Ethanol-tolerant bifunctional enzyme capable of degrading carbamide and ethyl carbamate (EC) and application of ethanol-tolerant bifunctional enzyme

A technology of urethane and bifunctional enzymes, applied in the field of bioengineering, can solve the problems of poor ethanol tolerance, far-fetched, limited production capacity, etc., and achieve the effect of high ethanol tolerance

Active Publication Date: 2015-08-05
JIANGNAN UNIV
View PDF1 Cites 13 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are only two EC hydrolases that have been obtained in the literature, and the amino acid sequence has been resolved: one is the Japanese Akutsu-Shigeno Y analyzed an amidase from Rhodococcus equi TB-60 in 2006, but it There is only a very weak degradation effect on EC, and the definition of EC hydrolase is somewhat far-fetched. At the same time, its ethanol tolerance is also very poor, so it cannot be applied to fermented foods such as rice wine; the second is to obtain an a

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Ethanol-tolerant bifunctional enzyme capable of degrading carbamide and ethyl carbamate (EC) and application of ethanol-tolerant bifunctional enzyme
  • Ethanol-tolerant bifunctional enzyme capable of degrading carbamide and ethyl carbamate (EC) and application of ethanol-tolerant bifunctional enzyme
  • Ethanol-tolerant bifunctional enzyme capable of degrading carbamide and ethyl carbamate (EC) and application of ethanol-tolerant bifunctional enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning of gene ureBL encoding ethanol-resistant and efficient degradation of EC urease

[0039] Genomic DNA of Bacillus licheniformis (Bacillus licheniformis 9945A) was extracted according to the instructions of the Bacterial Genome Extraction Kit (OMEGA). According to the urease gene sequence published by NCBI (GenBank: CP005965.1, GI: 521287266-521287273), design forward primer F and reverse primer R (sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively), Forward primer plus BamH I restriction site and its protective base, reverse primer plus Xho I restriction site and its protective base, as follows:

[0040] Forward primer F: 5'-CGCGGATCCGATGCAACTATTACCGCGTGAAGTAG-3'

[0041] Reverse primer R: 5'-CCGCTCGAGTTAAATCCAAAGGTTAAATAAACCC-3'

[0042] The ureBL gene (nucleotide sequence shown in SEQ ID NO.1) is amplified by using the Bacillus licheniformis genomic DNA as a template and the above-mentioned specific primer as a primer. Amplification s...

Embodiment 2

[0043] Embodiment 2: Construction of expression vector and expression system

[0044] The PCR product obtained by the method in Example 1 was digested with BamH I and Xho I, recovered after gel cutting, ligated with the pRSFDuet-1 plasmid digested with BamH I and Xho I, and transformed into E.coli BL21DE3 competent cells , spread on LB solid plates containing 50 μg / ml kanamycin, culture at 37°C for 12 hours, use forward primer F and reverse primer R, and identify positive clones by PCR. The positive clones identified correctly by colony PCR were cultured in LB liquid medium containing 50 μg / ml kanamycin, the plasmid was extracted, and the plasmid was double-enzymatically digested for verification. The correct plasmid was verified by BamH I and Xho I double digestion (such as figure 2 shown) were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Thus, the exogenous expression system BL21(DE3) / pRSFDuet-ureBL of recombinant urease was obtained.

Embodiment 3

[0045] Example 3: Recombinant expression of ethanol-resistant and efficient degradation of urethane urease

[0046] Pick a single colony of genetically engineered bacteria BL21(DE3) / pRSFDuet-ureBL, inoculate it in 25 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it overnight at 37°C with shaking. The next day, transfer to TB medium containing 50 μg / mL kanamycin according to 1% inoculum, and cultivate to bacterial concentration OD 600 When = 0.6, add IPTG to a final concentration of 1 mmol / L for induction, culture at 30°C for 10 h, collect the bacteria by centrifugation, resuspend the cells with 20 mM pH4.5 citric acid-citric acid buffer, break the wall by ultrasonication, and centrifuge to get the supernatant , Determination of enzyme activity, and detection of protein expression by SDS-PAGE. from Figure 4 It can be seen that, compared with the empty vector lane without the target band connected, the induced lane has target bands at 62kDa and 14KDa that ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses an ethanol-tolerant bifunctional enzyme capable of degrading carbamide and ethyl carbamate (EC) and application of the ethanol-tolerant bifunctional enzyme and belongs to the technical field of bioengineering. The enzyme is urease from bacillus licheniformis 9945A and is capable of tolerating high-concentration ethanol and degrading the ethyl carbamate efficiently. The ethanol-tolerant bifunctional enzyme capable of degrading the carbamide and the ethyl carbamate has the advantages that an escherichia coli expression system BL21 (DE3)/pRSFDuet-1 is adopted to achieve efficient expression of urease genes with EC urethanase, the enzyme activity reaches to 2.93U/ml when the EC serves as a zymolyte, and the enzyme activity reaches to 5.81U/ml when the carbamide serves as the zymolyte. Recombinase is retained in 20% ethyl alcohol at the temperature of 37 DEG C for 4 hours, and more than 50% of activity can be retained when the EC serves as the zymolyte. The prepared recombinase can eliminate the EC and the carbamide in yellow wine, lays the foundation for industrial production of the EC urethanase and has great economic and social benefits.

Description

technical field [0001] The invention relates to an ethanol-resistant bifunctional enzyme for degrading urea and ethyl carbamate and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Ethyl Carbamate (Ethyl Carbamate or Urethane, referred to as EC), is a genotoxic and strong carcinogenic substance (can cause lung tumors, lymphoma, liver cancer, skin cancer, etc.), widely present in a variety of fermented foods (such as soy sauce, vinegar, pickles) and alcoholic beverages (such as rice wine, white wine, wine, etc., Japanese sake, brandy). The human body absorbs ethyl carbamate mainly through the consumption of alcoholic beverages and food. Urethane has become a non-negligible factor affecting human health. There is an urgent need for effective methods to eliminate urethane from fermented food or beverages. [0003] There are two main approaches to eliminating EC from fermented foods: [0004] One is to reduce or eliminate...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N9/80C12N15/55C12N15/70C12H1/15
CPCC12H1/003C12N9/80C12Y305/01005C12Y305/01075
Inventor 康振陈坚刘庆涛堵国成
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap