Bile salt sulfotransferase in guinea pig liver

Bile salt sulfotransferase from guinea pig liver is purified by the procedures of ammonium sulfate fractionation, Sephadex G-100 column chromatography, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and polyacrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 6.8, an isoelectric point of 5.6 and a molecular weight of 7600 estimated by gel filtration technique. The apparent K_m values of the enzyme are 7.7·10^?5 M for taurolithocholate and 1.4·10^?6 M for 3′-phosphoadenosine 5′-phosphosulfate. It requires Mg²⁺ and free sulfohydryl group(s) for activity. The enzyme reacts with hydroxy groups of bile salts at both 3α and 3β positions. No activity is found in the kidney of guinea pig. The purified enzyme does not react with estrone, estradiol, testosterone, dehydroepiandrosterone, cholesterol, phenol, tyramine, and serotonin. The results indicate that bile salt sulfotransferase is distinct from other hepatic sulfotransferases.     

Partial purification of a human liver sulphotransferase active towards bile salts

An enzyme that catalyzes the transfer of sulphate from 3'-phosphoadenosine 5'-phosphosulphate to bile salts was purified from human liver cytosol by chromatography on DEAE-Sephadex and Sephadex G-200, by agarose suspension electrophoresis and by isoelectric focusing in free solution. The purified enzyme was also active towards oestrone, dehydroepiandrosterone and phenol. No other liver steroid sulphotransferases could be detected during this purification procedure.K_m values of 1.8 · 10⁻⁶ M and 3.3 · 10⁻⁶ M for glycolithocholate and 3'-phosphoadenosine 5'-phosphosulphate respectively were found. The sulphotransferase has an isoelectric point of 5.5. The enzyme was markedly activated by Mg²⁺, Mn²⁺ and Co²⁺ and inhibited by Cu²⁺, Fe²⁺ and Zn²⁺.Chenodeoxycholate and deoxycholate were sulphated at the 7-OH and 12-OH position, respectively.No bile salt disulphate formation was detected.A 30-fold increase in specific activity was obtained, although the purification based on ultraviolet light measurements was considerably higher.     

5′-Haloacetamido-5′-deoxythymidines: novel inhibitors of thymidylate synthase

5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [¹⁴C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I₅₀ values for BAT were 1·10⁻⁵ M and 1.2·10⁻⁶ M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The K_m for the enzyme was 9.2 μM, and the K_i determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min⁻¹, and the steady-state constant of inactivation, K_i, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Redox-linked Gating of Nucleotide Binding by the N-terminal Domain of Adenosine 5′-Phosphosulfate Kinase

Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine 5′-phosphosulfate (APS) to 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Crystallographic studies of APSK from Arabidopsis thaliana revealed the presence of a regulatory intersubunit disulfide bond (Cys⁸⁶–Cys¹¹⁹). The reduced enzyme displayed improved catalytic efficiency and decreased effectiveness of substrate inhibition by APS compared with the oxidized form. Here we examine the effect of disulfide formation and the role of the N-terminal domain on nucleotide binding using isothermal titration calorimetry (ITC) and steady-state kinetics. Formation of the disulfide bond in A. thaliana APSK (AtAPSK) inverts the binding affinities at the ATP/ADP and APS/PAPS sites from those observed in the reduced enzyme, consistent with initial binding of APS as inhibitory, and suggests a role for the N-terminal domain in guiding nucleotide binding order. To test this, an N-terminal truncation variant (AtAPSKΔ96) was generated. The resulting protein was completely insensitive to substrate inhibition by APS. ITC analysis of AtAPSKΔ96 showed decreased affinity for APS binding, although the N-terminal domain does not directly interact with this ligand. Moreover, AtAPSKΔ96 displayed reduced affinity for ADP, which corresponds to a loss of substrate inhibition by formation of an E·ADP·APS dead end complex. Examination of the AtAPSK crystal structure suggested Arg⁹³ as important for positioning of the N-terminal domain. ITC and kinetic analysis of the R93A mutant also showed a complete loss of substrate inhibition and altered nucleotide binding affinities, which mimics the effect of the N-terminal deletion. These results show how thiol-linked changes in AtAPSK alter the energetics of binding equilibria to control its activity.     

