Characterization of the smallest dimeric bile salt hydrolase from a thermophile Brevibacillus sp.
A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60 degrees C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K(M) and k(cat) values of 3.08 microM and 6.32 x 10(2) s(-1), respectively, for glycodeoxycholic acid. The NH(2)-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source.
Sridevi
N
N
Biochemical Sciences Division, National Chemical Laboratory, Pune 411008, India.
Srivastava
Sameer
S
Khan
Bashir Mohammad
BM
Prabhune
Asmita Ashutosh
AA
D000581:Amidohydrolases / Q000737:chemistry; D001406:Bacillaceae / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000737:chemistry; D002847:Chromatography, Agarose / Q000379:methods; D004275:DNA, Ribosomal / Q000737:chemistry; D019281:Dimerization; D005998:Glycine / Q000737:chemistry; D006002:Glycodeoxycholic Acid / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry*; D007700:Kinetics; D017434:Protein Structure, Tertiary; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D013696:Temperature
D001647:Bile Acids and Salts; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D006002:Glycodeoxycholic Acid; D006867:Hydrolases; D000581:Amidohydrolases; C023154:choloylglycine hydrolase; D005998:Glycine