scholarly journals Enzyme engineering and in vivo testing of a formate-reduction pathway

2021 ◽  
Author(s):  
Jue Wang ◽  
Karl Anderson ◽  
Ellen Yang ◽  
Lian He ◽  
Mary E. Lidstrom
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Specific Activity ◽  
Enzyme Engineering ◽  
Cell Extracts ◽  
Carbon Substrates ◽  
Natural Variants ◽  
In Vivo Testing ◽  
Fuels And Chemicals ◽  
Further Development

AbstractFormate is an attractive feedstock for sustainable microbial production of fuels and chemicals, but its potential is limited by the lack of efficient assimilation pathways. The reduction of formate to formaldehyde would allow efficient downstream assimilation, but no efficient enzymes are known for this transformation. To develop a 2-step formate-reduction pathway, we screened natural variants of acyl-CoA synthetase (ACS) and acylating aldehyde dehydrogenase (ACDH) for activity on one-carbon substrates and identified active and highly expressed homologs of both enzymes. We then performed directed evolution, increasing ACDH specific activity by 2.5-fold and ACS lysate activity by 5-fold. To test for in vivo activity of our pathway, we expressed it in a methylotroph which can natively assimilate formaldehyde. Although the enzymes were active in cell extracts, we could not detect formate assimilation into biomass, indicating that further improvement will be required for formatotrophy. Our work provides a foundation for further development of a versatile pathway for formate assimilation.

scholarly journals Enzyme Engineering and in vivo Testing of a Formate-Reduction Pathway

2021 ◽  
Author(s):  
Jue Wang ◽  
Karl Anderson ◽  
Ellen Yang ◽  
Lian He ◽  
Mary E Lidstrom
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Specific Activity ◽  
Enzyme Engineering ◽  
Cell Extracts ◽  
Carbon Substrates ◽  
Natural Variants ◽  
In Vivo Testing ◽  
Fuels And Chemicals ◽  
Further Development

Abstract Formate is an attractive feedstock for sustainable microbial production of fuels and chemicals, but its potential is limited by the lack of efficient assimilation pathways. The reduction of formate to formaldehyde would allow efficient downstream assimilation, but no efficient enzymes are known for this transformation. To develop a 2-step formate-reduction pathway, we screened natural variants of acyl-CoA synthetase (ACS) and acylating aldehyde dehydrogenase (ACDH) for activity on one-carbon substrates and identified active and highly expressed homologs of both enzymes. We then performed directed evolution, increasing ACDH specific activity by 2.5-fold and ACS lysate activity by 5-fold. To test for in vivo activity of our pathway, we expressed it in a methylotroph which can natively assimilate formaldehyde. Although the enzymes were active in cell extracts, we could not detect formate assimilation into biomass, indicating that further improvement will be required for formatotrophy. Our work provides a foundation for further development of a versatile pathway for formate assimilation.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

scholarly journals Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

2021 ◽  
Author(s):  
Kaifang Liu ◽  
Xiulai Chen ◽  
Yunlu Zhong ◽  
Jia Liu ◽  
Guipeng Hu ◽  
...  
Keyword(s):  
Catalytic System ◽  
Rational Design ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Disodium Salt ◽  
In Vivo Testing ◽  
Dynamics Simulations ◽  
Rate Limiting

The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.

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 Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

1993 ◽  
Vol 4 (1) ◽  
pp. 79-92 ◽  
Author(s):  
L Connell-Crowley ◽  
M J Solomon ◽  
N Wei ◽  
J W Harper
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Fold Increase ◽  
Cyclin A ◽  
Cyclin B ◽  
Activation Process ◽  
Cyclin Dependent Kinase ◽  
Cell Extracts

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

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.

Activity of NAD-Dependent Dehydrogenase Enzyme of Lactic Streptococci on Acetaldehyde and n-Hexanal1

1979 ◽  
Vol 42 (10) ◽  
pp. 778-779 ◽  
Author(s):  
R. H. SCHMIDT ◽  
L. K. FARRON ◽  
T. R. BATEH ◽  
P. E. ARAUJO
Keyword(s):  
Alcohol Dehydrogenase ◽  
Centrifugal Force ◽  
Aldehyde Dehydrogenase ◽  
Specific Activity ◽  
Enzyme System ◽  
Substrate Inhibition ◽  
Cell Extract ◽  
Cell Extracts ◽  
Dehydrogenase Enzyme

Alcohol dehydrogenase (alcohol:NAD oxidoreductase, E.C. 1.1.1.1) activity was observed on acetaldehyde and n-hexanal in homogenized cell extract of Steptococcus lactis C2. Substrate inhibition was apparent at levels of n-hexanal above 4.0 mM. Increased centrifugal force from 12.000 × g for 20 min to 350,000 × g for 1 h resulted in increased specific activity in the cell-extract supernatant fluids. Aldehyde dehydrogenase (aldehyde: NAD oxidoreductose; E.C. 1.2.1.3) was not detected in any of the cell extracts. A possible involvement of the enzyme system with flavor modification in lactic-fermented oilseed milk is suggested.

