Expression, purification, and characterization of arginine deiminase from Lactococcus lactis ssp. lactis ATCC 7962 in Escherichia coli BL21.
The arcA gene that encodes arginine deiminase (ADI, EC 3.5.3.6)--a key enzyme of the ADI pathway--was cloned from Lactococcus lactis ssp. lactis ATCC 7962. The deduced amino acid sequence of the arcA gene showed high homology with the arcA gene from Lactobacillus plantarum (99%) and from Lactobacillus sakei (60%), respectively. The arcA gene from Lc. lactis spp. lactis ATCC 7962 was expressed in soluble fraction of recombinant Escherichia coli BL21. ADI produced from Lc. lactis spp. lactis ATCC 7962 (LADI) in E. coli BL21 (DE3) was purified using sequential Q-Sepharose anion exchange and Sephacryl S-200 gel filtration column chromatography. The final yield of LADI in the purification procedure was 63.5%, and the specific activity was 140.27 U/mg. The presence of purified LADI was confirmed by N-terminal sequencing and determination of the molecular mass. The LADI had a molecular mass of about 140 kDa, and comprised a homotrimer of 46 kDa in the native condition. LADI exhibited only 35% amino acid sequence homology with ADI from Mycoplasma arginini. However, LADI shared a similar three dimensional structure. The K(M) and V(max) values for arginine were 8.67+/-0.045 mM (mean+/-SD) and 344.83+/-1.79 micromol/min/mg, respectively, and the optimum temperature and pH for the production of LADI were 60 degrees C and 7.2.
Kim
Jong-Eun
JE
Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea.
Jeong
Do-Won
DW
Lee
Hyong Joo
HJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011994:Recombinant Proteins; D006867:Hydrolases; C019723:arginine deiminase