-3-epimerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation of SfDAE enzyme mutants

[0028] (1) Construction of recombinant plasmid pET-22b(+)-SfDAE

[0029] According to the amino acid sequence of wild-type DAEase (SfDAE) of Sinorhizobium fredii CCBAU 83666 (accession number: ASY72161.1), its gene sequence (shown in SEQ ID NO: 2) was synthesized and ligated into the E. coli expression vector pET-22b(+). The recombinant plasmid pET-22b(+)-SfDAE was obtained between the restriction sites Nde I and BamH I.

[0030] (2) Construction of mutant expression vector

[0031]Using the recombinant plasmid pET-22b(+)-SfDAE obtained in step (1) as a template, a mutation primer for point mutation is designed, and then a site-directed mutation PCR amplification reaction is performed to construct a mutation expression vector.

[0032] The mutation primers included the following 4 pairs of primers: M15G-F / R, P40S-F / R, M245T-F / R and A254C-F / R. Wherein, M15G-F upstream primer and M15G-R downstream primer are shown as SEQ...

Embodiment 2

[0046] Example 2: Enzyme activity and thermostability assay

[0047] 1. Relative enzyme activity assay of wild-type SfDAE and enzyme mutants

[0048] Under standard reaction conditions (1 x PBS buffer (pH 7.4), 1 mM MgCl 2 , 10g / L D-fructose, 1μM pure enzyme, react at 70°C for 5min), the relative enzyme activity of the mutant is 100% of the wild-type enzyme activity, and the purified mutant is determined with reference to the aforementioned HPLC method for detecting the enzyme reaction system and the relative enzyme activity of the wild type, the results are as follows figure 1 As shown, the relative enzyme activities of the single point mutant enzymes M15G, P40S, M245T, A254C and the four point mutant M15G / P40S / M245T / A254C were 95%, 115%, 120%, 97% and 130% of the wild type, respectively ( figure 1 shown), in which the mutant M15G / P40S / M245T / A254C greatly improved the catalytic activity of fructose, and has great industrial application value.

[0049] 2. Thermostability a...

Embodiment 3

[0051] Example 3: Preparation of D-psicose using wild-type SfDAE and four point mutants

[0052] Add 500 mL of 50% D-fructose, 1 mM MgCl to a 1 L Erlenmeyer flask 2 , and 10 μM enzyme solution, react in a water bath at 70°C, take samples at different times, and finally terminate the reaction by boiling for 10 min, and detect the D-psicose concentration in the reaction product by HPLC. The yields of SfDAE mutants and quadruple mutants were shown in Table 3 by converting D fructose to D-psicose at different reaction times. After 1 h of reaction, the conversion rate of the wild type was 20.6%. The conversion rate of M245T / A254C was 33.3%, which was 1.6 times that of the wild type, and the yield of D-psicose reached 167 g / L.

[0053] Table 3. Productivity of D-psicose catalyzed by wild-type SfDAE enzyme and four-point mutant enzyme

[0054]

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Abstract

The invention provides a high-heat-resistance D-psicose-3-epimerase mutant as well as a preparation method and application of the high-heat-resistance D-psicose-3-epimerase mutant. The mutant is obtained by taking a wild type DAEase derived from a strain Sinorhizobium fredii as a parent and carrying out site-directed mutagenesis modification on a key site amino acid residue. Compared with existing similar enzymes, the thermal stability and catalytic activity of the high-heat-resistance D-psicose-3-epimerase mutant are further remarkably improved, the half-life period (t1 / 2) value at 65 DEG C of the obtained mutant is increased to 12.0 h compared with 4.0 h of wild enzymes, the half-life period (t1 / 2) value at 70 DEG C of the obtained mutant is increased to 4.5 h compared with 1.0 h of the wild enzymes, and the high-heat-resistance D-psicose-3-epimerase mutant has the advantages that the high-heat-resistance D-psicose-3-epimerase mutant can be used for preparing the high-heat-resistance D-psicose-3-epimerase mutant. The appropriate reaction temperature is 70 DEG C, the relative enzyme activity is 130% of that of a wild type, and the method has important industrial application value.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering, in particular to a mutant of high heat-resistant D-psicose-3-epimerase and its application. Background technique: [0002] In today's society where the concept of healthy diet is deepening, D-allulose, a safe and stable low-calorie drug, has attracted extensive attention of researchers because of its unique physiological functions. D-psicose is an important member of the rare sugar family. It exists only in a few plants and some bacteria, and the content is very low. It is a C-3 epimer of D-fructose. Due to its physiological properties It is becoming a potential functional ingredient for the pharmaceutical, health care and food industries. D-psicose has higher sweetness and lower energy, and is considered as an ideal sweetener and an effective substitute for sucrose, it has 70% sweetness of sucrose, but only contains about 0.2 calories kcal / g. In addition, D-psicose can increase the water h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N15/75C12N1/21C12P19/02C12P19/24C12R1/125C12R1/19
CPCC12N9/90C12Y501/03C12N15/70C12N15/75C12P19/02C12P19/24
Inventor 秦慧民路福平李超
Owner TIANJIN UNIV OF SCI & TECH