A kind of ethanol-resistant bifunctional enzyme for degrading urea and ethyl carbamate and its application

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...

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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 amide from Lysinibacillus fusiformis transferase, but its ethanol tolerance is poor, there is no activity at high ethanol concentration, and it is also inactive in pH4.5 environment, so it is difficult to realize the application in rice wine

Since the gene sequence of UH has not been effectively analyzed, the traditional method of purifying and producing UH enzyme from wild bacteria is expensive and has limited production capacity, making it difficult to apply it in large-scale commercial applications

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