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Genetically engineered bacterium for expressing sucrose phosphorylase and application of genetically engineered bacterium

A technology of sucrose phosphorylase and genetically engineered bacteria, which is applied in the fields of genetic engineering and enzyme engineering, and can solve the problems of unstable enzyme properties, low substrate concentration, unsuitable amylosucrase, etc.

Active Publication Date: 2017-10-10
金韵
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the unstable nature of the enzyme, the low concentration of the substrate, and the high ratio of donor and acceptor, amylosucrase is not suitable as an industrialized enzyme.

Method used

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  • Genetically engineered bacterium for expressing sucrose phosphorylase and application of genetically engineered bacterium
  • Genetically engineered bacterium for expressing sucrose phosphorylase and application of genetically engineered bacterium
  • Genetically engineered bacterium for expressing sucrose phosphorylase and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Optimization of the gene encoding Leuconostoc mesenteroides sucrose phosphorylase and the construction of the expression vector

[0036] (1) Acquisition of the gene: analyze and optimize the natural SPase gene (1473bp) derived from L. mesenteroides, obtain the gene shown in SEQ ID NO.1, and add two restriction sites (Nde I and Hind III) to the The two ends of the optimized sucrose phosphorylase were synthesized by Shanghai Jierui Bioengineering Co., Ltd. to obtain pGE-sp.

[0037] (2) Construction of expression vectors: the expression vectors pET-24a(+) and pGE-sp were digested with Nde I and Hind III respectively, and ligated with T4ligase. The ligation product was transformed into the expression host Escherichia coli BL21 (DE3) competent cells, and the transformation product was spread on a surface supplemented with 30 μg·mL -1 Grow on Kan's LB plate; Inoculate the normal growth bacteria on the plate to inactivated and add 30μg·mL -1 In Kan's liquid LB med...

Embodiment 2

[0038] Preparation and enzyme activity assay of embodiment 2 sucrose phosphorylase

[0039] (1) Preparation of sucrose phosphorylase

[0040]Inoculate the recombinant bacteria constructed in Example 1 to a concentration of 30 μg·mL -1 In Kan's LB liquid medium, transfer to TB medium (30 μg·mL -1 Kan), after culturing in a shaker at 37°C for 2 hours, IPTG with a final concentration of 0.2mmolL-1 was added to the fermentation broth and the temperature was adjusted to 25°C to induce enzyme production. The fermentation broth was centrifuged (4°C, 8000r / min, 15min) to precipitate the bacteria with 20mM pH7.0Na 2 HPO 4 -NaH 2 PO 4 The supernatant obtained by breaking the wall and centrifuging after buffer reconstitution was the crude enzyme solution.

[0041] from figure 1 It can be found that there is a clear band at 53KDa which is consistent with the size of sucrose phosphorylase, indicating that an optimized sucrose phosphorylase can be obtained.

[0042] (2) Enzyme activ...

Embodiment 3

[0045] The condition research of embodiment 3SPase synthesis α-arbutin

[0046] (1) Effect of pH on the synthesis of α-arbutin by SPase

[0047] Respectively carry out the reaction in the range of initial pH 6.0-8.0, other conditions remain unchanged. The result is given by figure 2 Can get, when pH is 7, hydroquinone (HQ) in the reaction has participated in enzymatic transformation and generates product α-arbutin, and the mass concentration of product is 89gL -1 ; When the pH continued to increase, the conversion rate of HQ began to decrease, and the closer to pH 7.0, the higher the yield of α-arbutin.

[0048] (2) Effect of reaction temperature on the synthesis of α-arbutin by SPase

[0049] Carry out the enzyme conversion reaction in the range of 25-45 ℃ respectively, keeping other conditions unchanged, the results are as follows image 3 As shown, as the temperature increases, the yield of α-arbutin increases, and the maximum conversion rate is obtained at 30-35°C, an...

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Abstract

The invention discloses a genetically engineered bacterium for expressing sucrose phosphorylase and application thereof, and belongs to the fields of gene engineering and enzyme engineering. The genetically engineered bacterium has the advantages that a gene shown in SEQ ID NO.1 is subjected to heterologous expression in escherichia coli, recombinant escherichia coli is constructed, and is fermented to produce recombinant sucrose phosphorylase, and enzyme activity in fermented supernatant reaches 40U.mL<-1>; when the recombinant sucrose phosphorylase is used for catalyzing saccharose and hydroquinone to generate alpha-arbutin, a conversion rate can reach 91%; the industrial application potential is higher.

Description

technical field [0001] The invention relates to a genetically engineered bacterium expressing sucrose phosphorylase and an application thereof, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Sucrose phosphorylase is mainly distributed in bacteria. According to literature reports, there are many production strains of this enzyme, including sources such as L. mesenteroides, Streptococcus mutans, Pseudomonas saccharophila, Bifidobacterium longum, B. adolescentis and B. megaterium. [0003] Sucrose phosphorylase (Sucrose phosphorylase, hereinafter referred to as SPase) can mainly catalyze two types of reactions: one is to transfer phosphorylated glucose as a donor to different substances, and sucrose can be converted and synthesized in this way; the other is The first catalytic method is to transfer the glucose group obtained by enzymatically decomposing sucrose to different types of acceptors, and in this way a variety of n...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N9/10C12P19/44C12R1/19
CPCC12N9/1051C12P19/44C12Y204/01007
Inventor 金韵
Owner 金韵
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