Biotechnological approach for the production of L-asparaginase from locally isolate

Background and objectives
L-asparaginase is a therapeutic enzyme used for the treatment of hematopoietic diseases, for example, acute lymphoblastic leukemia. It has many applications other than as an anticancer agent, which includes in the treatment of autoimmune diseases, infectious diseases, antimicrobial property, canine and feline cancers. The aim of this study is to increase the production level of L-asparaginase by the cloning and expression of L-asparaginase () gene in .
Materials and methods
PCR was used for the amplification of Asp gene of . gene was cloned with the blunt vector pJET1.2 under the control of or promoters. Transformation of both plasmids was done in JM 107. L-asparaginase was determined in JM 107 recombinant strains.
Results


gene was amplified using PCR and the primers were deduced from the published sequence. PCR program was optimized. The PCR product (1112 bp DNA fragment containing the gene) was detected, purified, and sequenced. The DNA sequence including the complete Asp CDS sequence was deposited in GenBank (GenBank accession number KJ642620.1). The amplicon was cloned using the blunt vector pJET1.2 under the control of or promoters. The recombinant plasmids containing the Asp gene were transformed and expressed into JM 107. The expression level of in the recombinant stains was increased up to 22 U/ml, which is 2.5-fold higher than that of JM 107 wild type. The vector promoters had regulatory effect on L-asparaginase production, where the activity under the control of promoter was increased by 47% compared with that of promoter.
Conclusion
L-asparaginase production could be improved by cloning and by the expression of its corresponding gene in . The promoter had a higher regulatory effect on L-asparaginase production level than promoter.