Provided is a method for preparing β-alanine, the method comprising: preparing a β-alanine product from a reactant containing fumaric acid and aqueous ammonia in the presence of a catalyst, wherein the catalyst contains a catalyst composition containing aspartase and L-aspartic acid-α-decarboxylase, and adding fumaric acid during the reaction, wherein the total moles of the fumaric acid added is equal to the initial moles of the aqueous ammonia in the reactant minus the initial moles of the fumaric acid in the reactant. Also provided are methods for preparing a β-alanine salt (in particular calcium β-alanine, sodium β-alanine, and potassium β-alanine) and a pantothenate (in particular calcium pantothenate, sodium pantothenate, and potassium pantothenate).

An R-3-aminobutyric acid preparation method with high efficiency and high stereoselectivity. The method comprises using aspartase with stereoisomerization catalytic activity derived from Escherichia coli to efficiently convert butenoic acid into R-3-aminobutyric acid. After only 24 h of reaction, the conversion rate is as high as ≥98%, and the ee value is ≥99.9%. The conversion efficiency is greatly improved, the reaction time is shortened, and the production costs are reduced. The method features a high yield, a high conversion rate, low costs, a short production cycle, a simple process, ease of enlargement, suitability for mass production and the like.

An R-3-aminobutyric acid preparation method with high efficiency and high stereoselectivity. The method comprises using aspartase with stereoisomerization catalytic activity derived from Escherichia coli to efficiently convert butenoic acid into R-3-aminobutyric acid. After only 24 h of reaction, the conversion rate is as high as ≥98%, and the ee value is ≥99.9%. The conversion efficiency is greatly improved, the reaction time is shortened, and the production costs are reduced. The method features a high yield, a high conversion rate, low costs, a short production cycle, a simple process, ease of enlargement, suitability for mass production and the like.

The present invention discloses an aspartase variant, method of preparing the same and use thereof. Compared with sequence 2 in the sequence listing, the amino acid sequence of the aspartase variant provided in the present invention has more than 96% identity, and there are mutations in T187I and N326C at positions 187 and 326, respectively, and has improved catalytic activity for the ammoniation of acrylic acid, compared with the aspartase shown in sequence 2. The experiment proves that the aspartase variant provided in the present invention does have improved catalytic activity for the ammoniation of acrylic acid compared with the wild type parent aspartase, and has better thermal stability and pH spectrum. The reaction can greatly increase the conversion ratio of acrylic acid.

The present invention provides an aspartase mutant, a recombinant expression vector and recombinant bacterium containing the aspartase mutant, and the use thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the aspartase mutant is as set forth in SEQ ID NO: 1. In the aspartase mutant of the present invention, on the basis of wild type aspartase (with an amino acid sequence as set out in SEQ ID NO: 3), glutamic acid at position 427 is mutated into glutamine. In the present invention, by mutating the amino acid residue at position 427 into glutamine, the polar environment near an active site is changed, and thus ammonia supply during substrate reaction is further facilitated, thereby improving an enzyme activity, enhancing the ability of the enzyme in synthesizing a -amino acid, and providing a practical and effective strategy for industrial production of the -amino acid.

The invention relates to the technical field of bioengineering, and discloses a method for synthesizing L-aspartic acid with maleic acid by whole-cell biocatalysis. In the invention, a recombinant strain co-expressing maleate cis-trans isomerase and L-aspartate lyase is constructed, and engineered and optimized to produce L-aspartic acid from maleic acid with a high conversion rate by whole-cell catalyzing. Relatively inexpensive maleic acid is utilized by the recombinant strain to produce L-aspartic acid, where maleic acid is reacted completely in 40-120 min, there is almost no buildup of the intermediate fumaric acid, and the conversion rate is up to 98% or more.

The invention relates to the technical field of bioengineering, and discloses a method for synthesizing L-aspartic acid with maleic acid by whole-cell biocatalysis. In the invention, a recombinant strain co-expressing maleate cis-trans isomerase and L-aspartate lyase is constructed, and engineered and optimized to produce L-aspartic acid from maleic acid with a high conversion rate by whole-cell catalyzing. Relatively inexpensive maleic acid is utilized by the recombinant strain to produce L-aspartic acid, where maleic acid is reacted completely in 40-120 min, there is almost no buildup of the intermediate fumaric acid, and the conversion rate is up to 98% or more.

An R-3-aminobutyric acid preparation method with high efficiency and high stereoselectivity. The method comprises using aspartase with stereoisomerization catalytic activity derived from Escherichia coli to efficiently convert butenoic acid into R-3-aminobutyric acid. After only 24 h of reaction, the conversion rate is as high as 98%, and the ee value is 99.9%. The conversion efficiency is greatly improved, the reaction time is shortened, and the production costs are reduced. The method features a high yield, a high conversion rate, low costs, a short production cycle, a simple process, ease of enlargement, suitability for mass production and the like.

The present invention discloses an aspartase variant, method of preparing the same and use thereof. Compared with sequence 2 in the sequence listing, the amino acid sequence of the aspartase variant provided in the present invention has more than 96% identity, and there are mutations in T187I and N326C at positions 187 and 326, respectively, and has improved catalytic activity for the ammoniation of acrylic acid, compared with the aspartase shown in sequence 2. The experiment proves that the aspartase variant provided in the present invention does have improved catalytic activity for the ammoniation of acrylic acid compared with the wild type parent aspartase, and has better thermal stability and pH spectrum. The reaction can greatly increase the conversion ratio of acrylic acid.

The present invention provides a computational method for designing artificial variants which have activity towards non-natural substrates. The present invention provides a special method to process stability evaluation results and creatively combines a process of calculating free energy barrier, which can improve the accuracy of the virtual screening of enzyme variants. The computational method disclosed by this invention greatly reduce the number of variants to be constructed and tested in the wet lab. In some cases, this method achieved the effect of enzyme engineering that cannot be achieved by traditional directed enzyme evolution methods.