47 results about "Heparinase I" patented technology

Modified heparinases having altered binding specificity and activity are provided. Isolated nucleic acids encoding the same as well as vectors and host cells are provided. Methods for using the modified heparinases are also provided.

Modified heparinases having altered binding specificity and activity are provided. Isolated nucleic acids encoding the same as well as vectors and host cells are provided. Methods for using the modified heparinases are also provided.

Modified heparinases having altered binding specificity and activity are provided. Isolated nucleic acids encoding the same as well as vectors and host cells are provided. Methods for using the modified heparinases are also provided.

The invention provides a preparation method of flavobacterium heparinum heparinases I, II and III, which comprises the following steps: inoculating flavobacterium heparinum used as the raw material into a seed culture medium for culture, then inoculating into a fermentation culture medium, centrifuging, collecting the precipitate, carrying out ultrasonication on the precipitate, and centrifuging to obtain a crude enzyme liquid of flavobacterium heparinum heparinases I, II and III; carrying out SP-Sepharose FF on the crude enzyme liquid to obtain an enzyme II and enzymes I and III through chromatographic separation; carrying out SP-Sepharose FF on the enzymes I and III to obtain a heparinase I and a heparinase III through separation; respectively carrying out SP-Sepharose FF on the obtained enzyme I and enzyme III twice to obtain high-purity heparinases I and III through purification; and respectively carrying out HEP-Sepharose 4B, HA-Ultrogel and SP-Sepharose FF on the obtained enzymeII to obtain a high-purity heparinase II.

The invention relates to a mutant that improves the thermal stability of heparanase I based on molecular dynamics analysis of enzyme flexibility, and the mutant is mutant BtHepI Q198R and / or mutant BtHepI K247W , the mutant BtHepI Q198R The amino acid sequence is SEQ ID NO.1, the mutant BtHepI K247W The amino acid sequence of is SEQ ID NO.2. The mutant of the present invention is a heparanase I mutant strain with excellent thermal stability, which has great potential for industrial application and economic value. Operational stability and batch reuse in catalytic processes, reducing production costs, etc., are of great importance.

The invention discloses a connecting peptide and an application thereof. The connecting peptide is mainly applied in preparation of MBP fusion polysaccharide lyases, and in particular, relates to MBP fusion heparinases I, II and III and MBP fusion chondroitinases B and AC. In an MBP fusion expression system, MBP solubilization assisting and folding assisting are improved through application of the connecting peptide, the target protein activity and thermal stability are increased, and the MBP fusion heparinases I, II and III and the MBP fusion chondroitinases B and AC having fermentation enzyme activity in escherichia coli preliminarily reaching an industrial application level and having excellent thermal stability are eventually obtained.

The invention relates to a high-expression water-soluble heparinase I fusion protein and a coding gene thereof. The amino acid sequence of the heparinase I fusion protein is as shown in SEQ ID NO. 2; the nucleotide sequence of the coding gene of the heparinase I fusion protein is as shown in SEQ ID NO. 1. According to the invention, a pColdTF vector is utilized to transform an expression gene of heparinase I, a section of nucleotide sequence expressing a pColdTF protein is added and the heparinase I fusion protein is obtained; the enzyme activity of the heparinase I fusion protein can reach 64000U / L of fermentation solution, the expression level can reach 320mg / L of fermentation solution and the specific enzyme activity can reach 200U / mg. Furthermore, the enzyme can further realize one-step purification of the fusion protein by nickel column separation.

The invention provides a method for preparing heparinase I. The method for preparing the heparinase I comprises the following steps of: inoculating flavobacterium heparinum serving as a raw material to a seed culture medium for culture; then inoculating the flavobacterium heparinum to a fermentation culture medium; centrifugally collecting precipitate; performing ultrasonication on the precipitate; performing centrifugation again to obtain crude enzyme liquid of the flavobacterium heparinum heparinase I; and performing SP-sepharose FF chromatographic purification on the crude enzyme liquid for three times to obtain the high-purity flavobacterium heparinum heparinase I, wherein the SP-sepharose FF chromatographic purification for three times is protected by calcium chloride in the whole course, so that the yield of pure enzymic activity is greatly increased. The method for preparing the heparinase I has the characteristics of simple process, easy amplification, large preparation amount of products at a time, low cost of reagents and the like. The specific activity of the prepared heparinase I reaches 223 IU / mg and the yield of the pure enzymic activity reaches 30 percent. Compared with the conventional newest method, the method for preparing the flavobacterium heparinum heparinase I has the advantages of increasing the specific activity by more than two times, and the purification yield by about one time.