Characterization of a lysosomal proteinase purified from haploid cells of Physarum flavicomum undergoing encystment

1983 ◽  
Vol 61 (5) ◽  
pp. 1357-1366 ◽  
Author(s):  
Henry R. Henney Jr. ◽  
Hiltrud U. White
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Acid Proteinase ◽  
Thiol Groups ◽  
Ph Optimum ◽  
Golgi Bodies ◽  
Cell Extracts ◽  
Thiol Reagent ◽  
Lysosomal Proteinase ◽  
Ph Range

Haploid cells of Physarum flavicomum differentiating to dormant microcysts exhibited prominent Golgi bodies, lysosomes, and autophagic vacuoles digesting intracellular materials. Total intracellular acid proteinase activities, as well as enzyme specific activity, increased during the encystment process. Lysosomes, isolated by fractionation of cell extracts on sucrose density gradients, were identified by their ultrastructural characteristics as well as their content of the following enzymes of high specific activity: acid proteinase, acid β-galactosidase, acid phosphatase, and β-glucuronidase. The lysosomal acid proteinase was chromatographically purified, and its molecular weight was estimated to be 32 000. The proteinase was most stable in the pH range of 2–3 which similarly corresponds to its pH optimum using hemoglobin and Azocasein, respectively, as substrates. Its isoelectric point was about 2. The enzyme exhibited little activity and was unstable at pH 7 and above. The rate of activity of the proteinase was maximal at 55 °C, and good stability of the enzyme was noted up to 45 °C. The proteinase required a thiol reagent for stability. Pepstatin, which specifically affects acid proteinases, inhibited the enzyme. Also, compounds reactive with enzyme thiol groups were highly effective inhibitors of the proteinase. The lysosomal enzyme is an acid (carboxyl) proteinase with essential thiol groups.

scholarly journals Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

2008 ◽  
Vol 74 (21) ◽  
pp. 6697-6702 ◽  
Author(s):  
Kathrin H�lsch ◽  
Jan Havel ◽  
Martin Haslbeck ◽  
Dirk Weuster-Botz
Keyword(s):  
Specific Activity ◽  
Acyl Carrier Protein ◽  
Asymmetric Reduction ◽  
Enantiomeric Excess ◽  
High Specific Activity ◽  
Lactobacillus Brevis ◽  
Ph Optimum ◽  
Synechococcus Sp ◽  
Prochiral Ketones ◽  
Pcc 7942

ABSTRACT A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44�C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 � 2.15 U mg−1) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 � 0.49 U mg−1) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).

scholarly journals Purification and some characteristics of an oestrogen sulphotransferase from guinea pig adrenal gland and its non-identity with adrenal pregnenolone sulphotransferase

1990 ◽  
Vol 268 (3) ◽  
pp. 759-764 ◽  
Author(s):  
R Hobkirk ◽  
M A Glasier ◽  
L Y Brown
Keyword(s):  
Adrenal Glands ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Protein Band ◽  
Thiol Groups ◽  
Reducing Conditions ◽  
Main Protein ◽  
Salt Fractionation

An oestrogen sulphotransferase, active towards both oestrone and oestradiol, and of high specific activity, is present in cytosol prepared from adrenal glands of both sexes of English Shorthair and Hartley guinea pigs. The ovarian and testicular cytosolic activities of this enzyme are markedly low in comparison with the adrenal activity. The adrenal enzyme is distinct from an accompanying pregnenolone sulphotransferase as judged by f.p.l.c. gel filtration, chromatofocusing, and differences in activation brought about by the addition of thiol groups. The oestrogen sulphotransferase behaved as a 67 kDa protein on a Sephadex G100 column and as a 48 kDa protein on f.p.l.c. gel-filtration columns. Two forms of the enzyme with apparent pI values of 6.1 and 5.5 were eluted during f.p.l.c. chromatofocusing. Sequential salt fractionation, f.p.l.c. gel filtration and elution from an agarose-hexane-adenosine-3′,5′-diphosphate affinity gel has resulted in a preparation which, when resubmitted to f.p.l.c. gel filtration, yields a considerably purified oestrogen sulphotransferase. When submitted to SDS/polyacrylamide-gel electrophoresis under reducing conditions, a main protein band of 34-36 kDa is observed. It is suggested that the enzyme may exist as a dimer in the cytosol.

