8 results about "Carboxypeptidase B" patented technology

The invention belongs to the technical field of walnut protein preparation, and particularly relates to walnut protein powder with high digestibility, low sensitization and low bitterness as well as a preparation method and application of the walnut protein powder. According to the preparation method of the walnut protein powder with high digestibility, low sensitization and low bitterness provided by the invention, proline specific endo protease, recombinant trypsin and carboxypeptidase B are cooperatively used, so that accurate damage to an anti-digestion structure and a sensitization epitope of walnut protein can be realized; according to the method, ultrahigh digestibility (greater than or equal to 92%), thorough desensitization (greater than 99.8%), bitterness inhibition and functional peptide enrichment are synchronously realized, so that the defects of low digestibility, sensitization residue, obvious bitterness, low yield of functional peptide, complex process and the like caused by uncontrollable cutting in the existing random enzymolysis technology are overcome, and meanwhile, the core technical bottleneck of high-value utilization of walnut protein is overcome.

The invention relates to the technical field of biology, in particular to a recombinant fusion protein, polynucleotide for coding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell and a method for preparing target polypeptide by using the recombinant fusion protein. The recombinant fusion protein comprises tag polypeptide-target polypeptide-(linker 1-linker x-linker 2-target polypeptide) n from N terminal to C terminal, x is the number of linkers, x is 0 or 1, n is a positive integer not less than 1, the tag polypeptide is SEQ ID NO.1 or SEQ ID NO.2, the linker 1 contains a Kex2 protease enzyme cutting site and a carboxypeptidase B enzyme cutting site, and the linker 1 contains a Kex2 protease enzyme cutting site and a carboxypeptidase B enzyme cutting site. And the connecting peptide 2 contains a WELQ protease enzyme cutting site. The recombinant fusion protein disclosed by the invention is expressed in an inclusion body form, the synthesis process is simplified, the target polypeptide can be prepared with high yield and high purity by using the recombinant fusion protein disclosed by the invention, and the production cost can be reduced from multiple dimensions.

The present application belongs to the field of biotechnology, and particularly relates to a carboxypeptidase B (CPB) precursor gene sequence, a recombinant carboxypeptidase B precursor and a preparation method of carboxypeptidase B. The carboxypeptidase B precursor gene sequence is shown in SEQ ID No. 6, the amino acid sequence is shown in SEQ ID No. 5, the gene sequence of the CPB enzyme is shown in SEQ ID No. 2, and the amino acid sequence is shown in SEQ ID No. 4. The preparation method of CPB comprises the following steps: (1) cloning and construction of a CPB enzyme expression vector; (2) expression of CPB precursor protein by using an engineering bacterium to form an inclusion body; (3) washing, crushing and centrifugation of the bacterium, collection of the inclusion body, denaturation and dissolution; (4) renaturation of the inclusion body; and (5) CPB activation and purification. The process steps of the present application are simple, animal pancreas is not used, and the possibility of infection is reduced; the specific activity of the obtained CPB enzyme is high, the yield per liter of fermentation liquid is high, and the present application is suitable for large-scale production and application.

The application discloses a fusion protein and application thereof in preparation of semaglutide precursor polypeptide, wherein N (N>=1) semaglutide fragments GLP-1 (11-37) are connected through KR, and then sequentially fused with an enterokinase enzyme cutting site and a fusion protein label to realize tandem expression, so that the fusion protein is a fusion protein label-enzyme cutting site-N associated semaglutide fragment GLP-1 (11-37), and the fusion protein label is an albumin affinity peptide (SEQ ID NO. 1). The fusion protein is expressed in a heterologous manner in an E. coli BL21 (DE3) competent cell, an engineering strain is constructed, and then fermentation expression is carried out by using a flask, so that the fermentation density of the bacterial body is greatly improved, expression of the target protein in an inclusion body is promoted, the yield of the target protein is improved, and the fusion protein has a good industrial application prospect. The fusion protein can be successfully recognized and cut by commercial enterokinase, KEX2 enzyme and carboxypeptidase B to release the semaglutide precursor polypeptide.

