Effect of nutrient deficiency on accumulation and relative molecular weight of poly-β-hydroxybutyric acid by methylotrophic bacterium,Pseudomonas 135

Summary Pseudomonas 135, a facultative methylotroph, was cultivated on methanol as a sole carbon and energy source for the accumulation of poly--hydroxybutyric acid (PHB). The cells grew fairly well on minimal synthetic medium containing 0.5% (v/v) of methanol at pH 7.0 and 30° C. The maximum specific growth rate was determined to be 0.26–0.28 h^–1 with a growth yield of 0.38 in the optimized growth medium. For stimulation of PHB accumulation in the cells, deficiency of nutrients such as NH _inf4 ^sup+ , Mg²⁺ and PO _inf4 ^sup3– was crucial even though cell growth was significantly suppressed. The PHB content of a 40-h culture was determined to be 37% of the total cell mass in NH _inf4 ^sup+ -limited medium, 42.5% on Mg²⁺-deficient medium, and 34.5% on PO _inf4 ^sup3– -deficient medium. The maximum content of PHB in the cells could reach 55% in NH _inf4 ^sup+ -limited fed-batch culture. The average relative molecular eight determined by gel permeation chromatography was 3.7 × 10⁵ in NH _inf4 ^sup+ -limited culture, 2.5 × 10⁵ in Mg²⁺-deficientmedium, and 3.1 × 10⁵ in PO _inf4 ^sup3– -deficient medium. Polydispersity determined in each culture was relatively high (about 10–11). The solid PHB had a melting temperature of 173° C. Correspondence to: J. M. Lebeault     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a beta-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN'-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The K(m) value for hydrolysis of p-nitrophenyl beta-2-acetamido-2-deoxy-d-glucopyranoside at 37 degrees C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Partial purification and comparative characterization of α-amylase secreted byLactobacillus amylovorus

An -amylase (E.C. 3.2.1.1.) secreted byLactobacillus amylovorus was partially purified and characterized. This high-molecular-weight enzyme [Imam SH, Burgess-Cassler A, Côté GL, Gordon SH, Baker FL (1991) Curr Microbiol 22:365–370] was quantified with a clinical -amylase assay adapted to a microplate format. It was isolated from concentrated cell-free culture medium by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The enzyme was not particularly thermostable, but like three other microbial -amylases tested for comparison, was renaturable following treatment with SDS and heat. The pH optimum and pI were 5.5±0.5 and 5.0, respectively; its temperature optimum was 60–65°C, and the molecular weight on SDS gels was 140±10 kDa.     

Partial purification and characterization of intracellular car☐ypeptidase ofCandida albicans

Intracellular car☐ypeptidase was partially purified from the yeast form ofCandida albicans H-317 by acid dialysis, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Peptidase activity was measured with an enzyme-coupled colorimetric assay. Fractionation by native polyacrylamide gel electrophoresis and Sephacryl S-200 gel filtration indicated the presence of a single peak of car☐ypeptidase, with the isoelectric point at pH 4.6 and a molecular weight of 100,000. The pH optimum and apparentK_m usingN-carbobenzoxy-l-phenylalanine-l-leucine (N-Cbz-l-Phe-l-Leu) as substrate were 6.5 and 2 × 10⁻⁴M, respectively. The best substrates for the enzyme wereN-Cbz-Ala-X peptides, withN-Cbz-Ala-Leu giving the highest rate of hydrolysis. Substrates that gave 7% or less of the control (N-Cbz-Phe-Leu) rate of hydrolysis were Gly-Leu, Gly-Phe, Ile-Phe, Ile-Met, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. There was no detectable hydrolysis of the followingN-Cbz peptides; Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride,p-chloromercuribenzoate, benzyloxy-carbonyl-l-phenylalanine chloromethyl ketone, and tosyl-l-phenylalanine chloromethyl ketone, but was not affected by EDTA, tosyl-l-lysine chloromethyl ketone, pepstatin A, leupeptin, bestatin, or antipain. Although secretory proteinases are thought to play a role in the pathogenesis of this organism, the role of this intracellular car☐ypeptidase has yet to be determined.     

β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.     

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.     

