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Sucrose isomerase mutant with improved thermal stability and catalytic efficiency

A technology of sucrose isomerase and mutants, which is applied in the fields of genetic engineering and enzyme engineering, and can solve problems such as reduced catalytic efficiency

Active Publication Date: 2015-07-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The thermal stability and secretion efficiency of the obtained mutants K576D and K576P have been improved to a certain extent (the half-lives of K576D and K576P at 45°C are 1.8 and 1.4 times that of the wild enzyme, respectively; the extracellular secretion efficiency of the two mutants during shake flask fermentation 4.6 and 4.7 times that of natural enzymes, respectively), but the catalytic efficiency has decreased to varying degrees

Method used

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  • Sucrose isomerase mutant with improved thermal stability and catalytic efficiency
  • Sucrose isomerase mutant with improved thermal stability and catalytic efficiency
  • Sucrose isomerase mutant with improved thermal stability and catalytic efficiency

Examples

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Embodiment 1

[0029] Example 1: Preparation of site-directed mutants of sucrose isomerase

[0030] (1) Construction of sucrose isomerase site-directed mutants

[0031] Two site-directed mutants of sucrose isomerase E175N and E175N / K576D from S. plymuthica:

[0032] In the present invention, with the highest similarity Protaminobacter rubrum CBS 574.77 sucrose isomerase (SmuA) crystal structure (PDB ID: 3GBD) as template, S.plymuthica AS9 sucrose isomerase (PalI AS9) was constructed by EMBI-EBL online server ) of the three-dimensional simulation structure ( picture 1). According to the amino acid primary sequence alignment, there is only one amino acid difference between SmuA and PalI AS9, and the similarity reaches 99.86%. Therefore, it can be considered that PalI AS9 has almost the same three-dimensional structure and B factor parameters of SmuA. Based on the above analysis, 6 amino acid residues with higher B factors in PalI AS9 were selected ( Table 1 ), which were then replaced wi...

Embodiment 2

[0063] Embodiment 2: enzyme activity analysis method

[0064] 1) Enzyme activity assay method

[0065] The enzyme activity of sucrose isomerase was determined by 3,5-dinitrosalicylic acid method (DNS method). Sucrose isomerase catalyzes sucrose to generate isomaltulose, and a small amount of trehalulose, glucose and fructose under certain conditions. Isomaltulose is a reducing sugar, sucrose is a non-reducing sugar, and 3,5-dinitrosalicylic acid is reduced to a brown-red amino complex after co-heating with a reducing sugar solution. The depth is proportional to the amount of reducing sugar, so colorimetry can be performed at a wavelength of 540nm to calculate the enzyme activity. Definition of enzyme activity unit: under the above conditions, the amount of enzyme that releases 1 μmol of isomaltulose per minute at the initial stage of the reaction is regarded as an activity unit.

[0066] Enzyme activity assay steps:

[0067] A. Preheating: Take 1.8ml of 10% sucrose solutio...

Embodiment 3

[0070] Example 3: Temperature Optimum and Thermostability of Sucrose Isomerase Mutants

[0071] Using citric acid-disodium hydrogen phosphate (50 mM) at pH 6.0 as buffer solution, the optimum temperature of native sucrose isomerase and mutants was determined in the temperature range from 20 to 50°C. Such as picture 4(a), the optimum temperature of E175N and E175N / K576D is 35°C, which is 5°C higher than that of natural enzymes; E175N and E175N / K576D retain 80.5% and 86.8% of their activities at 40°C, respectively, which are higher than that of natural enzymes. Natural enzymes retain higher activity at 40°C.

[0072] Dilute the purified natural sucrose isomerase and mutants with 50mM pH 6.0 citric acid-disodium hydrogen phosphate buffer to a protein content of 0.25mg / mL and a pH of 6.0, and place them in a constant temperature water bath at 45°C, every 20min Take a sample once, measure its residual enzyme activity, and compare its stability, such as picture 4(b). The half-...

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Abstract

The invention discloses a sucrose isomerase mutant with improved thermal stability and catalytic efficiency and belongs to the technical fields of genetic engineering and enzyme engineering. According to the sucrose isomerase mutant with improved thermal stability and catalytic efficiency, site-specific mutagenes is performed on the amino acid with relatively high B factor in the sucrose isomerase crystal structure, namely mutating E at the 175 site of the sucrose isomerase into N; at the same time, E at the 175 site is further mutated into N on basis of the mutant K576D, so that two sucrose isomerase mutants with both improved thermal stabilities and catalytic efficiencies are obtained. Compared with the natural sucrose isomerase, the half life periods of the mutant E175N and the mutant E175N / K576D disclosed by the invention are respectively improved by 130% and 665% at 45 DEG C and the catalytic efficiencies Kcat / Km are respectively improved by 38% and 19%. Compared with natural enzymes, when catalysis is performed on sucrose by the sucrose isomerase mutant to produce isomaltulose, the maximum isomaltulose conversion rates of the mutants are respectively improved by 1.8% and 1.6%. Compared with natural sucrose isomerases, the two mutants obtained by the invention are more suitable for industrial applications.

Description

technical field [0001] The invention relates to a sucrose isomerase mutant with improved thermal stability and catalytic efficiency, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Sucrose isomerase (EC 5.4.99.11), also known as isomaltulose synthase and sucrose mutase, is an isomerase with high industrial application value, belonging to the α-amylase 13 family, which can efficiently catalyze isomerase Isomaltulose and trehalulose can be produced by forming sucrose, and subsequent hydrogenation of isomaltulose can produce isomaltulose. Isomaltulose and isomaltulose have extremely high value in medicine. Sucrose is similar, especially the two are not easy to cause the rise of human blood sugar, which is very suitable for diabetic patients. Compared with the synthesis of isomaltulose by chemical method, the enzymatic conversion of sucrose by using sucrose isomerase has the advantages of high conversion rate, short...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/63C12N1/21C12N1/19C12N1/15C12P19/12C12P7/26
CPCC12N9/90C12P7/26C12P19/12C12Y504/99011
Inventor 吴敬段绪果程胜
Owner JIANGNAN UNIV
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