Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus.
Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.
Wang
Xiuwen
X
Experiment Medical Research Center, Binzhou Medical University, Yantai, People's Republic of China.
Zheng
Zhaojuan
Z
Dou
Peipei
P
Qin
Jiayang
J
Wang
Xiaochen
X
Ma
Cuiqing
C
Tang
Hongzhi
H
Xu
Ping
P
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019344:Lactic Acid / Q000096:biosynthesis*; D052201:Lacticaseibacillus rhamnosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment
D001426:Bacterial Proteins; D019344:Lactic Acid; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase; C021379:D-2-hydroxyacid dehydrogenase