211At-AntiCD33 in NMRI nu/nu mice

2011 ◽  
Vol 50 (05) ◽  
pp. 214-220
Author(s):  
S. Sriyapureddy ◽  
D. Krull ◽  
T. Petrich ◽  
G.-J. Meyer ◽  
W. H. Knapp ◽  
...  
Keyword(s):  
Animal Model ◽  
Specific Activity ◽  
Time Behaviour ◽  
Nacl Solution ◽  
Therapeutic Application ◽  
Intact Antibody ◽  
A Minor ◽  
Further Development ◽  
Unlabelled Antibody

SummaryThe aim of this study is to verify the in vivo stability, to determine the biodistribution and to estimate the unspecific radiotoxicity of an 211At-labelled CD33-antibody (211At-anti-CD33) in mice with a view to therapeutic application in treating leukaemia. Animals, methods: 211At was produced via the 209Bi(α,2n)211At reaction and was linked via 3-211At-succinimidyl-benzoate to the anti-CD33-antibody. The biodistribution and the in vivo stability in serum were determined after i.v.-injection in NMRI nu/nu-mice. For toxicity experiments, mice received either three times 315–650 kBq 211At-antiCD33 or unlabelled antibody and NaCl-solution respectively. Results: 211At-antiCD33 showed a characteristic biodistribution complying with the unspecific antibody retention in the reticular endothelial system. The largest proportion of radioactivity remained in blood and blood-rich tissues with a minor accumulation in the thyroid and stomach. After 21 h, > 85% of activity in serum still represented intact antibody. Mice showed no difference in unspecific toxicity of 211At-labelled antibodies over six months compared to those treated with unlabelled antibody and NaCl-solution respectively, with regard to histopathologic lesions, survival time, behaviour and haemograms. Conclusion: The radiolabelling method yielded adequate in vivo stability of 211At-antiCD33. Biodistribution with rapid elimination of free 211At via kidneys and urine complies with requirements for targeted therapy. Activity doses potentially required for treatment do not elicit radiotoxicity to normal organs in mice. Further development is required to enhance the apparent specific activity and to verify the efficacy in an adequate animal model before phase I clinical studies in leukaemia can be envisaged.

XV.—The metabolism of DNA in the liver during precocious induction of metamorphosis in Rana catesbeiana

1969 ◽  
Vol 70 (4) ◽  
pp. 295-310 ◽  
Author(s):  
Ailsa M. Campbell ◽  
Martita H. Corrance ◽  
J. N. Davidson ◽  
H. M. Keir
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Rana Catesbeiana ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Polymerase Activity ◽  
Nucleic Acid Metabolism ◽  
Cell Extracts

SynopsisStudies have been made on the incorporation of [8H]-deoxythymidine into the DNA of the livers of Rana catesbeiana tadpoles. When metamorphosis was induced with tri-iodothyronine, the specific activity of the nuclear DNA rose 5 days after administration of the hormone. In contrast, the specific activity of the DNA from the mitochondrial fraction rose grave–2 days after hormone administration.In order to determine whether the in vivo change was due to alterations in the pool sizes of the DNA precursors, in vitro studies on DNA polymerase were carried out. It was found that under conditions where the enzyme activity was not limited by availability of template or substrates, there was a rise in the DNA polymerase activity in crude cell extracts from the tadpole liver. Fractionation of the cell components showed that little of this increment in activity appeared to be located in the nucleus, but that a large percentage alteration in activity occurred in the mitochondrial and cell sap fractions.A possible interpretation of these results is that an increase in the mitochondrial DNA polymerase is one of the early effects of thyroid hormone. This possibility is discussed in relation to the other known effects of thyroid hormones in tadpoles, with particular reference to nucleic acid metabolism and also to mitochondrial hyperplasia.

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