42 results about "L asparaginase" patented technology

The invention discloses a method for producing a red blood cell drug containing L-ASPase II (L-asparaginase) by in vitro induction. The method comprises the steps of: 1) using a lentiviral vector system to transform induced pluripotent stem (IPS) cells: I. constructing a pLenti6.3 / V5-GW / Em-GFP VerA-L-ASPase II plasmid; II. using 293T cells to perform packaging so as to generate high titer virus particles containing L-ASPase II; III. using the virus particles to transfect induced pluripotent stem cells; and 2) inducing the induced pluripotent stem cells in vitro to generate L-ASPase II-containing red blood cells: I. subjecting IPS cells to in vitro differentiation induction to form an erythroid body EB; and II. performing induced differentiation on EB to obtain mature red cells. The method provided in the invention performs genetic modification on IPS cells by the lentiviral vector system, and induces them in vitro to generate red blood cells containing L-ASPase II. The generated red blood cells can be used as a sustained release carrier of L-ASPase II, and also can maintain the original shapes and functions of red blood cells. The toxic and side effects of L-ASPase II are further reduced, so that the method lays a solid foundation for future clinical application.

The invention relates to a macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and a method. In the papain polymer, a papain is embedded in the pore canal of a carrier the aperture of which is more than 0.5mu m; an inorganic macroporous material or an organic macroporous material the surface of which is provided with hydroxyl is taken as an immobilized enzyme carrier; synchronous complementation of the formation of the crosslinking papain polymer and the covalent connection of the crosslinking papain polymer and the macroporous carrier is finally realized through amino-group modification, enzyme adsorption, enzyme precipitation and synchronous crosslinking on the surface of the carrier; the enzyme carrying amout of the immobilized enzyme is higher than that of the immobilized enzyme of a common carrier; the enzyme leakage caused by applications of the macroporous carrier is avoided by adopting a covalent fixed method; the carrier shape can be adjusted according to the actual requirements; and the optimum catalysis temperature, pH value, solvent and heat stability are obviously improved. The papain polymer provided by the invention can be applied to other proteases, lipase, amylase, glucose isomerase, penicillin, acylase, pectinase, oxidase, L-asparaginase, aspartase and peroxidase and the like.

The invention discloses an L-asparaginase mutant with the improved enzyme activity and a construction method thereof, and belongs to the field of gene engineering. According to the L-asparaginase mutant, on the basis of amino acid shown in the SEQ ID NO.2, the 107th glycine is mutated into aspartic acid. The obtained mutant is expressed in bacillus subtilis, fermentation is performed in a shake flask for 24 h and then the enzyme activity is 961 U / mL; the enzyme activity of the mutant is improved by 80%, the appetency of a substrate is decreased by 50% compared with protoenzyme, the catalytic efficiency is improved by 84%, and meanwhile the specific enzyme activity is improved by 83%. According to the L-asparaginase mutant, it is shown that the 107th amino acid residue has a great influence on the enzyme catalytic action, a certain foundation is provided for research on the enzyme catalytic mechanism, and the enzyme industrial application potential is improved.

The invention provides deimmunized mutant proteins having reduced immunogenicity while exhibiting substantially the same or greater biological activity as the proteins of interst from which they are derived, as exemplified by mutant L-asparaginase that comprises amino acid substitutions compared to wild type L-asparaginase. The invention further provides methods for screening mutant deimmunized proteins that have substantially the same or greater biological activity as a protein of interest, and methods for reducing immunogenicity, without substantially reducing biological activity, of a protein of interest.The invention's compositions and methods are useful in, for example, therapeutic applications by minimizing adverse immune responses by the host mammalian subjects to the protein of interest. Thus, the invention further provides methods for treating disease comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising at least one of the mutant deimmunized proteins produced by the invention's methods.

The present invention discloses one kind of new compound, polyethylene glycol derivative modified L-asparaginase in the structure as shown. The present invention also provides the preparation of the polyethylene glycol derivative modified L-asparaginase and its medicine composition. The compound of the present invention has homogeneous component, high antitumor effect, high chemical stability, and low immunogenicity.

The disclosure provides a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, and methods of preparation and use thereof.

The disclosure provides a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, and methods of preparation and use thereof.

The invention discloses a method for improving secretory expression of L-asparaginase, and belongs to the field of genetic engineering. According to the method, by fusing a pelB signal peptide at an N terminal of asparaginase, the enzyme activity of the extracellular asparaginase of a recombinant bacterium is increased by 20 times, and further through coexpression of lepB signal peptidase, the enzyme activity of the asparaginase can be increased by 1.45 times on the above basis. The enzyme-producing capacity of a modified bacterial strain is significantly improved; the method is more suitable for industrial application; by the method, the production cost can be reduced and the production efficiency can be improved.

