Efficient preparation method and applications of food-grade acid ureases

An acid urease, food-grade technology, applied in the field of high-efficiency expression of acid urease, can solve problems such as limited application, and achieve the effect of solving residual problems and high-efficiency expression

Inactive Publication Date: 2014-02-12
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, many research groups have screened and obtained a variety of acid urease-producing strains from nature. Studies have shown that the strains are mostly Enterobacter, Helicobacter pylori, and Klebsiella pneumoniae. Since these bacteria are mostly pathogenic bacteria or opportunistic pathogenic bacteria, thus limiting its further application

Method used

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  • Efficient preparation method and applications of food-grade acid ureases
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  • Efficient preparation method and applications of food-grade acid ureases

Examples

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Embodiment 1

[0066] Embodiment 1: Cloning of acid urease gene

[0067] Genomic DNA of Lactobacillus reuteri CICC6124 was extracted according to the instructions of the Bacterial Genome Extraction Kit (OMEGA), and stored at -20°C for future use. According to the acid urease gene sequence published by NCBI (GenBank: AAPZ02000001.1, GI: 194454092-194454098), primers P1-P2 were designed to amplify the acid urease gene using Lactobacillus reuteri DNA as a template. The amplification conditions were as follows: 95°C, 5min, 1 cycle; 95°C, 30s, 55°C, 30s, 72°C, 5min30s, 30 cycles; 72°C, 5min, 1 cycle; 12°C, 5min, 1 cycle. Amplification system: Prepare a 50 μL system according to the instructions of the PrimeSTAR HS DNA Polymerase (TAKARA) kit. PCR products were recovered by 1% agarose gel electrophoresis, and the recovery method was carried out according to the instructions of the gel recovery kit (Thermo). The target gene obtained by PCR amplification was connected to the pMD19T-simple (TAKARA)...

Embodiment 2

[0068] Example 2: Construction of recombinant lactic acid bacteria and high expression of acid urease

[0069] (1) The pMD19T-ureLR plasmid obtained by cloning in Example 1 was double-digested with NcoI and SacI restriction endonucleases (Thermo), and the target fragment was recovered by agarose gel electrophoresis according to the method in Example 1 after digestion . Ligate the recovered fragment of interest to the recovered Lactococcus lactis expression plasmid pNZ8148 after digesting with the same restriction enzymes (NcoI and SacI), transform into E.coil MC1061 competent cells, and spread it on a medium containing 10 μg / mL chloramphenicol On the prime LB plate, after some colonies grow, randomly pick a few colonies, use P3-P4 as primers, and identify positive clones by PCR, inoculate the correct colonies in LB liquid after colony PCR verification, and extract plasmids for sequencing verification after cultivation. The acid urease plasmid that was successfully ligated was...

Embodiment 3

[0078] Embodiment 3: expression and purification preparation of food-grade acid urease

[0079] Fermentation experiments were carried out on the successfully constructed recombinant Lactococcus lactis strain NZ3900 (pNZ8149-UreLR). Seed liquid preparation: Inoculate the experimental strains into LM17 medium, fill 20 mL of liquid in a 50 mL shake flask, and culture at 30 ° C 16h; 1.5L medium in 3L fermenter (5% lactose, 1.5% peptone, 1% yeast extract, 1mM MgSO 4 , 0.1 mM MnSO 4 , 2.0mM NiCl 2 ), at a temperature of 30°C, cultured statically until the cell OD 600 1.0, add the inducer Nisin (final concentration 10ng / mL), induce the expression for 27h, and the enzyme activity reaches 10200U / L (such as image 3 shown). After collecting the bacteria by centrifugation, wash the bacteria twice with 300mL sterile distilled water, resuspend the bacteria in 200mL of 10mM Tris-HCl (pH7.4), 1mM EDTA solution, add 10mg of lysozyme, and treat in a water bath at 37°C for 3h, 300w, Sonica...

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Abstract

The invention discloses an efficient preparation method and applications of food-grade acid ureases, belonging to the technical field of biological engineering. The efficient expression and preparation methods and applications of acid ureases are realized successfully by using food-grade lactococcus lactis expression system, the enzyme activity of the acid ureases reaches 10200 U/L, and the enzyme activity of purified acid ureases is 325.6 U/mg. The specific enzyme activity of purified enzymes to an urethane substrate reaches 243.4 U/mg, and food-grade acid ureases which are prepared by using the method disclosed by the invention and added in a yellow rice or millet wine can efficiently degrade urea (the degradation rate per two days is 100%) and urethane (the degradation rate per three days is 72% %). Recombined acid ureases prepared by using the method disclosed by the invention can be applied to the removal of urea and urethane in fermented food, so that the residue problem of urethane in fermented food can be efficiently solved. The efficient preparation method and applications of food-grade acid ureases disclosed by the invention lay a foundation for the implementation of the industrialized production of food-grade acid ureases.

Description

technical field [0001] The invention relates to a method for efficiently expressing acid urease in lactic acid bacteria, and belongs to the technical field of bioengineering. Background technique [0002] Urease (Urease, EC3.5.1.5), also known as aminohydrolase, widely exists in animals, plants, bacteria, fungi, etc. in the biological world. It can specifically catalyze the hydrolysis of urea to produce two molecules of ammonia and one molecule of carbonic acid. Since Summer extracted crystalline urease from sword bean for the first time in 1926, various countries have conducted extensive research on urease. According to the optimum pH value of urease action, urease can be divided into acid urease, neutral urease and alkaline urease. [0003] After entering the 1980s, studies have confirmed that in the fermentation production of alcohol (such as rice wine in my country), the urea produced by arginine metabolism in yeast cells is secreted outside the cell due to the sequence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/74C12H1/15A23L1/015A23L5/20
CPCC12H1/14C12N9/80C12N15/746C12Y305/01005
Inventor 陈坚堵国成康振杨宇清
Owner JIANGNAN UNIV
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