N, O-diacetyl Muramidase R2 Gene Cloning And Expression In Bacillus Subtilis And Pichia Pastoris,

N, O-diacetymuramidase R2 gene was amplified by PCR from the N, O-diacetymuramidase R2 chromosome. The recombinant plasmid pMD18-T~R2 was successfully constructed. After restriction enzymes analyzing and PCR identification , it was confirmed that the inserted heterogeneous gene was muramidase R2 gene. R2 gene was sequenced and analyzed, the result showed that it was identical to the reported N, O-diacetymuramidase R2 gene which consisted of 655 bp. The restriction map of R2 gene was construed for further use.Hind Ⅲ and Kpn Ⅰ were used together to digest pMD18-T-R2 and plasmid pMA, then pMA5-R2 was constructed by linkage reaction. Recombinant plasmid pMA5-R2 was digested by Sst I to remove the sequence irrelative to gene expression. Then the digested pMA5-R2 self-linked and was transformed to BCL1050. Positive transformants were screened from Kanamycin plates. N, O-diacetymuramidase R2 gene was highly expressed by the acquired positive transformants, and the activity was up to 138U/ml. The activity can reach 240 U/ml after optimizing the fementation conditions. Compared with growing in LB culture medium , the activity increased 1.75 times.Digesting by FcoRI and Not I, R2 was inserted in MCS of pPIC9K according to the right reading frame translational direction. Recombinant plasmid pPIC9K-R2 was successfully constructed and transformed into His" phenotype SMD1168 by electroporation. First selecting transformants first by MD, then MM, MD and YPD of (0. 5-5mg/mL)G418. Only one Pichia pastoris transformant with multiple copies, which can resist 4mg/ml G418, was obtained. The activity can amount to 156U/mL after methanol inducing.Both of the recombinant muramidases indicated nearly the same properties as nature enzyme, except the lower activity.