Enzyme Composition, Product and Its Application in Preparing Ectoine

Abstract

The present invention relates to the field of bioengineering, and particularly to an enzyme composition, a product and their application in the preparation of ectoine. The present invention provides an enzyme composition, comprising: a mutant of 2-hydroxy-4-aminobutyric acid oxidase, a mutant of keto transaminase, a mutant of hydrogen peroxide hydrolase, a mutant of ectoine synthase, a mutant of pyruvate-CoA oxidase, and Lactobacillus helveticus lactate dehydrogenase. The present invention for the first time uses the combined transformation of multiple mutant enzymes to rapidly and efficiently produce ectoine with inexpensive 2-hydroxy-4-aminobutyric acid as the starting material; because the enzymatic method has characteristics such as being easy to control, short transformation time, and few generated impurities, this method has unique advantages in the inexpensive large-scale production of ectoine.

Claims

1. An enzyme composition, characterized in that include: mutants of 2-hydroxy-4-aminobutyrate oxidase, mutants of ketotransaminase, mutants of hydrogen peroxide hydrolase, mutants of ectoine synthetase, mutants of pyruvate-CoA oxidase, and Lactobacillus helveticus lactate dehydrogenase; The amino acid sequence of the mutant of 2-hydroxy-4-aminobutyrate oxidase is shown in SEQ ID NO: 1; The amino acid sequence of the mutant of the ketotransaminase is shown in SEQ ID NO: 2; The amino acid sequence of the mutant of the hydrogen peroxide hydrolase is shown in SEQ ID NO: 3; The amino acid sequence of the mutant of the ectoine synthase is shown in SEQ ID NO:4; The amino acid sequence of the mutant of pyruvate-CoA oxidase is shown in SEQ ID NO:5; The amino acid sequence of the Lactobacillus helveticus lactate dehydrogenase is shown in SEQ ID NO:

6.

2. An immobilized enzyme, characterized in that include: The enzyme composition according to claim 1.

3. A nucleic acid molecule encoding the enzyme composition according to claim 1 and / or the immobilized enzyme according to claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule encoding the mutant of 2-hydroxy-4-aminobutyrate oxidase is shown in SEQ ID NO: 7; The nucleotide sequence of the nucleic acid molecule encoding the mutant of the ketotransaminase is shown in SEQ ID NO: 8; The nucleotide sequence of the nucleic acid molecule encoding the mutant of the hydrogen peroxide hydrolase is shown in SEQ ID NO:9; The nucleotide sequence of the nucleic acid molecule encoding the mutant of the ectoine synthase is shown in SEQ ID NO: 10; The nucleotide sequence of the nucleic acid molecule encoding the mutant of the pyruvate-CoA oxidase is shown in SEQ ID NO: 11; The nucleotide sequence of the nucleic acid molecule encoding the Lactobacillus helveticus lactate dehydrogenase is shown in SEQ ID NO:

12.

5. Biomaterial, characterized in that Including at least one of the following (1) to (3): (1) A recombinant vector containing the nucleic acid molecule according to claim 3 or 4; (2) transforming or transfecting a host cell with the recombinant vector described in (1); (3) Cultivating the host cell as described in (2) to obtain the mixture.

6. Use of any one of the enzyme composition according to claim 1, the immobilized enzyme according to claim 2, the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5 in the preparation of ectoine.

7. The product, characterized in that include: The enzyme composition according to claim 1, the immobilized enzyme according to claim 2, the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5, and an acceptable auxiliary agent or carrier.

8. The preparation method of Ekedoin is characterized in that: Using 2-hydroxy-4-aminobutyric acid, pyruvic acid and β-alanine as raw materials, ectoine is prepared by any of the following conversions; (a), the enzyme composition according to claim 1; (b), the immobilized enzyme according to claim 2; (c) a nucleic acid molecule as described in claim 3 or 4; or (d) The biomaterial according to claim 5; (e) The product as claimed in claim 7.

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