Partial purification, characterization and nitrogen regulation of the lysine ɛ-aminotransferase of Streptomyces clavuligerus

The L-lysine ɛ-aminotransferase (LAT) of Streptomyces clavuligerus was partially purified and characterized. The 51.3-kDa enzyme exhibited optimal activity at pH 7.0–7.5 and 30°C. It catalyzed transfer of the terminal amino group of L-lysine or L-ornithine to α -ketoglutarate. Oxalacetate and pyruvate were also used as acceptors of the amino group but with very low efficiency. Increasing ammonium concentrations added to chemically-defined medium MM enhanced the formation of LAT and decreased production of cephalosporins by S. clavuligerus. In cultures grown in the absence of lysine, greater enhancement of LAT formation by ammonium and less repression of cephalosporin biosynthesis were observed. In the chemically-defined GSPG medium, ammonium ions decreased cephalosporin production without showing an effect on LAT formation. Received 20 August 1996/ Accepted in revised form 15 November 1996     

Interrelations between glycogen,poly-β-hydroxybutyric acid and lipids during accumulation and subsequent utilization in a Pseudomonas

A pleomorphic pseudomonad, V-19, was isolated from activated sludge on the basis of its floc-forming capacity in 0.1% casitone ?0.035% yeast extract (CYE) ?0.2% glycerol medium. In the late exponential phase of growth morphological changes and flocculation phenomena took place accompanied by a massive deposition of reserve granules in the cell. Chemical and electron-microscopical examination revealed 3 types of storage products: glycogen, poly-β-hydroxybutyric acid (PHB) and ether-extractable lipids. These products were isolated and chemically characterized. In CYE medium supplied with 0.5% glucose or glycerol as the carbon source mainly ether-soluble lipids and glycogen were synthesized. On continued incubation these materials were slowly utilized, which enabled the cells to survive for long periods of time. Growth in inorganic salts medium (0.1% ammonium sulfate; 1% carbon source) yielded cells containing different accumulated products, depending on the carbon source used. Glycerol-grown cells contained mainly glycogen, but also ether-soluble lipid, and no PHB. Glucose was largely converted into gluconic acid and excreted into the medium before being deposited in the form of PHB as the primary product of assimilation. Subsequently, PHB was metabolized thereby being partly transformed into glycogen and ether-soluble lipid. Addition of ammonium sulfate to nitrogen-starved cells caused a ready mobilization of the accumulated products, resulting in a net synthesis of reservefree cell material and an increase in the number of cells.     

AGI,a previously unreportedD. melanogaster α-glucosidase: Partial purification,characterization, and cytogenetic mapping

InbredDrosophila melanogaster stocks were surveyed for α-glucosidases with nondenaturing gel electrophoresis using a fluorogenic substrate to stain the gels. The glucosidase most active under these conditions is polymorphic. We established that the polymorphism is genetic in origin and that the glucosidase was not likely to be a previously characterized enzyme. The gene encoding the enzyme was mapped cytogenetically to 33 A1-2- 33A8-B1, confirming that this is an enzyme not yet reported inD. melanogaster. The enzyme was partially purified by elution from nondenaturing gels, which enabled us to establish that it has optimal activity at pH 6 and interacts most strongly with α-1–4 glucosides. A developmental and tissue survey suggested that this enzyme could have a purely digestive role or be involved in carbohydrate metabolism inside the organism. We propose that this enzyme is involved in either starch digestion or glycogen metabolism.     

α-glucosidase from grape berries: Partial purification and characterization

-Glucosidase (-D-glucoside glucohydrolase EC 3.2.1.20) was purified approximately 30-fold from grape berries (Vitis vinifera var. Riesling). Besides maltose the enzyme preparation hydrolyzes to a lesser extent maltotriose, isomaltose, and starch. It has a pH optimum of 5.1 and a molecular weight of about 100,000. Tris, glycerol, several mono-and disaccharides were tested as inhibitors. The kinetic behavior of ribose, fructose, cellobiose, sucrose, turanose, methylglucopyranoside, Tris, and glycerol was fully investigated. The inhibition studies suggest a Ping-Pong mechanism, with the second substrate concentration being constant, that can be treated as a Uni Bi system. The purified enzyme is stable when stored frozen at-20° C. The grape-berry -glucosidase may exist as multiple forms (pI 7.2 and 8.2 respectively), and it does not require ions for its activity.This work was supported by the consiglio Nazionale delle Ricerche, Roma, Italy     

One-step purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Stereum hirsutum

