The present application discloses a PEGylated asparaginase and use thereof. In this application, the polyethylene glycol (PEG) is coupled to the N-terminal amino of 1 or 2 subunits of L-asparaginase, and the molecular weight of the PEG is 30-40 KDa. The PEG is preferably branched and has an aldehyde serving as an activating group. The PEGylated asparaginase is useful in the preparation of anti-tumor drugs.

The disclosure provides, in various embodiments, polypeptides that upon administration to a mammalian subject, elicit a reduced immunogenicity in the subject, relative to a bacterial asparaginase. The disclosure also provides, in various embodiments, fusion proteins comprising one or more of the polypeptides, polynucleotides encoding the polypeptides, vectors and host cells suitable for expressing the polypeptides, methods for treating a disease (e.g., cancer) in mammalian subject (e.g., a human), and methods for reducing a level of asparagine or asparagine-containing product in a biological fluid from a mammalian subject.

The disclosure provides, in various embodiments, polypeptides that upon administration to a mammalian subject, elicit a reduced immunogenicity in the subject, relative to a bacterial asparaginase. The disclosure also provides, in various embodiments, fusion proteins comprising one or more of the polypeptides, polynucleotides encoding the polypeptides, vectors and host cells suitable for expressing the polypeptides, methods for treating a disease (e.g., cancer) in mammalian subject (e.g., a human), and methods for reducing a level of asparagine or asparagine-containing product in a biological fluid from a mammalian subject.

Provided herein are methods of production of recombinant E. coli asparaginase. Methods herein allow production of asparaginase in Pseudomonadales host cells at high expression levels and having activity comparable to commercially available asparaginase preparations.

Provided herein are methods of production of recombinant E. coli asparaginase. Methods herein allow production of asparaginase in Pseudomonadales host cells at high expression levels and having activity comparable to commercially available asparaginase preparations.

The present invention refers to polypeptides with asparaginase activity that have an increased rate of self-processing compared to human wild L-asparaginase (ASRGL1), with mutation in the ASRGL1 glycine rich loop called HGG loop (Histidine 8-Glycine 9-Glycine 10). Polynucleotides that encode the polypeptides of invention are also described here, expression cassettes comprising so-called polynucleotides, expression vectors, host cells, pharmaceutical compositions, uses of the invention polypeptide in the manufacture of a preventive medicine or cancer treatment and methods to produce the polypeptide of invention and to prevent or treat cancer.

The present disclosure discloses a thermophilic L-asparaginase mutant and screening and fermentation methods thereof, and belongs to the field of gene engineering, enzyme engineering and fermentation engineering. In Bacillus subtilis 168, a Pyrococcus yayanosii CH1-derived L-asparaginase encoding gene is used as a template, and a mutation library is constructed by an error-prone PCR (epPCR) technology. A mutant strain with improved specific enzyme activity is screened through a high-flux screening method of synchronous cell disruption and enzyme activity measurement. Mutated residues included in a positive mutant are analyzed to construct a composite mutant strain S17G/A90S/R156S/K272A with improved specific enzyme activity and specific enzyme activity of 3108 U/mg. An expression quantity of the composite mutant strain in the Bacillus subtilis 168 is increased through measures of a strong promoter P.sub.43 and RBS optimization. Finally, the Bacillus subtilis 168 with a gene of the L-asparaginase composite mutant strain is subjected to enzyme production fermentation in a 5 L fermentation tank through culture medium optimization and pH and feeding coupling strategies. The enzyme activity yield of the L-asparaginase is up to 6453+/−127 U/mL.

The present disclosure discloses a thermophilic L-asparaginase mutant and screening and fermentation methods thereof, and belongs to the field of gene engineering, enzyme engineering and fermentation engineering. In Bacillus subtilis 168, a Pyrococcus yayanosii CH1-derived L-asparaginase encoding gene is used as a template, and a mutation library is constructed by an error-prone PCR (epPCR) technology. A mutant strain with improved specific enzyme activity is screened through a high-flux screening method of synchronous cell disruption and enzyme activity measurement. Mutated residues included in a positive mutant are analyzed to construct a composite mutant strain S17G/A90S/R156S/K272A with improved specific enzyme activity and specific enzyme activity of 3108 U/mg. An expression quantity of the composite mutant strain in the Bacillus subtilis 168 is increased through measures of a strong promoter P.sub.43 and RBS optimization. Finally, the Bacillus subtilis 168 with a gene of the L-asparaginase composite mutant strain is subjected to enzyme production fermentation in a 5 L fermentation tank through culture medium optimization and pH and feeding coupling strategies. The enzyme activity yield of the L-asparaginase is up to 6453+/−127 U/mL.

A novel protein deamidase having an activity of directly acting on a side chain amide group of an asparagine residue in a protein to form a side chain carboxyl group and release ammonia, a microorganism that produces the same, a gene encoding the same, a method for producing the same, and use of the same are provided. A bacterium classified into the class Actinobacteria is cultured to generate protein deamidase, and the enzyme is collected from culture.

A novel protein deamidase having an activity of directly acting on a side chain amide group of an asparagine residue in a protein to form a side chain carboxyl group and release ammonia, a microorganism that produces the same, a gene encoding the same, a method for producing the same, and use of the same are provided. A bacterium classified into the class Actinobacteria is cultured to generate protein deamidase, and the enzyme is collected from culture.