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.

The present disclosure discloses an engineering strain and application thereof in joint production of Danshensu and alanine, and belongs to the technical field of bioengineering. The present disclosure constructs a three-enzyme co-expression genetic engineering strain, and realizes joint production of Danshensu and alanine. Further, the transport of a substrate is promoted and decomposition of products is reduced by knocking out or enhancing expression of related genes on E. coli genome. The genetic engineering strain provided by the present disclosure can produce optically pure D-danshensu and L-danshensu, and jointly produce pyruvic acid. The production process is simple, raw materials are easily available, impurities are fewer, and a good industrial application prospect is achieved.

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. 1-9, 11, 13, 15, 17-19 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 invention discloses a glutamate dehydrogenase mutant and an application thereof. The mutant is one of the following: a mutation of the 402th lysine of the amino acid sequence shown in SEQ ID NO. 1 to phenylalanine or aspartic acid; a mutation of the 406th isoleucine to phenylalanine or threonine; a combined mutation of the 121th threonine and the 123th leucine; a combined mutation of the 379th alanine and the 383th leucine. In the invention, the catalytic activity of glutamate dehydrogenase derived from Pseudomonas putida to 2-carbonyl-4-(hydroxymethylphosphonoyl)butanoic acid (PPO) is significantly improved by a molecular transformation method combining directed evolution and a semi-rational design; and the issue of low glutamate dehydrogenase activity in the process of preparing L-glufosinate by reductive amination is solved.

The present disclosure discloses a production method of Danshensu, belonging to the technical field of bioengineering. The present disclosure constructs a novel genetic engineering strain co-expressed by three enzymes, which can be applied to the production of optically pure 3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid. All of the (D/L)--hydroxycarboxylic acid dehydrogenase selected by the present disclosure have the characteristics of poor substrate specificity and strong optical specificity, and can produce optically pure D-danshensu and L-danshensu. Further, the production efficiency of the recombinant strain is improved by knocking out or enhancing the expression of a related gene on the E. coli genome to promote substrate transport and reduce product decomposition. The method for producing Danshensu and -ketoglutaric acid by using the transformation of the recombinant strain according to the present disclosure is simple, has easily available raw materials, few impurities, and has good industrial application prospects.

A method for producing an active-form mutant enzyme, by specifying a protein of which a native form exhibits an enzyme activity but which has 10% or less enzyme activity of the native form when a gene of the protein is expressed to provide an inactive-form enzyme; determining a sequence conservation of amino acid residues in an amino acid sequence of the inactive-form enzyme and specifying amino acid residue(s) for which sequence conservation in the inactive-form enzyme is lower than sequence conservation of other amino acid(s) of the same residue; preparing a gene having a base sequence that codes for the amino acid sequence of the inactive-form enzyme in which at least one said specified amino acid residue is substituted by another amino acid with a higher sequence conservation; and expressing the gene to obtain an enzyme that exhibits an enzyme activity of the native form protein.

Provided herein are methods for selection of circular aptamers using a circular nucleic acid library. Also provided are circular aptamers, circular aptamer probes, biosensor systems, and the methods for their use in detecting a microorganism target, or a target molecule present on or generated from a microorganism or a virus in a test sample, including C. difficile glutamate dehydrogenase and methods for determining whether a subject has a C. difficile infection.

An object of the present invention is to provide a novel method for producing L-cysteine in place of a conventional fermentation method. More specifically, the object is to provide a method for producing L-cysteine by the combination of heat-resistant enzymes. In particular, the object is to provide a method for efficiently producing a pathway for synthesizing O-phosphoserine from 3-phosphoglyceric acid (3PG) via phosphohydroxypyruvic acid (HPV). The present invention solved the problem by a method for producing O-phosphoserine including acting phosphoserine aminotransferase (PSAT) and 3-phosphoglycerate dehydrogenase (PGDH) that are each derived from a thermophilic bacterium on 3PG to generate O-phosphoserine, and a method for producing L-cysteine including the step described above.

A method for expressing, as a soluble protein or an active-form mutant enzyme, an enzyme that cannot be expressed as a soluble protein or an active-form enzyme in a heterologous expression system or that is obtained in a minute amount even when an active-form enzyme is expressed, the method including a technique for selecting an effective mutation site and a mutated amino acid. A new active-form mutant enzyme is also disclosed. The method involves: specifying an insoluble protein or an inactive-form enzyme; specifying a hydrophilic amino acid in a hydrophobic domain and/or a hydrophobic amino acid in a hydrophilic domain of an -helix structure portion of the insoluble protein or the inactive-form enzyme and preparing a gene that codes for an amino acid sequence in which a substitution is made to the hydrophilic amino acid in the hydrophobic domain and/or the hydrophobic amino acid in the hydrophilic domain.

A method of the bio-production of methionine and/or of its derivatives thereof from a reduced source of sulfur, such as MeSH or MeSNa including genetically modified yeasts, having an increased ability to produce methionine and/or its derivatives thereof, as compared to the parent yeasts.