The invention relates to a L-asparaginase variant with increased activity, and is a variant of escherichia coli wild type L-asparaginase shown in a SEQ ID NO:3, and the variant comprises an one or a plurality of amino acid substituted amino acid sequence on 48th site, 49th site, 152nd and 283rd by corresponding to a SEQ ID NO:3. The invention also provides the separated nucleic acid containing the nucleotide sequence which codes the L-asparaginase variant, a recombinant expression construct containing the nucleic acid and a recombinant host cell containing the expression construct. In addition, the invention also provides a method for generating the L-asparaginase variant. The invention also provides a pharmaceutical composition used for treating tumor containing the L-asparaginase variant.

The invention discloses an activity-improved L-asparaginase amidohydrolase mutant and a construction method thereof, and belongs to the field of genetic engineering. The mutant is characterized in that the 166th serine is mutated into alanine on the basis of the amino acid shown in SEQ ID No.2. The mutant is expressed in bacillus subtilis, the activity is 657.1 U / ml after flask fermentation is carried out for 24 h, the mutation activity is improved by 23%, the substrate affinity is reduced by 20% compared with original amidohydrolase, the catalytic efficiency is improved by 8.4%, and meanwhile the specific activity is improved by 25%. The mutant and the construction method show that the 166th serine residue has large influences on the catalytic action of the amidohydrolase, a certain foundation is provided for researching the catalytic mechanism of the amidohydrolase, and the industrial application potential of the amidohydrolase is improved.

The invention discloses a preparation method of L-asparaginase. The preparation method comprises the following steps of: carrying out wall breaking on Erwinia thalli by using an extracting solution containing lysozyme; and obtaining high-purity L-asparaginase through ion-exchange chromatography and affinity chromatography. The preparation method of the L-asparaginase is simple and easy to apply and has high efficiency, the yield can reach higher than 60 percent, and the purity of the prepared L-asparaginase can is higher than 99 percent and meets the requirement of the Chinese Pharmacopoeia (2005 Version). The preparation method has no pollution to the environment, is economical and environmenta friendly and can be applied to industrial production.

The present invention relates to the construction and optimization of synthetic genes from the Zymomonas mobilis L-asparaginase gene, the method for cloning same and expression thereof in Escherichia coli. The purpose of the production of said enzyme is for producing high levels of a novel L-asparaginase that can be used in L-asparaginase-based pharmaceutical compositions for treating cancer, tumours and diseases involving cell proliferation, as well as for other medical applications.

The invention discloses L-asparaginase as well as an encoding gene and application thereof. A protein disclosed by the invention is shown in the following formula (1) or (2): (1) the protein is a protein shown in SEQ ID No.2; and (2), the protein is a protein which is obtained through substitution and / or deficiency and / or addition of one or more amino acid residues on an amino acid sequence shown in SEQ ID No.2 and has the same function. The L-asparaginase disclosed by the invention is suitable for being used as a food additive applied to bakery products so that the production amount of acrylamide as a cancerogen in the product is reduced, and is also suitable for being used as a medicine applied to adjuvant therapy of leucocythemia.

The present invention provides a mutant with enhanced secretion of L-asparaginase and its application, which relates to the field of enzyme engineering. This invention successfully constructed a mutant with enhanced secretion of L-asparaginase through N-terminal deletion on amino acid sequence of L-asparaginase, and then expressed the mutant via a recombinant strain. Compared with the wild L-asparaginase, the secretion ability of L-asparaginase mutant in present invention significantly improves by 3.14 times compared to that of wild type. The recombinant strain that constructed in this prevention has an L-asparaginase yield of 407.6 U / mL and a production efficiency of 9.26 U / (mL / h), which reaches to the highest yield been reported.

The invention discloses a preparation method of D-asparagine, which is characterized in that the method comprises the following steps: (1) allowing L-asparagine to react with phthalic anhydride at 130 DEG C-180 DEG C so as to generate phthaloyl-L-aspartic acid; (2) allowing the phthaloyl-L- asparagine obtained in step (1) to perform reflux reaction with acetic anhydride and a racemization reagentfor 20-60 min so as to generate phthaloyl-DL-aspartic anhydride; (3) allowing the phthaloyl-DL- aspartic anhydride obtained in step (2) to react with an ammonia tetrahydrofuran solution so as to obtain phthaloyl-DL-asparagine; (4) allowing the phthaloyl-DL-asparagine obtained in step (3) to react with hydrazine hydrate to remove the phthaloyl group so as to obtain the product of DL-asparagine; (5) specifically hydrolyzing L-asparagine in the DL-asparagine obtained in step (4) by microbial L-asparaginase for separation so as to obtain D-asparagine and L-asparagine.