The effect of thyroxine on transparency and PAPS synthesis in the avian cornea

The normal onset of developing corneal transparency, which begins on Day 14 of chick embryogenesis, can be accelerated by the in vivo application of exogenous thyroxine, as originally demonstrated by Coulombre and Coulombre (1964, Exp. Eye Res.3, 105–114). When thyroxine (5 μg) is injected at Day 6, measurements of ³⁵SO^2?₄ incorporation by corneal homogenates indicate that synthesis of 3′-adenosine phosphosulfate (APS) in the cornea is increased at Day 8, but not that of 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Injection of hormone at Day 9 results in a precocious increase in corneal transparency at Day 12 and a corresponding increase in the synthesis of APS and PAPS.     

Adenosine 5'-triphosphate-sulfurylase in corn roots and its partial purification

ATP-sulfurylase (ATP-sulfate adenyltransferase, EC 2.7.7.4) was found in nonparticulate fractions of both roots and leaves of Zea mays L. seedlings using two detection methods. Addition of exogenous pyrophosphatase was essential for maximum rates of conversion of ³⁵SO₄²⁻ to labeled adenosine phosphosulfate in unpurified root extracts, but not in unpurified leaf extracts. In the presence of exogenous pyrophosphatase, the enzyme from roots exhibited specific activities as high as those obtained with the leaf enzyme. The root enzyme was purified 33-fold by centrifugation and column chromatography procedures. Its molecular weight obtained by Sephadex gel filtration was about 42,000. Its Km for pyrophosphate was 7 μm, while for adenosine phosphosulfate, the Km was 1.35 μm. None of the enzyme fractions studied converted adenosine phosphosulfate into detectable amounts of 3′-phosphoadenosine-5′-phosphosulfate. ATP-sulfurylase was also found in roots of corn seedlings grown aseptically. The data suggest that at least the first reaction in sulfate reduction might proceed as effectively in roots as in shoots.     

Crystal Structures of the Kinase Domain of the Sulfate-Activating Complex in Mycobacterium tuberculosis

In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5’-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3’-phosphoadenosine-5’-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.     

Localization of cerebroside-sulfotransferase activity in the Golgi apparatus of rat kidney

A Golgi-rich fraction that contains both uridine diphosphogalactose: N-acetylglucosamine galactosyltransferase activity and 3′-phosphoadenosine-5′-phosphosulfate:cerebroside sulfotransferase activity has been isolated from rat kidney. Both activities are increased about 80-fold in the Golgi fraction compared to the homogenate. Little or no galactosyltransferase or sulfotransferase activity was found in purified nuclei, mitochondria, rough endoplasmic reticulum, plasma membranes and supernatant. The results indicate that both galactosyltransferase and sulfotransferase are localized in Golgi apparatus from rat kidney. This is the first evidence that Golgi apparatus functions to modify a lipid component of the cell.     

Studies of sulfate utilization by algae 18. identification of glutathione as a physiological carrier in assimilatory sulfate reduction by Chlorella

