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Bacillus subtilis capable of efficiently expressing Fe<3+>-dependent food-grade acid urease

A technology of Bacillus subtilis and acid urease, applied in the field of bioengineering, can solve the problems of hidden safety hazards and hidden dangers of human health impact, and achieve the effect of improving expression yield

Active Publication Date: 2016-09-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the reported ureases are Ni 2+ Dependent urease, Ni 2+ As a heavy metal, there are also hidden dangers to human health
Therefore, strictly speaking, Ni 2+ Dependent urease is a non-food enzyme, which has potential safety hazards when used in food

Method used

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  • Bacillus subtilis capable of efficiently expressing Fe&lt;3+&gt;-dependent food-grade acid urease
  • Bacillus subtilis capable of efficiently expressing Fe&lt;3+&gt;-dependent food-grade acid urease
  • Bacillus subtilis capable of efficiently expressing Fe&lt;3+&gt;-dependent food-grade acid urease

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Fe 3+ Cloning of dependent food-grade acid urease gene ureBL

[0040] Bacillus licheniformis (Bacillus licheniformis9945a) genomic DNA was extracted according to the instructions of the bacterial genome extraction kit (OMEGA). According to the relevant information of the patent CN201510218597.1, the forward primer BL-F and the reverse primer BL-R were designed (sequences are shown in Table 1, respectively), the forward primer was added with KpnI restriction site, and the reverse primer was added with XhoI enzyme cut site. Using Bacillus licheniformis genomic DNA as a template and the above-mentioned specific primers as primers, the ureBL gene was amplified. Amplification system: Prepare a 50 μl reaction system according to the instructions of the PrimerSTAR HS DNA Polymerase (TAKARA) kit. The amplification conditions were: 98°C for 30s for 1 cycle, 98°C for 10s, 54°C for 5s, 72°C for 5min and 48s, 30 cycles, and 72°C for 10min as a cycle. The PCR product ...

Embodiment 2

[0043] Embodiment 2: Construction of food-grade recombinant engineering bacteria Bacillus subtilis168 (pTTB-ureBL)

[0044] 1) The ureBL fragment recovered in Example 1 (containing the urease gene whose nucleotide sequence is shown in SEQ ID NO: 1) and the plasmid pTTB were double-digested with KpnI and XhoI respectively, and were recovered by T4DNA ligase after gel cutting. Ligation was performed overnight at 16°C, and the ligation product was directly transformed into Bacillus subtilis168.

[0045] 2) After the transformation of the recombinant plasmid, it was spread on LB solid medium containing 10 g / L xylose, cultured statically at 37°C, and identified by colony PCR to obtain positive transformants. Successfully obtained Fe 3+ Dependent food-grade acid urease recombinant strain Bacillus subtilis168 (pTTB-ureBL).

Embodiment 3

[0046] Example 3: Fe 3+ Food-grade high-efficiency expression of dependent food-grade acid urease

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Abstract

The invention discloses bacillus subtilis capable of efficiently expressing Fe<3+>-dependent food-grade acid urease, and belongs to the technical field of biological engineering. Ni-dependent urease has potential safety hazards due to Ni and is a non-food enzyme, and Fe is essential trace elements of a human body and is beneficial for the human body. Accordingly, food-grade efficient expression of the Fe<3+>-dependent food-grade acid urease is successfully achieved in bacillus subtilis 168 (pTTB) through a food-grade bacillus subtilis expression system, and the enzyme activity of the Fe<3+>-dependent food-grade acid urease reaches 5.8 U / mL and reaches 1.3 U / mL when EC is taken as a substrate; compared with an original bacillus licheniformis strain, the expression yield of the food-grade urease is greatly increased, and a foundation is laid for industrialized application of the food-grade acid urease.

Description

technical field [0001] The present invention relates to a kind of efficient expression Fe 3+ The invention relates to a food-grade acid urease-dependent Bacillus subtilis, which belongs to the technical field of bioengineering. Background technique [0002] Urease (Urease, EC3.5.1.5), widely present in animals, plants, bacteria, fungi, etc., can specifically catalyze the hydrolysis of urea to produce two molecules of ammonia and one molecule of carbonic acid. Urease is the first crystalline enzyme successfully obtained in the world. It was first extracted from sword bean by Summer in 1926. According to the optimum pH value of urease action, urease can be divided into acid urease, neutral urease and alkaline urease. [0003] Ethyl Carbamate (Ethyl Carbamate or Urethane, referred to as EC), has genotoxicity and carcinogenicity, and is widely present in a variety of fermented foods (such as soy sauce, vinegar, pickles) and alcoholic beverages (such as rice wine, white wine, w...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N9/80C12R1/125
CPCC12N9/80C12N15/75C12N2800/101C12Y305/01005
Inventor 康振陈坚堵国成刘庆涛杨森
Owner JIANGNAN UNIV
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