A highly efficient β-1,4-glucosidase (BGL) secreting strain, Stereum hirsutum SKU512, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. A BGL containing a carbohydrate moiety was purified to homogeneity from S. hirsutum culture supernatants using only a single chromatography step on a gel filtration column. The relative molecular weight of S. hirsutum BGL was determined as 98 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. S. hirsutum BGL showed the highest activity toward p-nitrophenyl-β-D-glucopyranoside (V _max = 3,028 U mg-protein⁻¹, k _cat = 4,945 s⁻¹) ever reported. The enzyme also showed good stability at an acidic pH ranging from 3.0 to 5.5. The BGL was able to promote transglycosylation with an activity of 42.9 U mg-protein⁻¹ using methanol as an acceptor and glucose as a donor. The internal amino acid sequences of the isolated enzyme showed significant homology with hydrolases from glycoside hydrolase family 1 (GH1), indicating that the S. hirsutum BGL is a member of GH1 family. The characteristics of S. hirsutum BGL could prove to be of interest in several potential applications, especially in enhancing flavor release during the wine fermentation process.     

Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium

The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer.     

Fungal thermostable α-dialkylamino acid aminotransferase: occurrence,purification and characterization

-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K _m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K _i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyrate - PLP pyridoxal 5-phosphate     

Genetics and physiological expression of β-hydroxy acid dehydrogenase in Drosophila

A mutant Had ^nl was induced in Drosophila melanogaster and found to be deficient in -hydroxy acid dehydrogenase. This mutation was utilized to study the genetics and physiological expression of Had ⁺. Had⁺ was mapped to the X chromosome at 54.4 and seems to be the structural gene for the enzyme. Enzyme activity in male and female flies indicates that the gene shows both dosage compensation independent from dose effect and differential activity during ontogeny. Electrophoretic mobility data indicate that the enzyme is a dimer which forms by random association of subunits. The fact that the mutant shows no detrimental effect implies that the enzyme is dispensable, at least under laboratory conditions. The biological and technical implications of this gene-enzyme system are discussed.This research was sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. J. E. T. was a postdoctoral investigator supported by USPHS Fellowship No. 1-F02-GM53673-01 during a portion of this work.     

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L: -Arabinofuranosidase (β-Xyl I-α-L: -Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L: -Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L: -Ara exhibited a specific β-Xylosidase I activity of 1.25?U?mg(-1) to oNPX and a specific α-L: -Arabinofuranosidase activity of 0.47?U?mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45?°C, and a high degree of stability was verified over 240?min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89?±?0.13?mM and 1.4?±?0.04?μM?min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.     

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells: purification, characterization and subcellular localization

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells was purified to homogeneity as judged by SDS-PAGE and silver staining of a single 32 kD band. A second, more hydrophobic product of the purification protocol contained additional bands at 45 and 80 kD. The 17β-estradiol dehydrogenase activities of both products exceeded those for 17-one reduction by more than 260-fold. Activities of 3-, 3β- and 20-dehydrogenases were absent in either fraction. Polyclonal and monoclonal antibodies raised against the 32 kD protein and the more hydrophobic product precipitated the enzymatic activity and reacted with the 32 and 80 kD bands, but not with the 45 kD band in Western blots. The subcellular localization of the enzyme was studied in sections of intact cells and of isolated organelles using gold sol coated with F(ab′)₂ fragments of monoclonal antibody F1. Gold particles were found exclusively over cytoplasmic vesicles of 120–150 nm diameter with electron-dense contents.     

Reduction of β-thiopyruvic acid by lactate dehydrogenase: a kinetic study

Summary In this paper a steady-state kinetic study on the system lactate dehydrogenase--thiopyruvate--thiolactate is presented and the possible mechanistic and physiological implications are discussed.At pH 7.4 the equilibrium between -thiopyruvate and -thiolactate, in the presence of NADH and lactate dehydrogenase is largerly shifted towards the formation of -thiolactate as in the case of pyruvate and lactate. This can be relevant in connection with the mixed disulfide between cysteine and -thiolactate that is observed to be present in the mammalian body fluids.The catalytic mechanism is of the bi-bi compulsory order type, and rapid equilibrium conditions for the binding of the first substrate (NADH) are shown to apply. A complex inhibition pattern of inhibitions by both substrates, however, prevents simple suggestions about the nature of the dead-end species involved.Abbreviations TPA -thiopyruvic acid - TLA -thiolactic acid - E enzyme This work is gratefully dedicated to Prof. Alessandro Rossi Fanelli on the occasion of his 75th birthday.