In some embodiments, the present invention a mutated FAD-GDH protein, wherein the mutated FAD-GDH protein is mutated from a wild-type first species to contain at least one point mutation, wherein the mutated FAD-GDH protein comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of X, S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is selected from the group consisting of S and X; and d) X.sup.5 is selected from the group consisting of L and X.

The present invention provides for a mechanism to reduce glycerol production and increase nitrogen utilization and ethanol production of recombinant microorganisms. One aspect of this invention relates to strains of S. cerevisiae with reduced glycerol productivity that get a kinetic benefit from higher nitrogen concentration without sacrificing ethanol yield. A second aspect of the invention relates to metabolic modifications resulting in altered transport and/or intracellular metabolism of nitrogen sources present in corn mash.

The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineered Escherichia coli co-expresses epoxide hydrolase, alcohol dehydrogenase, -transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADP.sup.+ and an amino doner L-Glu; alcohol dehydrogenase expressed by the engineered Escherichia coli is RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course.

An aqueous composition includes (i) glutamate dehydrogenase from a bacterium of the Clostridium genus, (ii) a stabilizing compound that is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two COOH groups, or a salt thereof, and (iii) any of a monosaccharide polyol, disaccharide polyol, or polymeric macromolecule in addition to the glutamate dehydrogenase. A process for stabilizing the glutamate dehydrogenase in order to maintain antigenic properties of the glutamate dehydrogenase includes stabilizing the glutamate dehydrogenase in the aqueous composition and maintaining the antigenic properties of the glutamate dehydrogenase during storage of the aqueous composition.

A recombinant microorganism includes a gene encoding a pyridoxine dehydrogenase, a gene encoding a pyridoxamine synthetase having an enzymatic activity of synthesizing pyridoxamine from pyridoxal, and a gene encoding an amino acid regeneration enzyme having an enzymatic activity of regenerating an amino acid consumed by the pyridoxamine synthetase, in which at least two of the gene encoding the pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthetase, or the gene encoding the amino acid regeneration enzyme, are introduced from outside of a bacterial cell, or are endogenous to the bacterial cell and have an enhanced expression. In addition, a recombinant microorganism into which a gene encoding a pyridoxine dehydrogenase is introduced is provided.

The present invention relates to glutamate dehydrogenase mutants and their application in preparation of L-phosphinothricin. The amino acid sequences of the glutamate dehydrogenase mutants are as shown in SEQ ID NO. 19, 11, 13, 15, 1719 and 22. By means of molecular engineering, mutating the specific alanine in glutamate dehydrogenase substrate-binding pocket into glycine and/or mutating the specific valine in glutamate dehydrogenase substrate-binding pocket into alanine, the present invention has obtained NADPH-specific glutamate dehydrogenase mutants with high enzyme activity in catalyzing the substrate 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid or its salt for L-phosphinothricin preparation or NADH-specific glutamate dehydrogenase mutants with catalytic activity toward PPO; this has significantly improved substrate conversion, and increased the product concentration of the L-phosphinothricin preparation process.

The present invention provides for a mechanism to reduce glycerol production and increase nitrogen utilization and ethanol production of recombinant microorganisms. One aspect of this invention relates to strains of S. cerevisiae with reduced glycerol productivity that get a kinetic benefit from higher nitrogen concentration without sacrificing ethanol yield. A second aspect of the invention relates to metabolic modifications resulting in altered transport and/or intracellular metabolism of nitrogen sources present in com mash.

A process for stabilizing glutamate dehydrogenase (GDH) from a bacterium of the Clostridium genus, in an aqueous solution, in order to maintain the antigenic properties thereof, includes the step of mixing the glutamate dehydrogenase and a stabilizing composition which is a carboxylic acid having a carbon-based chain of at least 3 carbon atoms and comprising at least 2 COOH groups, or a salt thereof. GDH compositions thus stabilized and a method of detecting the presence of bacteria of the Clostridium genus are also disclosed.

Disclosed are a gene mining method combining functional sequence and structure simulation, an NADH-preferring phosphinothricin dehydrogenase mutant and an application thereof. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence which an NADH-type glutamate dehydrogenase should have; (2) searching a gene library based on the characteristic sequence; (3) performing clustering analysis and protein structure simulation on genes obtained by the searching; (4) selecting genes that feature high gene aggregation and a protein structure similar to that of the known phosphinothricin dehydrogenase as candidate genes. A wild-type phosphinothricin dehydrogenase with an amino acid sequence as set forth in SEQ ID No.2 derived from Lysinibacillus composti is obtained through the gene mining, and then mutated, and an NADH-preferring phosphinothricin dehydrogenase mutant is screened out, which has a mutation site selected from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; (3) A144G. This mutant enzyme can be used for catalytic reaction with an inexpensive coenzyme NAD.

Disclosed in the present invention is an L-glutamate dehydrogenase mutant, the sequence of the L-glutamate dehydrogenase mutant being a sequence in which amino acid residue A at position 175 in SEQ ID NO: 1 is mutated to be G, and amino acid residue V at position 386 is mutated to be an amino acid residue having less steric hindrance. Further disclosed in the present invention is an application of the described L-amino acid dehydrogenase mutant in the preparation of L-glufosinate-ammonium or a salt thereof. When the L-glutamate dehydrogenase mutant of the present invention is used to prepare L-glufosinate-ammonium or a salt thereof, compared to an L-glutamate dehydrogenase mutant in which only position 175 or 386 is mutated, the specific enzyme activity is higher. Therefore, the action efficiency of the enzyme is improved, reaction costs are reduced, and industrial production is facilitated.