Adenosine 5′-phosphosulfate-sulfotransferase is the first enzyme in the pathway of assimilatory sulfate reduction in Chlorella, and transfers the sulfo group from APS (adenosine 5′-phosphosulfate) to a thiol acceptor forming an organic thiosulfate. In vitro, adenosine 5′-phosphosulfate-sulfotransferase transfers to a variety of thiol acceptors, the best among the monothiols is glutathione, the only one to show a regulatory interaction with adenosine 5′-phosphosulfate-sulfotransferase. To identify the physiological acceptor, adenosine 5′-phosphosulfate-sulfotransferase is assayed without thiols; the cell fraction which stimulates adenosine 5′-phosphosulfate-sulfotransferase activity is expected to contain the physiological acceptor. Boiled Chlorella extract contains physiological acceptor when assayed in the presence of adenosine 5′-phosphosulfate-sulfotransferase and NADPH. Physiological acceptor is dialyzable but is retained by filters of 1000 daltons cut off. After passage through DEAE—Sephadex A-25 and Sephadex G-25, physiological acceptor is found to be ninhydrin-positive and shows co-electrophoresis with oxidized glutathione. Upon reduction with dithiothreitol, physiological acceptor moves with reduced glutathione and on acid hydrolysis yields amino acids identical with those from authentic oxidized glutathione. Physiological acceptor, with adenosine 5′-phosphosulfate-sulfotransferase and AP³⁵ S, yields labeled glutathione—S—SO₃⁻. Thiosulfonate reductase from Chlorella reduces glutathione—S—SO₃⁻ to bound sulfide. Only one active accepting component is found in the boiled extracts.     

Substrate and inhibitor complexes of dihydrofolate reductase from amethopterin-resistant L1210 cells.

Dihydrofolate reductase (EC 1.5.1.3), purified to homogeneity from an amethopterin-resistant subline (R6) of cultured L1210 murine leukemia cells, has been used to study enzyme-substrate and enzyme-inhibitor complexes. NADPH, NADP⁺acid-modified NADPH (λ_max at 265 nm, elevated absorbance at 290 nm), 2′-phosphoadenosine-5′-diphosphate ribose, dihydrofolate, and amethopterin formed binary complexes with the enzyme. Ternary complexes could be formed by admixing the enzyme with: (a) NADPH and amethopterin; (b) NADP⁺ and tetahydrofolate; and (c) acid-modified NADPH and dihydrofolate. All of these complexes migrated as stable well-defined bands on polyacrylamide gel electrophoresis at pH 8.3. The bands could be visualized by staining both for enzyme activity and for protein. These binary and ternary complexes were also stable to extensive dialysis. Spectra of the dialyzed enzyme complexes indicated that each ligand was present at an equimolar ratio with the enzyme.     

Purification and properties of galactokinase from Tetrahymena thermophila

Galactokinase (EC 2.7.1.6; ATP: d-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50 000-55 000. The holoenzyme consists of two subunits of approx. 28 000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1 Optimal activity was obtained at pH 7.8 and 41°C, with no added monovalent salt. d-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2′-dATP and 3′-dATP as phosphate donors; ADP and adenosine-5′-[γ-thio]triphosphate are inhibitory. The K_m values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg²⁺ >Co²⁺ >Mn²⁺ >Fe²⁺. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.     

Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L⁻¹ OD⁻¹. The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L^-1 OD⁻¹ of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3′-phosphoadenosine-5′-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.     

Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394

Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni⁺²-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.     

A novel thermophilic β-mannanase with broad-range pH stability from Lichtheimia ramosa and its synergistic effect with α-galactosidase on hydrolyzing palm kernel meal

β-Mannanase is the key enzyme in the hydrolysis of mannan which has been widely applied in diverse industrial fields such as biobleaching pulps, food and feed industry, bioethanol and pharmaceutical applications. In this study, a novel GH5 family β-mannanase gene (LrMan5B) with 381 amino acid residues was identified from Lichtheimia ramosa, and highly expressed in Pichia pastoris X33. The amino acid sequence shares the highest identity (64%) with the β-mannanase from Rhizomucor miehei. Purified recombinant LrMan5B showed the optimal activity at pH 5.0 and 65 °C. It had broad-range pH stability (retaining >65% activity after incubation at pH 3.0–8.0 at 37 °C for 24 h) and was highly thermostable (retaining >80% activity after incubation at 60 °C for 30 min). LrMan5B displayed the highest catalytic efficiency for locust bean gum and the k_cat/K_m value was 1357.47 mL·mg⁻¹·s⁻¹, followed by guar gum (512.82 mL·mg⁻¹·s⁻¹), konjac glucomannan (454.21 mL·mg⁻¹·s⁻¹), and palm kernel meal (137.00 mL·mg⁻¹·s⁻¹). In order to evaluate the synergistic effect of LrMan5B and α-galactosidase LrAgal36A from L. ramosa, LrAgal36A was supplemented to hydrolyze palm kernel meal with LrMan5B together, showing that the reducing sugar release significantly increased by 21% (compared with the sum of that by hydrolysis of single Lrman5B or LrAgal36A). Due to its favorable enzymatic properties, LrMan5B might own potential applications in the area of food and feed processing.     

