β-Galactosidase of Pediococcus species: induction,purification and partial characterization

Summary Six strains of Pediococcus pentosaceus and two of P. acidilactici had intracellular -galactosidase (-gal) activity when grown in the presence of lactose; all but two strains of P. pentosaceus and one of P. acidilactici had such activity when grown in the presence of glucose. Synthesis of -gal by P. pentosaceus ATCC 25745 was inducible with lactose, galactose, melibiose, lactobionic acid and possibly cellobiose but not with glucose, sucrose, maltose, glycerol, fructose or mannose. Lactose, galactose and possibly maltose, melibiose and lactobionic acid but not glucose, sucrose, glycerol, cellobiose, fructose or mannose induced -gal synthesis by P. acidilactici ATCC 25740. Synthesis of -gal was partially inhibited in P. pentosaceus ATCC 25745 and P. acidilactici ATCC 25740 by glucose added to the medium during growth in the presence of galactose or lactose. Isopropyl -d-thiogalactopyranoside failed to induce synthesis of -gal by either strain during growth on glucose. -Gal from P. pentosaceus ATCC 25745 had a molecular weight of 66,000 and activity optima of pH 6.5 and 45° C. Activity of the enzyme was stimulated by reducing agents, Mg²⁺, Mn²⁺, Zn²⁺ and Co²⁺ but not by Ca²⁺, and was markedly inhibited by ethylenediaminetetraacetate (EDTA), HgCl₂, 1,10-phenanthroline, and an oxidizing agent. The K _mvalues of the enzyme for o-nitrophenol--d-galactopyranoside and lactose were 3.07 and 7.0 mM, respectively, suggesting its low affinity for lactose. Offprint requests to: E. H. Marth     

Aquaporins – Expression,purification and characterization

Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.     

Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α

Summary The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK ) as reported by others (2). In the absence of calcium the K_m of PhK for phosphorylase b was 53 + 10 U/ ml with a V_m of 17 = 1 U/gm of tissue. The K_m of PhK for phosphorylase b was 20 + 2 U/ml with a V_m of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase(A_0.5 0.03 M). In the presence of 0.1 M calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the V_m of both forms, but the effect was greater for PhK b than for PhK . The K_m of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts.Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 × 106. The pII profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.     

Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Aims: This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. Methods and Results: The β‐glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l^?1) and Cu²⁺ (5 mmol l^?1) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β‐glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K_m and V_max values for 4‐nitrophenyl‐β‐d ‐glucopyranoside were 0·38 mmol l^?1 and 5·21 nmol min^?1, respectively. The enzyme was stable at pH 5·0 with a value of t_1/2 = 50 days for the crude extract. Conclusions: The β‐glucosidase of O. oeni ST81 is substantially different from those characterized from other wine‐related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β‐glucosidase from O. oeni ATCC BAA‐1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine‐related compounds on the enzymatic activity and long‐term stability of the enzyme could be of interest for winemaking. Significance and Impact of the Study: Information regarding a β‐glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β‐glucosidase purified from crude extracts of O. oeni ST81.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.     

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct

Coronavirus envelope (E) proteins are short (～100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.     

Isolation and characterization of a lytic bacteriophage φKp-lyy15 of Klebsiella pneumoniae

<正>Dear Editor,Bacteriophages(phages)are viruses that specifically infect and kill bacteria.They are ubiquitous throughout all environments that bacteria inhabit.Following their discovery by F.W.Twort in 1915 and F.d'Herele in 1917,bacteriophages were recognized as potential agents to treat bacterial diseases and phage therapy has been used     

High-cell-density cultivation of recombinant Escherichia coli,purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase

We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5′ and 3′ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available. Received: 24 August 2000 / Accepted: 15 December 2000     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Identification, purification, and characterization of a secretory serine protease in an Indian strain of Leishmania donovani

An aprotinin sensitive serine protease was identified in the culture supernatant of the Indian strain of Leishmania donovani (MHOM/IN/1983/AG83). The protease was subsequently purified and characterized. The apparent molecular mass of the enzyme was 115 kDa in SDS-PAGE under non-reducing condition, while on reduction it showed a 56 kDa protein band indicating that the protease is a dimeric protein. The purified enzyme was optimally active at the pH and temperature of 7.5 and 28 degrees C, respectively. Assays of thermal stability indicated that the enzyme preserved 59% of activity even after pretreatment at 42 degrees C for 1 h. The purified protease was not glycosylated and its isoelectric pI was 5.0. N-alpha-p-tosyl-L-arginine methylester (TAME) appeared to be relatively better substrate among the commonly used synthetic substrates. The enzyme was inhibited by Ca(2+) and Mn(2+), but activated by Zn(2+). The protease could play important role(s) in the pathogenesis of visceral leishmaniasis or kala-azar.     

Isolation and characterization of a gene encoding α-tubulin from Candida albicans

A gene encoding the α-tubulin of Candida albicans has been cloned and characterized. Nucleotide sequence analysis reveals the presence of an intron within the structural gene and predicts the synthesis of a polypeptide of 448 amino acid residues. Comparison of nucleotide and amino acid sequences with the Saccharomyces cerevisiae α-tubulin encoding genes shows a 75% homology and about 92% similarity respectively. In contrast to S. cerevisiae, C. albicans appears to possess only one gene for α-tubulin which is able to functionally complement a S. cerevisiae cold-sensitive tub1 mutant.     

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture

Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme α-glucuronidase. A gene (deg75-AG) encoding a putative α-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45?°C. Unlike other α-glucuronidases, the DEG75-AG had a more basic pH optimum of 7-8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.     

Purification and partial characterization of a gibberellin 2β-hydroxylase fromPhaseolus vulgaris

A gibberellin 2β-hydroxylase has been purified from mature seeds ofPhaseolus vulgaris. The enzyme is of molecular weight 36,000 and has the characteristics of a dioxygenase; the cofactors areα-ketoglu-tarate, Fe²⁺ and ascorbate, and activity is stimulated by catalase. The V_max of the enzyme is 6.86 nmole h^?1 mg^?1, and the Km values for [1,2-³H₂]GA₁ andα-ketoglutarate are 0.085 μM and 21 μM, respectively. The purified enzyme preparation catalyzes hydroxylation of GA₁, GA₄, GA₉, and GA₂₀ but exhibits a marked preference for the 3-hydroxylated gibberellins as substrate.     

Isolation and purification ofβ-galactosidase ofLactobacillus murinus CNRZ 313

β-Galactosidase has been isolated inLactobacillus murinus CNRZ 313, and its properties have been studied. The enzyme was purified 292-fold by chromatography on Ultrogel ACA 34, DEAE-Sephadex A-50 columns, and by affinity chromatography in agarose-p-aminophenyl-β-d-thiogalactoside. The purified extract exhibited a single band following polyacrylamide gel electrophoresis (PAGE). Molecular weight was estimated to be 170,000 on an Ultrogel ACA 34 column. Maximum enzymatic activity was observed at 45°C and pH 7 in 50 mM phosphate buffer. The Km for ONPG and ONPG + 20 mM of lactose were 480 μM and 870 μM, respectively. The effect of different salts on the enzymatic activity was studied. An inhibitory effect was observed when using 10 mM CaCl₂, and a stimulating effect when using 10 mM MgCl₂. The latter protected the enzyme from thermal denaturation. Among the protective agents belonging to the sulfhydryl group that was tested, mercaptoethanol and dithiotreitol acted as activators, while glutathione and cysteine acted as inhibitors, of the enzymatic activity.p-β-Galactosidase activity was not observed.