Expression of Aspergillus aculeatus β-mannanase in Pichia pastoris Yeast and Analysis of Industrially Important Properties of Enzyme

2019 ◽  
Vol 35 (1) ◽  
pp. 38-44
Author(s):  
M.N. Lazareva ◽  
E.I. Semenko ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
High Specific Activity ◽  
Yeast Cells ◽  
Aspergillus Aculeatus ◽  
Gene Encoding ◽  
Pichia Pastoris Yeast ◽  
Efficient Expression ◽  
Ph Range ◽  
First Time

β-Mannanases are enzymes for the industrial application and they can be used, in particular, in the feed industry. The most important requirements for feed enzymes are broad pH range, thermal stability and high specific activity. The efficient expression of the man1 gene encoding Aspergillus aculeatus β-1,4-mannanases in Pichia pastoris yeast cells has been obtained for the first time. The industrially valuable properties of the enzyme were confirmed. The obtained data indicate that the man1 gene from A. aculeatus is potentially useful for the construction of industrial mannanase producers on the basis of the Pichia pastoris yeast. recombinant β-mannanase, Pichia pastoris, Aspergillus aculeatus, overexpression. The work was financially supported by State project №595-00004-18 PR and used the help of the National Bioresource Center - Russian National Collection of Industrial Microorganisms NRC «Kurchatov Institute» - GosNIIgenetika (Moscow, Russia).

Thymidine-requiring mutants of Dictyostelium discoideum

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

The methylating system for 3-sn-phosphatidylcholine biosynthesis in Fusarium oxysporum

1980 ◽  
Vol 26 (7) ◽  
pp. 774-777 ◽  
Author(s):  
Alan C. Wilson ◽  
Leslie R. Barran
Keyword(s):  
Fusarium Oxysporum ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Phospholipid Fraction ◽  
Ph Optimum ◽  
Methyl Group ◽  
Cell Extracts ◽  
Phosphatidylcholine Biosynthesis

Cell extracts of hyphae of Fusarium oxysporum f. sp. lycopersici rapidly transferred the methyl group of S-[methyl-3 H]adenosyl-L-methionine (Ado-Met) to endogenous phosphatidylethanolamine (PE). About 80% of the radioactivity incorporated into the phospholipid fraction was found in phosphatidylcholine (PC) while the rest of the radioactivity was present in the intermediates monomethylphosphatidylethanolamine (MePE) and dimethylphosphatidylethanolamine (DiMePE). The phospholipid methylating system had a pH optimum of 8.5, a Km of 30 μm for Ado-Met, and a Vmax of 10 nmol/h per milligram protein. The specific activity of the methylating system was highest in early log phase and lowest in the late log phase of growth.The activity of the cell-free methylating system was reduced by incubation at temperatures above 25 °C, and at 37 °C about 50% of the initial methylating activity remained after incubation for 15 min. In contrast, the activity of the in vivo methylation system almost doubled when the incubation temperature was raised from 25 to 37 °C.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

Oxidation of primary bile acids by a 7α-hydroxysteroid dehydrogenase elaborating Clostridium bifermentans soil isolate

1987 ◽  
Vol 33 (8) ◽  
pp. 663-669 ◽  
Author(s):  
J. Derek Sutherland ◽  
C. Noel Williams ◽  
Donna M. Hutchison ◽  
Lillian V. Holdeman
Keyword(s):  
Bile Acids ◽  
Chenodeoxycholic Acid ◽  
Deoxycholic Acid ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Ph Optimum ◽  
Reactive Blue 2 ◽  
Cell Extracts ◽  
Initial Drop ◽  
Clostridium Bifermentans

