Kim Geun-Bae, Miyamoto Carol M, Meighen Edward A, Lee Byong H
Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore Rd., Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada.
Appl Environ Microbiol. 2004 Sep;70(9):5603-12. doi: 10.1128/AEM.70.9.5603-5612.2004.
Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.
对来自两歧双歧杆菌ATCC 11863的纯化胆盐水解酶（BSH）进行的生化特性分析揭示了一些在其他双歧杆菌物种中未观察到的独特特征。从两歧双歧杆菌中克隆了bsh基因，并对bsh基因两侧的DNA进行了测序。将克隆基因推导的氨基酸序列与先前已知序列进行比较，发现与几种微生物的BSH酶和球形芽孢杆菌的青霉素V酰胺酶（PVA）具有高度同源性。PVA的假定活性位点高度保守，包括Cys-1残基的活性位点。通过用化学和结构相似的残基Ser或Thr取代Cys，证实了N端半胱氨酸中SH基团的重要性，这两种取代均导致酶失活。通过引物延伸分析确定了bsh基因的转录起始点。与长双歧杆菌bsh不同，两歧双歧杆菌bsh作为单顺反子单元转录，这通过Northern印迹分析得到证实。用类型特异性引物组进行PCR扩增显示，在两歧双歧杆菌物种内其bsh基因具有高度的序列同源性。