The invention belongs to the technical field of biology, and particularly relates to a carboxypeptidase B (CPB) precursor gene sequence, a recombinant carboxypeptidase B precursor and a preparation method of carboxypeptidase B. The gene sequence of the carboxypeptidase B precursor is as shown in SEQ ID No.6, the amino acid sequence of the carboxypeptidase B precursor is as shown in SEQ ID No.5, the gene sequence of the CPB enzyme target gene is as shown in SEQ ID No.2, and the amino acid sequence of the CPB enzyme target gene is as shown in SEQ ID No.4. The preparation method of the CPB comprises the following steps: (1) cloning and constructing a CPB enzyme expression vector; (2) expressing CPB precursor protein by using engineering bacteria to form an inclusion body; (3) thalli are washed, crushed and centrifuged, inclusion bodies are collected, and denatured dissolution is performed; (4) inclusion body renaturation; and (5) activating and purifying CPB. The process is simple and convenient in steps, does not use animal pancreas, and reduces the possibility of infection; the obtained CPB enzyme has high specific activity, and the yield of each liter of fermentation liquor is high, so that the method is suitable for large-scale production and application.

The invention discloses a carboxypeptidase B protein monoclonal antibody, a kit based on the monoclonal antibody and application, and belongs to the technical field of antibody preparation and biological detection. Specifically, two monoclonal antibodies, namely, BE-1E12B8 and BE4-2B6A6, which have binding characteristics with carboxypeptidase B are obtained through screening, the BE-1E12B8 antibody has high titer, and the BE4-2B6A6 antibody has high titer. The BE-1E12B8 and the BE4-2B6A6 are matched for use and are used for sandwich ELISA (enzyme-linked immunosorbent assay), and the carboxypeptidase B can be efficiently detected. Therefore, a double-antibody sandwich ELISA method is established by taking the two monoclonal antibodies as a capture antibody and a detection antibody respectively, the monoclonal antibodies can be bound to different epitopes of carboxypeptidase B respectively, the binding positions are proper, so that the monoclonal antibodies can be jointly used for realizing the detection of the double-antibody sandwich method, and the monoclonal antibodies have better sensitivity and specificity compared with the existing detection method; the method has the advantages of high repeatability and high flux.

The application discloses a preparation method of balsam pear seed peptides by enzymolysis, and belongs to the technical field of peptide preparation. The preparation method comprises the following steps: after balsam pear seeds are crushed, water is added for soaking treatment to obtain material A; cellulase and pectinase are added to the material A for pretreatment, enzyme inactivation is performed, and a primary enzymolysis liquid is obtained; compound protease is added to the primary enzymolysis liquid for step-by-step enzymolysis, enzyme inactivation is performed, solid-liquid separation is performed, and an enzymolysis liquid is obtained; the enzymolysis liquid is dried to obtain polypeptide powder; the polypeptide powder is extracted by ethanol, filtered, and dried to obtain the balsam pear seed peptides; and the compound protease in step (3) is bromelain, pepsin and carboxypeptidase B. Compared with the prior art, the balsam pear seed peptides prepared by the application have better alpha-glucosidase inhibiting capacity, are close to chemical hypoglycemic drugs, can be used as natural alpha-glucosidase inhibitor raw materials, and have lower side effects.

The present invention relates to a composition in the form of an injectable aqueous solution having a pH of 3.5 to 4.4 and comprising at least human insulin A21G and at least one prandial acting glucagon inhibitor. In one embodiment, the prandial acting glucagon inhibitor is selected from an amylin analogue, or an amylin receptor agonist, or a GLP-1 analogue, or a GLP-1 receptor agonist (GLP-1 RA). In one embodiment, the prandial acting glucagon inhibitor is an amylin analogue or an amylin receptor agonist. In one embodiment, the prandial acting glucagon inhibitor peptide is pramlintide. The present invention also relates to a process for obtaining human insulin A21G comprising at least the step of reacting human insulin A21G, B31R, B32R (insulin glargine) with rat carboxypeptidase B in a ratio of insulin / carboxypeptidase of 500 to 2000 at a pH of 7.5 to 8.5 and a temperature of 20 to 30 °C for 10 to 20 hours.