Cloning and characterization of a gene encoding phosphoketolase in a Lactobacillus paraplantarum isolated from Kimchi.
A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria (LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgamo sequence (aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7 (LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate (TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase (GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32 mg/400 ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78 mg/400 ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The K(M) and Vmax values for fructose-6-phosphate were 5.08 +/- 0.057 mM (mean +/- SD) and 499.21 +/- 4.33 micromol/min/mg, respectively, and the optimum temperature and pH for the production of acetyl phosphate were 45 degrees C and 7.0, respectively.
Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
Do-Won
DW
Jeong
Jung Min
JM
Lee
Hyong Joo
HJ
Lee
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D017124:Conserved Sequence / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005516:Food Microbiology; D005636:Fructosephosphates / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007723:Korea; D007778:Lactobacillus / Q000145:classification* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013835:Thiamine Pyrophosphate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D005636:Fructosephosphates; D011993:Recombinant Fusion Proteins; C027618:fructose-6-phosphate; D000446:Aldehyde-Lyases; C022367:phosphoketolase; D013835:Thiamine Pyrophosphate