The invention discloses a recombinant escherichia coli efficiently transforming fumaric acid into L-asparagine as well as a construction method and an application thereof. Fumarase encoding genes fumA, fumB, fumC in an ammonium-tolerant escherichia coli BEW308 are inactivated, and then L-aspartase encoding and L-asparaginase encoding genes are inserted into the positions of the fumarase encoding fumAC genes, thereby obtaining the recombinant escherichia coli having no malic acid by product, less L-aspartic acid accumulated, and a major product L-asparagine. The invention also discloses a construction method and an application of the bacterial strain. The recombinant escherichia coli can realize constitutive high-activity expression of L-aspartase and L-asparaginase, and ultimately achieve transformation of fumaric acid into L-asparagine.

The invention relates to a recombinant virus and method for producing L-asparaginase II by using the virus, which belong to the technical field of bioscience. The invention has the purpose of providing the recombinant virus. The virus comprises a polyhedrin gene and an L-asparaginase II gene. Therefore, the recombinant virus is transferred to a silkworm to produce the L-asparaginase II connected with the polyhedron by a covalent bond; and then the L-asparaginase II is separated and purified by a polyhedral body; the recombinant virus has the characteristic of high expression level in the silkworm body; and the L-asparaginase II separated and purified by the polyhedral body has the characteristic of high purity.

The invention relates to a pharmaceutical composition containing PEGylated asparaginase. The composition comprises PEGylated asparaginase, sorbitol, a protective agent, a buffering agent and a surfactant; the protective agent is selected from mannitol, sucrose or lactose; and the buffering agent is selected from phosphate or Tris-HCl. The pharmaceutical composition containing PEGylated L-asparaginase is advantageous in that: after refrigeration, samples are not easy to degrade.

The invention discloses a thermophilic L-asparaginase mutant and a screening and fermentation method thereof, belonging to fields of gene engineering, enzyme engineering and fermentation engineering.In bacillus subtilis 168, an L-asparaginase coding gene from thermophilic bacterium Pyrococcus yayanosii CH1 is used as a template, a mutant library is built through an error-prone PCR technology, anda mutant strain with improved specific enzyme activity is screened out through a high-throughput screening method 'synchronized cell disruption and enzyme activity assay'. Mutant residues included ina positive mutant strain are analyzed, a composite mutant strain S17G / A90S / R156S / K272A with improved specific enzyme activity is built, wherein the specific enzyme activity reaches 3108U / mg. The expression level of the composite mutant in bacillus subtilis 168 is improved through a strong promoter P43 and an RBS optimization manner. Finally, the bacillus subtilis 168 containing the L-asparaginasecomposite mutant strain gene produces enzyme and is fermented in a 5L fermentation tank through medium optimization and a pH feeding coupling policy, and the enzyme activity yield of the L-asparaginase reaches 6453+ / -127U / mL.

The invention discloses L-asparaginase XiDL and a coding gene and application thereof. The protein, L-asparaginase XiDL, provided herein is originated from Xinfangfangia sp., and is named as XiDL protein, which is a protein composed of an amino acid sequence shown as a sequence 1 in a sequence table, or a protein composed of an amino acid sequence shown as a sequence 3 in the sequence table. Invention also claims application of the XiDL protein as L-asparaginase, as well as application of the XiDL protein in treatment of acute lymphocytic leukemia, lymphoid malignancies, Hodgkin lymphoma and the like. The invention also claims a preparation to prevent formation of acrylamide in oil-fried or baked foods, with the preparation having the XiDL protein as an active ingredient. The L-asparaginase XiDL has a major application prospect in the related medical field, food industry field and other fields.

The invention discloses a novel nanometer immobilization L-asparaginase reactor and a preparation method thereof. The enzyme reactor is prepared through a method including the following steps that nanometer gold prepared through a sodium citrate method is used as an enzyme immobilization material, characteristics of large specific surface area and good biocompatibility of the nanometer gold are used, enzyme is absorbed to the surface of the nanometer gold, and aggregation of enzyme-nanometer gold is prepared; and sulfydryl silane coupling agent is decorated on an inner wall of a capillary pipe, effects of sulfydryl and the nanometer gold are used, the aggregation of enzyme-nanometer gold is immobilized on an inner wall of the capillary pipe, and immobilization of L-asparaginase is achieved. The method combines the characteristics of large specific surface area of the nanometer gold, and enzymolysis efficiency of L-asparaginase on L-glutamine is effectively improved. The immobilization enzyme reactor is simple in preparation process, improves use rate and efficiency of enzyme, is favorable for developing a novel dosing method of the L-asparaginase, and provides a research basis for diagnosis and treatment of diseases.

The present invention relates to a composition for breaking down L-asparagine comprising L-asparaginase, and to a production method for L-asparaginase. The L-asparaginase of the present invention differs from existing L-asparaginase in that it has improved heat stability and exhibits high activity even at high temperatures, and thus it improves upon shortcomings of existing L-asparaginase and so can be used to advantage industrially.

