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Preparation method and application of endo-inulinase

An inulin endonuclease and Escherichia coli technology, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of difficult separation of internal and external inulinase, complicated processes, high cost, etc., and achieves improved industrialization efficiency, short fermentation time, and high processing efficiency. handy effect

Inactive Publication Date: 2016-04-20
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the complex process of extracting endo-inulinase directly from wild fungi, the extraction rate is low, it is difficult to separate the endo-inulinase and the cost is high, and it is currently an important task to find a cheap and efficient exogenous gene expression method. Research Hotspots of Powder Enzyme

Method used

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  • Preparation method and application of endo-inulinase
  • Preparation method and application of endo-inulinase
  • Preparation method and application of endo-inulinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1E

[0044] The construction of embodiment 1 EscherichiacoliBL21pET28a-pelB-NSinu2 genetically engineered bacteria

[0045] 1. Amplification and codon optimization of the endo-inu2 gene sequence of Aspergillus fig ATCC16882

[0046] According to the endo-inu2 gene sequence of Aspergillus fig ATCC16882 (GenBank: AJ006951.1), a pair of primers for amplifying the inu2 gene were designed with SnapGene, and the inu2 sequence encoding endo-inulinase was cloned from the whole genome of Aspergillus figus and sent Completed the codon optimization of the inu2 sequence at Nanjing GenScript Biotechnology Co., Ltd.

[0047] (1) Extraction of Aspergillus fig genome DNA

[0048] a) Aspergillus ficuum ATCC16882 preserved in a glycerol cryopreservation tube was streaked and activated, inoculated with spores of Aspergillus ficuum and cultured in 50 mL potato sucrose medium at 24°C for 2 to 3 days;

[0049] b) The freshly cultured Aspergillus figum cells were collected in a sterile centrifuge tube,...

Embodiment 2

[0103] Embodiment 2 EscherichiacolipET-28a-pelB-NSinu2 Genetic Engineering Bacteria Expression

[0104] 1. IPTG induces the expression of EscherichiacolipET-28a-pelB-NSinu2 genetic engineering bacteria

[0105] Utilize recombinant Escherichia coli to express exogenous protein and use IPTG as an inducer, and the fermentation medium contains the following components: peptone 12g / L, yeast powder 24g / L, K 2 HPO 4 72mmol / L, MgSO 4 10mmol / L, thiamine 0.034g / L, kanamycin sulfate 0.03g / L, trace elements (CaCl 2 ·6H 2 O0.74g / L, ZnSO 4 ·7H 2 O0.18g / L, MnSO 4 ·H2O20g / L,Na 2 EDTA20.1g / L, CuSO 4 0.1g / L, CoCl 2 0.104g / L,FeSO 4 ·7H 2 (2g / L) 2mL / L.

[0106] (1) Pick a single colony of the recombinant bacteria and culture it overnight in LB medium, and use the bacterial sample containing the empty vector and E.coliBL21(DE3) as a control;

[0107] (2) The next day, the experimental group and the control group were inoculated into fresh 50mL fermentation medium according to the volu...

Embodiment 3

[0124] Example 3 Effects of Different Induction Timings, Different IPTG Concentrations, Different Induction Times and Different Induction Temperatures on the Secretion and Expression of Endo-inulinase Recombinant Protein

[0125] The expression conditions of genetically engineered bacteria were optimized through further research on induction timing, IPTG induction concentration, induction time and induction temperature.

[0126] 1. The influence of different induction timing

[0127] (1) Inoculate 1% inoculum of glycerol cryopreservation tube bacterial solution in LB medium overnight;

[0128] (2) The next day, the seed solution was inoculated into fresh 50mL fermentation medium according to the volume ratio of 1%, and cultured with shaking at 37°C and 200r / min;

[0129] (3) 1h after inoculation (OD 600 About 0.1), 2h (OD 600 About 0.26), 3h (OD 600 About 0.7), 4h (OD 600 About 2.0), 5h (OD 600 About 3.1), 6h (OD 600 About 3.9) Add IPTG to a final concentration of 0.5mm...

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Abstract

The invention discloses a preparation method and application of endo-inulinase. An endo-inu2 gene of Aspergillus ficuum ATCC 16882 serves as the source sequence, the gene sequence is optimized through the gene engineering technology, the optimized gene sequence of encoded endo-inulinase is cloned to a carrier pET-28a(+), and recombined Escherichia coli BL21pET28a-pelB-NSinu2 for efficiently secreting and expressing endo-inulinase is established. Recombined Escherichia coli serves as a fermenting strain, endo-inulinase is efficiently secreted and expressed, and finally prepared endo-inulinase is purified and successfully used for hydrolyzing inulin to prepare fructo-oligose.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a method for efficiently preparing endo-inulinase after expressing an endo-inulinase gene optimized by genetic engineering technology through recombinant Escherichia coli and its application. Background technique [0002] Inulin is a linear straight-chain polysaccharide composed of β-2,1-glucosidic bonds connecting D-fructofuranose molecules, often with a glucose at the end, and the degree of polymerization is usually between 2 and 60. It usually exists in chicory, Jerusalem artichoke and In the root tubers of plants such as yacon. Inulinase is a class of hydrolase that can hydrolyze β-2,1-D-fructan glycosidic bonds, scientific name β-2,1-D fructan hydrolase (EC3.2.1), also known as β-fructan Glycanase, 2,1-D-fructan hydrolase. Microbial inulinase can catalyze the hydrolysis of inulin in plants into fructose or fructo-oligosaccharides. Inulinase...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/70
CPCC12N9/2402C12Y302/01007C12N2800/101C12N2800/22
Inventor 姜岷王培培戴仲雪马江锋吴昊
Owner NANJING UNIV OF TECH
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