Cloning Of L-lactate Dehydrogenase Gene From Streptococcus Bovis And Expression In Escherichia Coli

Lactic acid is an important organic acid. It can be used in food, medicine, tanning, textile, cigarette industry, chemical industry, environmental protection, agriculture and many other applications. Using L-lactate to produce a new-type polyester: polylactate (PLA). PLA is a high-quality plastic that has the advantages, compared to existing plastics, of being biodegradable and good process capability. So it aroused the attention from world for eliminating "white polluting" and protecting the environment.At present, the annual requirement of lactic acid in the world increases progressively at the speed of 15%. In the traditional lactic acid production methods, DL-lactic acid can be synthesized by chemosynthesis method, its cost is too high and its raw materials are venomousness; Optical purity lactate can be produced by Enzyme catalysis. Due to it's more complicated technology and it's high demanding for working condition, microbial fermentation has become the important method to produce lactic acid.The gene encoding L-(+)-lactate dehydrogenase (LDH) was amplified from genomic DNA of streptococcus bovis by PCR. The gene of IdhL was inserted into expression vector pSE380 and then transformed into E.coli JM109. The recombinant organism was induced to express LDH with IPTG. SDS-PAGE showed that the relative molecular weights of the IdhL expression product was about 36KD. The LDH activity was detected by ultraviolet spectrophotometric method. The activity of L-(+)-Lactate Dehydrogenase of recombinant strain was 168.2U/mL, is higher than 74.1 U/mL of contrast. The optimal temperature of the recombinant enzyme was 20℃~25℃and the optimal PH was 5.0-5.5. The stability of the plasmid pSE-ldh in E.coli JM109 was 99.9%.Replacement plasmid for homologous recombination was constructed by inserting tet~R gene into D-(-)-lactate dehydrogenase gene. We plan to get the homologous recombination of idhA gene in E.coli chromosomes achieved by using nature linearized DNA fragments, and then the D-lactate dehydrogenase deficient strain was obtained.This research successfully introduce the novel pathway for fermenting glucose to L-lactate in E.coli by expressing streptococcus bovis gene encoding LDH, which was reported for the first time in China.