Expression of the Pediococcus acidilactici ldh L gene in Propionibacterium freudenreichii var. shermanii LAG16424T

As a cloning vector for transformation in Propionibacterium sp., pPK705 shuttle vector was experimented,
where it consists of the indigenous plasmid of Propionibacterium acidipropionici E214 (pRG01),
plasmid pUC18 and the hygromycin resistance gene (hyg B). A 1.2 Kb BamHI-EcoRI DNA fragment encoding
ldh L gene of Pediococcus acidilactici DG302 was cut from plasmid pGID150 and further subcloned in
pPK705. Transformation and expression of the ldh L gene of pediococci in NAD+-independent LDH Propionibacterium
freudenreichii var. shermanii LAG16424T was confirmed. The absolute L (+) LDH enzyme activity
was found to be (25.67 to 42.79 IU/ml) for Propionibacterium transformants cell free extract. Twenty Propionibacterium
transformants were further inoculated in skimmed milk, where curdling was detected after 48 h
with one of the prominent transformants. After 144 h, all transformants coagulated skimmed milk as compared
to parental bacterial strain. Maximum % of lactic acid produced by Propionibacterium transformants was
0.79 % as lactic acid after 96 h. Further proof for the expression of NAD+-dependent L (+) LDH through the
transformed ldh L gene was confirmed by polyacrylamide gel electrophoresis