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A genetically engineered bacteria producing sucrose phosphorylase

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 low expression level, low yield of sucrose phosphorylase, and difficulty in meeting the requirements of industrial applications.

Active Publication Date: 2021-03-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, sucrose phosphorylase is mainly distributed in bacterial microorganisms, and a small amount is distributed in plant cells. The enzyme is mainly obtained through biological fermentation. The complex metabolic regulation mechanism in wild-type strains makes the yield of sucrose phosphorylase low. Type strains ferment and produce sucrose phosphorylase, which is difficult to meet the requirements of industrial applications
Existing fermentation production of sucrose phosphorylase by constructing genetically engineered bacteria, heterologous expression of sucrose phosphorylase in Escherichia coli and Bacillus subtilis has been reported in the literature, but there are problems such as low expression level and low enzyme activity

Method used

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  • A genetically engineered bacteria producing sucrose phosphorylase
  • A genetically engineered bacteria producing sucrose phosphorylase
  • A genetically engineered bacteria producing sucrose phosphorylase

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Experimental program
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Effect test

Embodiment 1

[0023] The construction of embodiment 1 Escherichia coli SPLO1 genetically engineered bacteria

[0024] 1. Primer design

[0025] According to the SPase gene sequence (GenBank Accession NO.D90314) of Leuconostoc mesenteroides ATCC12291, a pair of primers for amplifying the SPase gene were designed with DNAMAN, NcoI and XhoI restriction enzyme sites (underlined) were introduced respectively, and protective bases were added. The upstream and downstream primers are as follows:

[0026] Upstream primer: 5'-AATTACCG CCATGG ATGGAAATTCAAAACAAAGC-3'

[0027] Downstream primer: 5'-AATTACCG CTCGAG TTAGTTCTGA GTCAAATTAT C-3’

[0028] 2. PCR amplification of Leuconostoc enteromenis SPase fragment

[0029] ⑴ Leuconostoc enterolis genome DNA extraction

[0030] ① Streak and activate Leuconostoc mesenteroides ATCC 12291, inoculate a single colony in 5ml MRS medium and culture overnight at 30°C;

[0031] ②Collect 1 mL of the bacterial solution by centrifugation to collect the bacteri...

Embodiment 2

[0040] The preparation of embodiment 2 recombinant sucrose phosphorylase

[0041] Inoculate a single colony of the recombinant strain in the LB liquid medium containing Kana, cultivate overnight at 37°C, 200r / min, and insert it into the TB liquid medium containing Kana at 37°C, 200r / min Min culture to bacterial density OD 600 After reaching 0.6, add IPTG inducer and induce at 25°C, 200r / min for 24h, then collect the bacterial liquid. Centrifuge the bacterial liquid in a low-temperature refrigerated centrifuge at 7000 r / min at 4°C for 15 minutes to collect the bacterial cells. Bacteria use 50mmol / L K 2 HPO 4 / KH 2 PO 4 The bacterial cells were collected after washing twice with buffer solution (pH6.5). Add the collected wet bacteria to 50mmol / L K 2 HPO 4 / KH 2 PO 4 Buffer solution (pH 6.5) was used to prepare bacterial suspension, placed on ice and fixed, and then the bacterial cells were disrupted by ultrasonic waves. Ultrasonic breaker working time: 2s, intermitten...

Embodiment 3

[0043] The enzyme activity assay of embodiment 3 recombinant sucrose phosphorylase

[0044] In phosphate buffer, sucrose phosphorylase can catalyze sucrose and inorganic phosphoric acid to generate glucose-1-phosphate and D-fructose. The sucrose hydrolysis reaction can be carried out first, and then the content of the generated D-fructose can be detected by DNS to determine the sucrose phosphate [Choi H.C.,Seo D.H.,Jung J.H.,et al.Developmemt of new assay for sucrose phosphorylase and its application to the characterization of Bifidobacterium longumSJ32 sucrose phosphorylase[J].Food Science and Biotechnology,2011,20(2):813 .].

[0045] The enzyme activity determination method refers to [Wu Jing, Wu Dan, Zhu Jie, et al. A recombinant Bacillus subtilis expressing sucrose phosphorylase derived from L. mesenteroides. Chinese invention patent application, application number: 201710637427.6, publication number: CN107236696A]. The assay steps include: 500 μL of 5% sucrose solution, ...

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Abstract

The invention discloses a gene engineering bacterium for producing sucrose phosphorylase, and belongs to the technical field of gene engineering and enzyme engineering. The nucleotide sequence shown in SEQ ID NO.2 is converted into escherichia coli BL21(DE3) through a recombinant plasmid pET-28a-SPase constructed with pET-28a as a carrier, recombinant escherichia coli is constructed, recombinant sucrose phosphorylase is produced through fermentation, and an escherichia coli strain for producing the high-yield sucrose phosphorylase is obtained, wherein the enzyme activity of fermentation intracellular wall-breaking supernatant is 504.19 U / ml, and the specific enzyme activity is 145.19 U / mg. The stable enzyme production performance of the strain can lay a foundation for further theoretical research and practical production application, and the strain has great practical application significance.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing sucrose phosphorylase, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7, 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, such as D-fructose as an acceptor Another catalytic method is to transfer the glucose group obtained by decomposing sucrose by sucrose phosphorylase to different types of acceptors, such as inorganic phosphoric acid, substances containing phenolic hydroxyl groups, alcoholic hydroxyl groups and carboxyl groups, and catalyze the synthesis of various glycosides . [0003] Sucrose phosphorylase is mainly distributed in bacteria. According to literature reports, the enzyme mainly exists in microorganisms such as Leuconostoc mesenteroides, Strept...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/10C12R1/19
CPCC12N9/1051C12N15/70C12Y204/01007
Inventor 陈献忠李晓玉沈微
Owner JIANGNAN UNIV
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