5results about How to "High specific activity" patented technology

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. Wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, the enzyme specific activity of the T7 RNA polymerase variant is remarkably improved compared with that of the wild type T7 RNA polymerase, the addition amount of the polymerase in the in-vitro transcription reaction process can be reduced, and the cost is saved; in addition, the mutant also effectively reduces the generation of incomplete fragment impurities, and the mRNA integrity is significantly improved to 99%. The invention further provides a method for preparing RNA through in-vitro transcription.

The application discloses an S-methyl-5'-thioribose kinase mutant and application thereof in ribose-1-phosphate synthesis, relates to the technical field of genetic engineering and enzyme engineering, and specifically discloses a high-activity S-methyl-5'-thioribose kinase mutant. The mutant is obtained through directional mutation, key mutations occur at the 128th position and the 240th position, and the catalytic efficiency on ribose-1-phosphate is significantly improved. The mutant is suitable for industrial production of ribose-1-phosphate and derivatives thereof.

The application discloses a two-stage high-density fermentation method for producing active nickel-iron hydrogenase. The method comprises the following steps: performing aerobic high-density fed-batch fermentation on recombinant Escherichia coli co-expressing nickel-iron hydrogenase structural genes and mature auxiliary protein genes, so that the cell density reaches OD 600 ≥40; then switching to anaerobic conditions, adding an inducer and metal ion cofactor, and inducing expression and active center assembly of the enzyme; and finally, breaking the cells in an anaerobic environment to obtain active enzymes. Through the time-decoupling strategy, the volume yield is improved to more than 900 mg / L under the premise of ensuring high specific activity (≥20 U / mg) of the enzyme, and the technical bottleneck of low cell density and limited yield in traditional anaerobic fermentation is solved, which is suitable for industrial production of nickel-iron hydrogenase and other oxygen-sensitive metal enzymes.

The invention provides chymotrypsinogen and application thereof, and particularly relates to a protein tag for improving the activation efficiency of recombinant chymotrypsinogen. Compared with naturally extracted human chymotrypsinogen, the recombinant chymotrypsinogen fused with the protein tag has significantly improved activation efficiency. The invention also relates to a recombinant chymotrypsinogen containing the protein tag, a nucleic acid molecule for coding the recombinant chymotrypsinogen, a vector containing a nucleotide sequence of the nucleic acid molecule, a nucleic acid construct and a host cell. In addition, the invention also relates to a method for expressing the recombinant chymotrypsinogen.

The application provides a Bst DNA polymerase domain, a mutant and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the Bst DNA polymerase domain is shown in SEQ ID NO:1. The Bst DNA polymerase mutant is obtained by replacing the natural DNA binding domain of the wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO:1. The Bst DNA polymerase mutant exhibits extremely high catalytic efficiency on dUTP, and also has excellent hot-start characteristics, and can reduce the false positive rate of LAMP technology to close to zero. The Bst DNA polymerase mutant also has extremely strong complex sample tolerance, and the detection sensitivity of the pathogen is 20 times higher than that of the wild-type Bst enzyme, and can meet the early and accurate diagnosis requirements of trace infection.