A gram-positive, rod-shaped anaerobe (strain F-6) was isolated from soil. This organism was identified by cellular morphology as well as fermentative and biochemical data as Clostridium bifermentans. Strain F-6 formed 7-ketolithocholic acid from chenodeoxycholic acid and 7-ketodeoxycholic acid from cholic acid in whole cell cultures, but did not transform deoxycholic acid, ursodeoxycholic acid, or ursocholic acid. This reaction is reversible. The structures of 7-ketolithocholic acid and 7-ketodeoxycholic acid were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. When incubated with the strain F-6 glycine and taurine conjugates of the primary bile acids were partially hydrolyzed and transformed to 7-keto products. Optimal yields of 7-ketolithocholic acid and 7-ketodeoxycholic acid were obtained after 78 h of incubation. Culture pH changed with time and was characterized by an initial drop (1.1 pH units) and a gradual increase back to the starting pH (7.3). Corroborating these observations, an inducible, NADP-dependent, 7α-hydroxysteroid dehydrogenase was demonstrated in cell extracts of strain F-6. A trace of NAD-dependent 7α-hydroxysteroid dehydrogenase was also found. A substantial increase in the specific activity of the NADP-dependent 7α-hydroxysteroid dehydrogenase was observed when either 7-ketolithocholic acid, chenodeoxycholic acid, or deoxycholic acid was included in the growth medium. Optimal induction of the NADP-dependent 7α-hydroxysteroid dehydrogenase was achieved with 0.3–0.4 mM 7-ketolithocholic acid. Production of the enzyme(s) was optimal at 6–8 h of growth and the 7α-hydroxysteroid dehydrogenases had a pH optimum of approximately 11. The 7α-hydroxysteroid dehydrogenase from strain F-6 was purified 12-fold by triazine dye affinity chromatography with reactive blue 2 (Cibacron blue) agarose (95% yield). It was successfully lyophilized into a stable powder form.

scholarly journals Thymidine-requiring mutants of Dictyostelium discoideum.

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791 ◽  
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

scholarly journals Application Potential of Cyanide Hydratase from Exidia glandulosa: Free Cyanide Removal from Simulated Industrial Effluents

Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1410
Author(s):  
Anastasia Sedova ◽  
Lenka Rucká ◽  
Pavla Bojarová ◽  
Michaela Glozlová ◽  
Petr Novotný ◽  
...  
Keyword(s):  
Specific Activity ◽  
Copper Ions ◽  
High Specific Activity ◽  
Industrial Effluents ◽  
Free Cyanide ◽  
Highly Active ◽  
Physico Chemical ◽  
Application Potential ◽  
Ph Range ◽  
Few Data

Industries such as mining, cokemaking, (petro)chemical and electroplating produce effluents that contain free cyanide (fCN = HCN + CN−). Currently, fCN is mainly removed by (physico)chemical methods or by biotreatment with activated sludge. Cyanide hydratases (CynHs) (EC 4.2.1.66), which convert fCN to the much less toxic formamide, have been considered for a mild approach to wastewater decyanation. However, few data are available to evaluate the application potential of CynHs. In this study, we used a new CynH from Exidia glandulosa (protein KZV92691.1 designated NitEg by us), which was overproduced in Escherichia coli. The purified NitEg was highly active for fCN with 784 U/mg protein, kcat 927/s and kcat/KM 42/s/mM. It exhibited optimal activities at pH approximately 6–9 and 40–45 °C. It was quite stable in this pH range, and retained approximately 40% activity at 37 °C after 1 day. Silver and copper ions (1 mM) decreased its activity by 30–40%. The removal of 98–100% fCN was achieved for 0.6–100 mM fCN. Moreover, thiocyanate, sulfide, ammonia or phenol added in amounts typical of industrial effluents did not significantly reduce the fCN conversion, while electroplating effluents may need to be diluted due to high fCN and metal content. The ease of preparation of NitEg, its high specific activity, robustness and long shelf life make it a promising biocatalyst for the detoxification of fCN.

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