The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase isolated from Bacillus laterosporus J1 is capable of hydrolyzing GL-7-ACA and GL-7-ADCA to glutaric acid and the corresponding beta-lactam rings. Traditionally, J1 acylase was classified as class V of GL-7-ACA acylase (GCA). However, the amino acid sequence of J1 acylase has lower than 5% homology to acylases isolated from Pseudomonas strains. J1 acylase consists of a single peptide of molecular weight ∼78 kDa, in contrast with the heterodimeric nature of other cephalosporin acylases. Previous studies on this enzyme described only the specific activity, substrate preference, pH optimum and thermostability. Its tertiary structure and catalytic mechanism were not investigated in detail. It is interesting that the J1 acylase showed totally different structure from other classes of acylases but possessed the same hydrolytic activity towards cephalosporins. Homolog search revealed that J1 acylase showed 25% to 35% sequence identity to several alpha/beta-hydrolases including cocaine esterase (CocE) and alpha-amino acid ester hydrolases (AEHs). The putative catalytic triad residues conserved in J1 acylase were S125, D264 and H309, while the oxyanion-hole residues were Y57, Y126 and W173. The putative catalytic S125 was located within a highly conserved motif GXS&barbelow;YXG observed among S-15 peptidases. Secondary structure analysis had revealed alpha/beta-hydrolase fold at the N-terminal region. The catalytically important residues were located at positions where corresponding residues were found in alpha/beta-hydrolases. Tertiary structure was elaborated by homology modeling based on the X-ray structures of CocE and Acetobacter turbidans AEH (Pdb entries: 1JU3 and 1NX9, respectively). The models had demonstrated the three structural domains observed in CocE, with the putative catalytic triad residues positioned at the bottom of the active site cleft. Other catalytically important residues were identified according to the amino acid sequence alignments and residue superimpositions in tertiary structural model. Parallel site-directed mutagenesis experiments on these sites were performed to validate their functions. The mutants S125A, D264A, H309A, Y57A and Y57F were completely inactive to GL-7-ACA. Substituting Y126, V158, W173, W240 and L266 with an alanine resulted in decrease in catalytic efficiency. The two inflection points observed in the pH rate profile with pKa of 5.9 and 9.0 had indicated, respectively, that H309 and Y57 were involved in catalysis. Further kinetic and substrate spectrum studies had elucidated the substrate binding mechanism of J1 acylase. This study had demonstrated the previous classification of J1 acylase in the GCA classes is improper. I propose that this enzyme should be included in the alpha/beta-hydrolase superfamily evolved from the same origin of CocE and AEHs with catalytic activity towards cephalosporins. / by Yau Ming-hon. / "August 2005." / Adviser: Wang Jun. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3605. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 113-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

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Three different types (A, B, and C) of bile salt hydrolase from different Bifidobacterium strains revealed during the purification study showed the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by SDS-PAGE was around 35 kDa and the native molecular mass in all types of BSH was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, pI value of 4.45 was obtained with type B, but type A and C BSHs showed similar pI values of around 4.65. While the N-terminal amino acid sequences of types A and B were highly homologous (19/20), six out of twenty amino acid residues were different in the N-terminal sequences of types A and C. / As the type A bsh gene was cloned from a strain of B. longum and the nucleotide sequence became available from the GenBank, our study has focused on the cloning and characterization of the type B and C bsh genes from Bifidobacterium strains. / The type B bsh gene was cloned from B. bifidum ATCC11863 and the DNA flanking the bsh gene was sequenced. The 951 by-long bsh gene encoded a 316-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.48. For the first time in the genus Bifidobacterium, the transcriptional start point of the bsh gene was identified by primer extension analysis. Furthermore, Northern blot analysis revealed that B. bifidum bsh was transcribed as a monocistronic unit, contrary to that of B. longum bsh. Despite a high level of sequence similarity among the bsh genes, a BSH type-specific primer set based on the variable regions of bsh genes was designed in order to differentiate B. bifidum strains from the other species of Bifidobacterium commonly detected in the human gut. / The type C bsh gene was cloned from a bile salt tolerant strain of Bifidobacterium and the DNA flanking the bsh gene was further identified by a thermal asymmetric interlaced PCR (TAIL-PCR) technique. The 945 by-long bsh gene encoded a 314-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.71. A predicted BSH promoter (Pbsh) sequence was experimentally verified and the transcriptional start point of the type C bsh gene was determined by primer extension analysis. An operonic structure including the type C bsh gene and two more ORFs, which were found within a complete set of a promoter and a transcription terminator, was identified in this study for the first time in the genus Bifidobacterium, and the polycistronic bsh transcript was revealed by RT-PCR and Northern blot analysis.