The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase

The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro456Pro457Ala458 of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.     

Expression, Purification, and Characterization of Histidine-Tagged Rat and Human Flavoenzyme Dihydroorotate Dehydrogenase

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in thede novosynthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system inTrichoplusia nicells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria ofSpodoptera frugiperdacells infected with the recombinant virus using Triton X-100 were loaded onto Ni²⁺-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of ourpreviously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130– 150 μmol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8–1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 μmol/min/mg and the rat enzyme with 83 μmol/min/mg, respectively, at pH 8.0–8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate,K_m= 14.6 μM; electron acceptor decylubiquinone,K_m= 9.5 μM) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8–1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.     

Overexpression of an alkaline lipase gene from Proteus vulgaris K80 in Escherichia coli BL21/pKLE

Over-expression of Proteus vulgaris K80 lipase gene in Escherichia coli BL21 (DE3)/pKLE was achieved, with the enzyme being produced in an as active soluble form, using the T7 RNA polymerase system in a modified M9 salt or M9ZB medium. d-Lactose (55 mM) was used to induce gene expression and gave twice the lipase activity achieved with 0.4 mM IPTG. The expression of the lipase gene depended on the feeding rate of glucose being optimal at 12 g l^–1 h^–1.     

Induction of ureogenesis in perfused liver of a freshwater teleost, Heteropneustes fossilis, infused with different concentrations of ammonium chloride

The induction pattern of urea cycle enzymes and the rate of urea-N excretion were studied with relation to ammonia load in the perfused liver of a freshwater ammoniotelic teleost, Heteropneustes fossilis, when infused with different concentrations of ammonium chloride for 60 min. Both urea-N excretion and uptake of ammonia by the perfused liver were found to be a saturable process. The V_max of urea-N excretion (0.45 μmol/g liver/min) was obtained at ammonium chloride addition of 1.18 μmol/g liver/min. The maximum induction of carbamyl phosphate synthetase (ammonia dependent), 200%, and of ornithine transcarbamylase, 120%, was seen by the addition of 0.58 μmol/g liver/min, and for argininosuccinate synthetase and argininosuccinate lyase of 150% and 115%, respectively, by the addition of 2.8 μmol/g liver/min of ammonium chloride. However, arginase activity did not alter in any of the concentrations of ammonium chloride added. An increase of ammonia load of 3–5 μmol/g wet wt from the physiological level in the perfused liver was sufficient to initiate and to cause maximum induction of most of the urea cycle enzymes activitty. These results further confirm the capacity of transition from ammoniotelism to ureotelism in this unique freshwater air-breathing teleost to tolerate a very high ambient ammonia.     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

Determination of the anticonvulsant felbamate and its three metabolites in brain and heart tissue of rats

An automated, internal standard high-performance liquid chromatographic method for the simultaneous quantitation of felbamate and its three metabolites in adult and neonatal rat brain and heart tissue homogenates was developed and validated. The homogenates prepared from one part of the tissue and four parts of water were extracted with ethyl acetate, and the extract was evaporated to dryness and redissolved in mobile phase. Separation was accomplished on a Waters Resolve C₁₈, 5 μm, 300 mm × 3.9 mm I.D. column with a mobile phase consisting of 0.01 M phosphate buffer, pH 6.8—acetonitrile—methanol (800:150:50, v/v/v). Eluting peaks were monitored with an ultraviolet detector at 210 nm. The linear range of the assay for felbamate and the metabolites was 0.20–50.00 μg/ml of homogenate or 1–250 μg/g of brain or heart tissue. The lower limit of quantitation for all four analytes was 0.20 μg/ml of homogenate or 1.00 μg/g of tissue.     

ATP and ADP hydrolysis in the kidney and liver of fish, chickens and rats

We investigated NTPDase-like activity [ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases)] in liver and kidney membrane from silver catfish (Rhamdia quelen), chicken (Gallus gallus) and rat (Rattus norvegicus) under different conditions and in the presence of several inhibitors. The cation concentration required for maximal activity was 0.5, 1.5 and 2.0 mM for fish, chicken and rat liver, respectively (with ATP and ADP as substrates). The maximal activity in the kidney was observed at calcium concentrations of 0.5, 2.0, 1.5 mM (ATP) and 0.5, 1.5, 1.0 (ADP) for fish, chickens and rats, respectively. The results showed that the pH optimum for all animals and for the two tissues was close to 8.0. The temperature chosen was 25 °C for fish and 36 °C for chicken and rat preparations. Ouabain had no effect on the NTPDase-like activity of fish, chickens or rats. NTPDase activity was decreased in the presence of lanthanum in the chicken (ADP) and rat (ATP and ADP) liver. In the kidney, lanthanum inhibited fish ATP and rat ATP and ADP (0.2 mM) hydrolysis. N-ethylmaleimide (NEM) had an inhibitory effect on the kidney of all species at the concentration of 3.0 mM (ADP). Orthovanadate only inhibited fish membrane NTPDase; azide only inhibited the preparation at high concentrations (10 mM) and fluoride inhibited it at 10 mM (fish and chicken) and 5 mM (rat). Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. These results suggest that NTPDase-like activity shows a different behavior among the vertebrate species and tissues studied. Additionally, we propose that NTPDase1 is the main enzyme present in this preparation.     

Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica

An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4°C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in tryptone—soya broth yielded better results than in—frequently used—phosphate buffer, pH 7.6.While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, DHL agar was most satisfactory: after 20 h incubation at 29°C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results.It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives as potential inhibitors of inflammatory factors in sepsis

Synthesis and anti-inflammatory effects of certain furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives 12–18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydoxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3′,2′:3,4]naphtho[1,2-d]imidazole (12) was inactive (IC₅₀ value of >30 μM) while its 5-phenyl derivative 13, with an IC₅₀ value of 16.3 and 11.4 μM against lysozyme and β-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC₅₀ value of 19.5 and 11.3 μM against lysozyme and β-glucuronidase release, respectively. An electron-withdrawing NO₂ substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC₅₀ value of 7.4 and 5.0 μM against lysozyme and β-glucuronidase release, respectively.For the LPS-induced NO production, the phenyl derivatives 12–15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC₅₀ value of 1.5 and 1.3 μM, respectively, which are more active than that of the positive 1400 W. Compounds 16–18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC₅₀ of 0.52 μM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.     

Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP)

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1 L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals.The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 μmol min⁻¹ mg⁻¹) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.     

Production enhancement and refolding of caprine growth hormone expressed in Escherichia coli

This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2–3 times higher cell densities were obtained in shake flask cultures when induction was done with lactose showing almost same level of expression as in case of IPTG induction. With lactose induction highest cell densities were achieved in TB (OD₆₀₀ 16.3) and M9NG (OD₆₀₀ 16.1) media, producing 885 and 892 mg cGH per liter of the culture, respectively. Lactose induction done at mid-exponential stage resulted in a higher cell density and thus higher product yield. cGH over-expressed as inclusion bodies was solubilized in 50 mM Tris–Cl buffer (pH 12.5) containing 2 M urea, followed by dilution and lowering the pH in a step-wise manner to obtain the final solution in 50 mM Tris–Cl (pH 9.5). The cGH was purified by Q-Sepharose chromatography followed by gel filtration with a recovery yield of 39% on the basis of total cell proteins. The product thus obtained showed a single band by SDS–PAGE analysis. MALDI-TOF analysis showed a single peak with a mass of 21,851 dalton, which is very close to its calculated molecular weight. A bioassay based on proliferation of Nb2 rat lymphoma cells showed that the purified cGH was biologically active.     

Active Recombinant Rat Dipeptidyl Aminopeptidase I (Cathepsin C) Produced Using the Baculovirus Expression System

An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC).In vivoactivation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5°C for 18–40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide–p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.     

Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia, Viviani

Summary Several amino acid requiring auxotrophs have been isolated from unsupplemented protoplast cultures of haploid Nicotiana plumbaginifolia following incubation with BUdR (1-5x10^-5, 2 days) and recovery on complete medium. The auxotrophic lines required the following amino acid(s) for growth: his, ile, leu, ile+val, met or try. Met^– is a new type isolated in higher plants. The same absolute amino acid requirement was observed in plants regenerated from auxotrophic cultures. Precursor feeding tests, enzyme assays, and/or metabolic complementation through protoplast fusion were used to identify the genetic lesion leading to auxotrophy. Mutant seeds were obtained from supplemented Met^– plants. Seeds were also collected from selfed plants regenerated from various complementing fusion products, and a His^– revertant. Genetic analysis indicated that under natural conditions of seed formation amino acid auxotrophy-in contrast to NR deficiency-failed to segregate in progeny tests.Abbreviations and definitions BUdR and FUdR 5-bromo- and fluoro-deoxyuridine respectively - AP imidazole acetol phosphate - IGP imidazole glycerol phosphate - NR nitrate reductase - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - TIP total isolation procedure - ER Escape rate—the proportion of the selected cell population surviving the BUdR treatment - BR Recovery rate—the proportion of clones identified as amino acid auxotrophs from total escaping clones - TS Total surviving colonies—the number of inoculated protoplasts/variant x plating efficiency - TST Total starvation time—the number of days on minimal medium (preincubation time+BUdR incubation time). The relationship days vs. number of divisions is as follows: 3- to 4-day-old protoplasts, 1 division; 5–6 days, 2 divisions; 7–8 days, 3 divisions Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Induction strategies in fed-batch cultures for recombinant protein production in Escherichia coli: Application to rhamnulose 1-phosphate aldolase

Different induction strategies for fed-batch recombinant protein production under the control of the strong T5 promoter in Escherichia coli have been investigated. Since the production of recombinant rhamnulose 1-phosphate aldolase is growth-related, the productivity of the process can be strongly reduced due to the negative effect of protein expression on cell growth. IPTG pulse induction as well as inducer dosage have been applied and their advantages and drawbacks highlighted. Both strategies led to high levels of the recombinant protein, 1000 AU g DCW⁻¹. Inducer concentration and inducer to biomass ratio were identified as the parameters influencing the rate of protein production and final enzymatic activity per gram of biomass. In pulse induction, the maximum enzymatic activity was found at inducer concentration of 70 μM. In continuous induction experiments, inducer concentrations between 4 and 12 μM were identified as the working range in which cell growth and recombinant protein accumulation occurred simultaneously. On the other hand, the amount of IPTG per gram of biomass required was 1.6 μmol IPTG gDCW⁻¹ in pulse induction and between 0.3 and 0.5 μmol IPTG g DCW⁻¹ in continuous induction.     

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0–11.0 and 25–40 °C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24 h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Chloroplast and Cytoplasmic Enzymes: IV. Pea Leaf Fructose 1,6-Diphosphate Aldolases

Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 μM for the chloroplast, 21 μM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 μM for the chloroplast enzyme, 18 μM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf.