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Construction and application of ureido amidohydrolase display strain

A technology of ureidoamide and hydrolase, applied in the direction of hydrolase, microbial-based methods, polypeptides containing positioning/targeting motifs, etc., can solve the problems of dissociation, instability, etc., and achieve the effect of wide application prospects

Pending Publication Date: 2021-02-09
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Claims
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AI Technical Summary

Problems solved by technology

The pir protein is covalently bound to the cell wall through an ester bond, but is unstable and easily dissociated from the cell wall by a weak base

Method used

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  • Construction and application of ureido amidohydrolase display strain
  • Construction and application of ureido amidohydrolase display strain
  • Construction and application of ureido amidohydrolase display strain

Examples

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Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Construction of cwp2 as an anchor protein displaying UA recombinant strain (ACCD)

[0047] The starting strain used in this example is Saccharomyces cerevisiae CICC32315. The YPD medium is a universal complete medium, and the solid medium contains 2% imported agar powder.

[0048] The main construction process of the strain is as follows:

[0049] (1) Construction of Yep352-PGK-Cas9-gRNA-KAN plasmid

[0050] The recombinant plasmid Yep352-PGK-Cas9-gRNA-KAN of the CAR1 gene locus was constructed using Yep352-PGK-Cas9-KAN as the base plasmid. The construction process is as follows: figure 1 shown. Using the Yep352-PGK-Cas9-KAN plasmid as a template, respectively, using primer SNR52 p -F (SEQ ID NO: 8) and SNR52 p -R (SEQ ID NO: 9) PCR amplification to obtain SNR52 p fragment and using primer SUP4 t -F (SEQ ID NO: 10) and SUP4 t -R (SEQ ID NO: 11) was amplified to obtain SUP4 t Gene, the 20bp guide sequence that guides Cas9 protein cleavage in the CAR1 ...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to breeding of industrial microorganisms, in particular to a strain construction method for displaying ureido amidohydrolaseon the surface of saccharomyces cerevisiae cells by taking cwp2 as anchoring protein and application. The ACCD display strain is constructed by fusing the cwp2 anchoring protein and target protein UA, so that the urea degradation rate of yellow rice wine fermentation liquor reaches 89.9% or above, and the EC degradation rate is 56.4% or above; when the ACCD strain is cultured for 72 hours, the enzyme activity of about 12,000 U / g (stem cells) can be achieved, and compared with commercial urease, displayed enzyme has higher thermal stability and tolerance; and when the ACCD is used as an enzymepreparation to treat the yellow rice wine fermentation liquor, the urea degradation rate and EC degradation rate are highest, the initial urea degradation rate can still be kept at 80% or above afterthe ACCD is repeatedly used ten times, and flavor substances of the yellow rice wine are not obviously changed before and after treatment. Therefore, the ACCD display enzyme strain has a wide marketprospect.

Description

technical field [0001] Construction and application of a ureido amidohydrolase display strain for reducing urea and ethyl carbamate in rice wine fermentation broth. Background technique [0002] Ethyl carbamate (abbreviated as EC) widely exists in various fermented foods and beverages, and has been classified as a 2A carcinogen, which is potentially harmful to the human body. Along with the raising of people's living standard, alcoholic beverages have become an indispensable part in life. On June 15, 2016, the Hong Kong Consumer Council announced the test results of 36 alcoholic beverages. 34 of the 36 alcoholic beverages contained EC, and the EC content in rice wine was relatively high. The high attention of the industry has also pushed the food safety issue of rice wine to the forefront. Therefore, effectively reducing the ethyl carbamate in rice wine ensures food safety and maintains the health of consumers, ensures the stability of the rice wine market, and promotes th...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N11/16C12N9/00C12N9/80C07K19/00C12G3/022C12H1/15C12Q1/58C12R1/865
CPCC12N9/78C12N9/93C12N9/80C12Y305/03001C12Y305/01054C12Y603/04006C12N15/81C12N11/16C12G3/022C12H1/003C12Q1/58C07K2319/02C07K2319/035
Inventor 陈叶福邓庆博王欢江森李蕊蕊张果石文琪武晓乐郭学武肖冬光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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