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°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 μM and 6.32 × 10² s⁻¹, respectively, for glycodeoxycholic acid. The NH₂-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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrolysis of 3′,4′-dichloropropionanilide by an aryl acylamidase from Taraxacum officinale

An aryl acylamidase (aryl-acylamine amidohydrolase, E.C. 3.5. 1.a) which hydrolyses the herbicide propanil (3′,4′-dichloropropionanilide), was isolated from dandelion roots and partially purified and characterized. Specificity tests on the enzyme revealed that it could hydrolyse various chlorine ring-substituted propionanilides and 3,4-dichloroanilide alkyl compounds. The partially purified enzyme was inhibited by several sulfhydryl reagents and metal ions. The pH optimum was broad, between 7·4 and 7·8. The apparent activation energy, determined from an Arrhenius plot, was 9·0 kcal/mol (37 700 J/mol) for the hydrolysis of 3′,4′-dichloropropionanilide. The apparent K_m was 1·7 × 10⁻⁴ M with propanil as substrate.     

Carbohydrate and sulfate conjugations of p-nitrophenol by hepatopancreas of Homarus americanus

1. Glucosyltransferase activity is present in hepatopancreas of Homarus americanus. The enzyme appears to have a specific requirement for UDP-glucose, and ADP-, CDP- or GDP-glucose do not substitute for it. The activity is mainly microsomal, exhibits a pH optimum at 7.9–8.1, and its apparent K_m values are 2 mM and 0.3 mM for UDP-glucose and p-nitrophenol respectively. Microsomal glucosyltransferase activity increases with increasing temperature up to 45°.2. Hepatopancreas possesses a very active sulfotransferase which utilizes 3′-phosphoadenosine-5′-phosphosulfate for sulfoconjugation of p-nitrophenol. The activity is associated chiefly with the soluble fraction and amounts to about 16 nmoles/mg protein/30 min.3. No detectable glucuronidation of p-nitrophenol occurred when preparations of hepatopancreas fortified with UDP-glucuronic acid were incubated with p-nitrophenol.     

Adenosine 5‘-Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Properties*

Biochemical and physiological properties of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of assimilatory sulfate reduction, from spruce trees growing under field conditions were studied. The apparent K_m for adenosine 5′-phosphosulfate (APS) was 29 ± 5.5μM, its apparent M_r was 115,000. 5′-AMP inhibited the enzyme competitively with a K_i of 1 mM, but also stabilized it. MgS0₄ at 800 mM increased adenosine 5′-phosphosulfate sulfotransferase activity by a factor of 3, concentrations higher than lOOOmM were inhibitory. Treatment of isolated shoots with nutrient solution containing 1 or 2 mM sulfate, and 3 or 10 mM glutathione, respectively, induced a significant decrease in extractable adenosine 5′-phosphosulfate sulfotransferase activity over 24h, whereas GSH as well as S^2- up to 5mM cysteine and up to 200 mM SO₃^2- had no effect on the in vitro activity of the enzyme. As with other enzymes involved in assimilatory sulfate reduction, namely ATP sulfurylase (EC 2.7.7.4), sulfite reductase (EC 1.8.7.1) and O-acetyl-L.-serine sulfhydrylase (EC 4.2.99.8), adenosine 5′-phosphosulfate sulfotransferase was still detected at appreciable activities in 2- and 3-year-old needles. Adenosine 5′-phosphosulfate sulfotransferase activity was low in buds and increased during shoot development, parallel to the chlorophyll content. The enzyme activity was characterized by an annual cycle of seasonal changes with an increase during February and March.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.