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Sucrose isomerase mutant with high activity and high conversion rate and application thereof

A technology of sucrose isomerase and high conversion rate, which is applied in the direction of isomerase, application, transferase, etc., can solve the problems of low enzyme utilization rate, limited pace, inevitable by-products, etc., and achieve the improvement of activity and conversion rate , Improve production efficiency, broaden the effect of actual production and application prospects

Pending Publication Date: 2021-07-27
苏州朗邦营养科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of producing isomaltulose with sucrose isomerase, low enzyme utilization and unavoidable by-products are two important unfavorable factors, and because of these two factors, the production cost of isomaltulose remains At a high level, and the high cost also limits the pace of isomaltulose to replace sucrose

Method used

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  • Sucrose isomerase mutant with high activity and high conversion rate and application thereof
  • Sucrose isomerase mutant with high activity and high conversion rate and application thereof
  • Sucrose isomerase mutant with high activity and high conversion rate and application thereof

Examples

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Effect test

Embodiment 1

[0040] A sucrose isomerase mutant Y246L with high activity and high conversion rate, the specific steps of its preparation method are as follows:

[0041] (1) Use the vector pET-28a(+)-pal-2 carrying the sucrose isomerase gene as a template to carry out site-directed mutation to construct the mutant plasmid pET-28a(+)-Y246L, and the mutation primers are as follows (the underline is the mutation point) :

[0042] The forward mutation primer is shown in SEQ ID NO.9, wherein CG TTG CG, the underline is the mutant base;

[0043] The reverse mutation primer is shown in SEQ ID NO.10, wherein GC AAC GC, the underline is the mutant base.

[0044] (2) Perform PCR amplification under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 5 min, 35 cycles, and finally incubation at 68°C for 10 min. PCR amplified products were detected by agarose gel electrophoresis, recovered and purified by ta...

Embodiment 2

[0050] A sucrose isomerase mutant H287R with high activity and high conversion rate, the specific steps of its preparation method are as follows:

[0051] (1) Use the vector pET-28a(+)-pal-2 carrying the sucrose isomerase gene as a template to carry out site-directed mutation to construct the mutant plasmid pET-28a(+)-H287R, and the mutation primers are as follows (the underline is the mutation point) :

[0052] The forward mutation primer is shown in SEQ ID NO.11, wherein GT CGC GT, the underline is the mutant base;

[0053] The reverse mutation primer is shown in SEQ ID NO.12, wherein AC GCG AC, underlined are mutant bases.

[0054] (2) Perform PCR amplification under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 5 min, 35 cycles, and finally incubation at 68°C for 10 min. PCR amplified products were detected by agarose gel electrophoresis, recovered and purified by tappin...

Embodiment 3

[0060] A sucrose isomerase mutant H481P with high activity and high conversion rate, the specific steps of its preparation method are as follows:

[0061] (1) Use the vector pET-28a(+)-pal-2 carrying the sucrose isomerase gene as a template to carry out site-directed mutation to construct the mutant plasmid pET-28a(+)-H481P, and the mutation primers are as follows (the underline is the mutation point) :

[0062] The forward mutation primer is shown in SEQ ID NO.13, wherein TC CCG GT, the underline is the mutant base;

[0063] The reverse mutation primer is shown in SEQ ID NO.14, wherein AG GGC CA, the underline is the mutant base.

[0064] (2) Perform PCR amplification under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 5 min, 35 cycles, and finally incubation at 68°C for 10 min. PCR amplified products were detected by agarose gel electrophoresis, recovered and purified by t...

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Abstract

The invention discloses a sucrose isomerase mutant with high activity and high conversion rate and an application thereof, and belongs to the technical field of gene engineering. The mutants comprise mutants Y246L, H287R and H481P, and belong to the technical field of gene engineering. According to the sucrose isomerase mutant disclosed by the invention, a gene for coding the mutant is obtained by designing a site-directed mutagenesis primer and carrying out site-directed mutagenesis on the gene, and the gene is expressed in an escherichia coli expression system, so that the activity and the conversion rate of the obtained sucrose isomerase mutant are remarkably improved. The sucrose isomerase mutant prepared by the invention is a more efficient biocatalyst, and can improve the production efficiency and reduce the production cost when being applied to industrial production of isomaltulose.

Description

technical field [0001] The invention relates to a sucrose isomerase mutant with high activity and high conversion rate and its application, belonging to the technical field of genetic engineering. Background technique [0002] Isomaltulose, also known as palatinose, has the molecular formula C 12 h 22 o 11 , is a functional disaccharide, which belongs to the isomer of sucrose, and is a reducing sugar, which can be reduced by hydrogenation. Isomaltulose crystals are orthorhombic crystals. When water is present, the relative molecular mass of isomaltulose crystals becomes 360.32, the melting point is 120-128°C, and the water solubility is 250g / L at 25°C. Compared with sucrose, the sweetness is natural and The sweetness is 52%, so it can be used as a new sweetener to replace sucrose. [0003] The US FDA approved isomaltulose as a safe food additive without intake restrictions. Its physical and chemical properties and physiological functions also enable isomaltulose to repl...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N15/77C12N1/21C12P19/12C12P19/24C12R1/19C12R1/15
CPCC12N9/90C12Y504/99011C12N15/70C12N15/77C12P19/12C12P19/24
Inventor 费颖李舒宇程慧君樊启磊
Owner 苏州朗邦营养科技有限公司
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