The invention provides a method for efficiently expressing L-asparaginase II by recombinant escherichia coli, and an L-asparaginase II. The method for efficiently expressing L-asparaginase II by the recombinant escherichia coli comprises the steps of acquiring the recombinant escherichi for expressing L-asparaginase II; culturing a seed solution; carrying out fermenting cultivation; and acquiring the L-asparaginase II, wherein the fermenting cultivation is carried out by step by step. The method for efficiently expressing L-asparaginase II by the recombinant escherichia coli is simple in process and convenient for operations, has high output and reduces production cost.

The disclosure provides a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, and methods of preparation and use thereof.

The invention relates to application of rosmarinic acid in an escherichia coli L-asparaginase enzyme activator, which belongs to the technical field of biomedicine. The application comprises the following steps: firstly, preparing an L-asparaginase enzyme extract from escherichia coli; secondly, adding the rosmarinic acid and an L-asparaginase which have different concentrations into an enzyme activity measuring system of the L-asparaginase respectively; thirdly, measuring the reaction speed of each reaction system, calculating the michaelis constant under the concentration of each rosmarinic acid, and judging the activation of the michaelis constant on the L-asparaginase enzyme. The rosmarinic acid can significantly improve the degradation activity of the L-asparaginase enzyme to catalyze the L-asparagine when the concentration of the rosmarinic acid is between 0.375 x 10<-4> and 3.0 x 10<-4> g / L. The application can reduce the dosage of the L-asparaginase enzyme in the process of tumor therapy clinically, and reduce side effects caused by immunogenicity of the L-asparaginase enzyme, or a rosmarinic acid derivative is designed aiming at a target point of the bacterium L-asparaginase enzyme and is used as a new antibacterial drug.

The invention relates to an acinetobacter Y-3L-asparaginase gene as well as expression and application thereof. The L-asparaginase gene disclosed by the invention is derived from a strain of acinetobacter soli Y-3 (preservation number of the strain: CGMCC NO:14831) screened from soil, and a nucleotide sequence is as shown in SEQ. ID NO.1. The L-asparaginase has good catalytic activity, effectivelyinhibits the formation of acrylamide in fried foods, can fundamentally control the production of potential carcinogenic substance acrylamide in the high-temperature processing of starch-containing foods, and has a broad application prospect in the field of food processing.

The present invention discloses one kind of new compound, polyethylene glycol derivative modified L-asparaginase in the structure as shown. The present invention also provides the preparation of the polyethylene glycol derivative modified L-asparaginase and its medicine composition. The compound of the present invention has homogeneous component, high antitumor effect, high chemical stability, and low immunogenicity.

Variant Erwinia chrysanthemi L-asparaginases with reduced L-glutaminase activity and enhanced in vivo circulation are described as are fusion proteins containing an L-asparaginase and three tandem soluble domains of TRAIL for use in the treatment of cancers such as acute lymphoblastic leukemia and acute myeloid leukemia.

The invention discloses a method for producing a red blood cell drug containing L-ASPase II (L-asparaginase) by in vitro induction. The method comprises the steps of: 1) using a lentiviral vector system to transform induced pluripotent stem (IPS) cells: I. constructing a pLenti6.3 / V5-GW / Em-GFP VerA-L-ASPase II plasmid; II. using 293T cells to perform packaging so as to generate high titer virus particles containing L-ASPase II; III. using the virus particles to transfect induced pluripotent stem cells; and 2) inducing the induced pluripotent stem cells in vitro to generate L-ASPase II-containing red blood cells: I. subjecting IPS cells to in vitro differentiation induction to form an erythroid body EB; and II. performing induced differentiation on EB to obtain mature red cells. The method provided in the invention performs genetic modification on IPS cells by the lentiviral vector system, and induces them in vitro to generate red blood cells containing L-ASPase II. The generated red blood cells can be used as a sustained release carrier of L-ASPase II, and also can maintain the original shapes and functions of red blood cells. The toxic and side effects of L-ASPase II are further reduced, so that the method lays a solid foundation for future clinical application.

The invention discloses a recombinant escherichia coli efficiently transforming fumaric acid into L-asparagine as well as a construction method and an application thereof. Fumarase encoding genes fumA, fumB, fumC in an ammonium-tolerant escherichia coli BEW308 are inactivated, and then L-aspartase encoding and L-asparaginase encoding genes are inserted into the positions of the fumarase encoding fumAC genes, thereby obtaining the recombinant escherichia coli having no malic acid by product, less L-aspartic acid accumulated, and a major product L-asparagine. The invention also discloses a construction method and an application of the bacterial strain. The recombinant escherichia coli can realize constitutive high-activity expression of L-aspartase and L-asparaginase, and ultimately achieve transformation of fumaric acid into L-asparagine.