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

Abstract

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 Bacillus subtilis L-asparaginase (Asp) gene in Escherichia coli. Materials and methods PCR was used for the amplification of Asp gene of B. subtilis. Asp gene was cloned with the blunt vector pJET1.2 under the control of T7 or lacUV5 promoters. Transformation of both plasmids was done in E. coli JM 107. L-asparaginase was determined in E. coli JM 107 recombinant strains. Results Bacillus Asp gene was amplified using PCR and the primers were deduced from the published ansA sequence. PCR program was optimized. The PCR product (1112 bp DNA fragment containing the Asp 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 T7 or lacUV5 promoters. The recombinant plasmids containing the Asp gene were transformed and expressed into E. coli JM 107. The expression level of Asp in the recombinant stains was increased up to 22 U/ml, which is 2.5-fold higher than that of E. coli JM 107 wild type. The vector promoters had regulatory effect on L-asparaginase production, where the activity under the control of T7 promoter was increased by 47% compared with that of lacUV5 promoter. Conclusion L-asparaginase production could be improved by cloning and by the expression of its corresponding gene in E. coli. The T7 promoter had a higher regulatory effect on L-asparaginase production level than lacUV5 promoter.