Ultralight ultrafast enzymes

Abstract Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physico-chemical properties from their isotopically natural (native) counterparts; however, in biotechnology such effects have not been investigated yet. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in ultralight media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase showed a 40-250% increase in activity compared to the native counterparts. The efficiency of ultralight Pfu DNA polymerase in polymerase chain reaction was also significantly higher than that of the normal enzyme. At 10 °C, the advantage factor of ultralight enzymes typically increased by 50%, which points towards the reduction in structural entropy as the main factor explaining the kinetic effect of heavy isotope depletion. Ultralight enzymes may find an application where extreme reaction rates are required.

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Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes

Biochemical Journal ◽

10.1042/bj3500429 ◽

2000 ◽

Vol 350 (2) ◽

pp. 429-441 ◽

Cited By ~ 79

Author(s):

Stuart M. PITSON ◽

Richard J. D'ANDREA ◽

Lucianne VANDELEUR ◽

Paul A. B. MORETTI ◽

Pu XIA ◽

...

Keyword(s):

Conformational Changes ◽

Mammalian Cells ◽

Active Form ◽

Chemical Properties ◽

Sphingosine Kinase ◽

Exchange Chromatography ◽

Recombinant Enzymes ◽

Post Translational Modifications ◽

E Coli ◽

Physico Chemical

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 106-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

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Development of the technology for preparation of enzymatic hydrolysate of waste chick embryos

BIOpreparations Prevention Diagnosis Treatment ◽

10.30895/2221-996x-2021-21-3-200-205 ◽

2021 ◽

Vol 21 (3) ◽

pp. 200-205

Author(s):

Yu. S. Ovsyannikov ◽

M. S. Dursenev

Keyword(s):

Nutrient Medium ◽

Mass Fraction ◽

Chemical Properties ◽

Chick Embryos ◽

Nutrient Media ◽

Enzymatic Hydrolysate ◽

E Coli ◽

Physico Chemical ◽

Growth Properties ◽

Solid Nutrient Medium

The development of technologies for preparation of protein nutritional bases for microbiological nutrient media, from production waste of mainly readily available or non-food products, is a promising area in biotechnology. Researchers of Vyatka State Agrotechnological University assume that non-food secondary raw materials, such as waste chick embryos (WCEs) used in the production of anti-influenza products, could be used for these purposes, after removal of the virus-containing allantoic fluid. The aim of the study was to develop a technology for preparation of WCE enzymatic hydrolysate (WCEEH), and to evaluate growth properties of the hydrolysate-based solid nutrient medium, using Escherichia coli M-17 and Pseudomonas alcaligenes IP-1 test strains. Materials and methods: the authors offer methodological approaches to obtaining WCEEH and substantiate hydrolysis parameters. The obtained WCEEH was characterised in terms of physico-chemical properties: pH, amine nitrogen, total nitrogen, sodium chloride, degree of protein cleavage. The growth properties of the hydrolysate-based nutrient medium were studied using E. coli M-17 and Ps. alcaligenes IP-1 test strains. Results: the experiments demonstrated the feasibility of performing enzymatic hydrolysis of WCEs, and assessed physico-chemical properties of the prepared WCEEH batches. The study demonstrated the possibility of using the prepared hydrolysate as a component of solid nutrient media for growing the selected test strains. Conclusions: the study substantiated the optimal technological parameters for WCE enzymatic hydrolysis: pH (7.6 ± 0.3), duration (48 ± 2 h), temperature (49 ± 1) °C. The loading of hydrolysis components was optimised: mass fraction of the substrate—500 g/L, mass fraction of the hydrolysing agent—100 g/L. The physico-chemical properties of WCEEH make it suitable for preparation of microbiological media; the hydrolysate-based solid nutrient medium consistently ensures the growth of E. coli M-17 and Ps. alcaligenes IP-1 test strains with standard properties. The growth properties of the experimental medium are comparable to those of the meat-peptone broth-based nutrient medium.

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CHANGES IN MICROBIOLOGICAL AND PHYSICO-CHEMICAL PROPERTIES DURING COMPOSTING OF SIAM WEED (Chromolaena odorata) AND COWDUNG

Journal of Natural Sciences Engineering and Technology ◽

10.51406/jnset.v14i2.1753 ◽

2018 ◽

Vol 14 (2) ◽

pp. 40-48

Author(s):

A K AKINTOKUN ◽

P O AKINTOKUN ◽

A O OBAWUSI ◽

O R LAWAL

Keyword(s):

Micrococcus Luteus ◽

Chemical Properties ◽

Fungal Species ◽

Cow Dung ◽

Chromolaena Odorata ◽

E Coli ◽

Standard Procedures ◽

Physico Chemical ◽

Three Samples ◽

Phosphorus And Potassium

Three compost samples were prepared in this study from Siam weed (Chromolaena odorata) and cowdung. Sample A was prepared from Cow dung and siam weed at ratio 100g: 100g, Sample B was prepared from 200g chopped siam weed and sample C contained 200g cowdung. These three sam-ples were composted in plastic drums perforated for aeration and each sample were replicated three times. The content in the drums were regularly turned and monitored at 1, 10, 30 and 60 days for mi-crobiological and physicochemical properties. The microbiological and physicochemical analyses of the compost were carried out using standard procedures. Bacterial, Coliform and Fungal count in-creased from day 1 to the 30th day and thereafter decreased from 30th day to the 60th day in all the composting samples. The bacteria species isolated and identified were Pseudomonas fragilis, Pseu-domonas nitrificans, Proteus mirabilis, E. coli, Streptococcus faecium, Micrococcus luteus, Clostridium perfringes, Bacillus cereus, Proteus morganii, Micrococcus acidophilus. Fungal species were Aspergil-lus flavus, Aspergillus fumigatus, Fusarium oxysporium, Penicillum chrysogenum, Aspergillus niger, Mucor sp. and Saccharomyces cerevisiae. The pH of the composted samples ranges between 5.8 to 6.9. The nitrogen, phosphorus and potassium content increased with days of composting but the heavy metals decreased with days of composting. The sulfatase, phosphatase, dehydrogenase, amyl-ase and cellulose enzymes in the three samples increased from day 1 to the 60th day. Sulfatase en-zyme which was the highest ranged from 25 to 76.5% in the three sample, phosphatase (14 to 60.5%), dehydrogenase (20.5 to 55.0%), cellulose (16.5 to 49%) and amylase which was the least enzyme recorded ranged from 5.0 to 38%.

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Physico-chemical properties of actin cleaved with bacterial protease from E. coli A2 strain

FEBS Letters ◽

10.1016/0014-5793(91)80247-z ◽

1991 ◽

Vol 279 (1) ◽

pp. 49-51 ◽

Cited By ~ 38

Author(s):

S.Yu. Khaitlina ◽

J.H. Collins ◽

I.M. Kuznetsova ◽

V.P. Pershina ◽

I.G. Synakevich ◽

...

Keyword(s):

Chemical Properties ◽

E Coli ◽

Bacterial Protease ◽

Physico Chemical

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A benchmark of protein solubility prediction methods on UDP-dependent glycosyltransferases

10.1101/2020.02.28.962894 ◽

2020 ◽

Author(s):

Fatemeh Ashari Ghomi ◽

Tiia Kittilä ◽

Ditte Hededam Welner

Keyword(s):

Escherichia Coli ◽

Protein Solubility ◽

Chemical Properties ◽

Prediction Methods ◽

Solubility Prediction ◽

E Coli ◽

Physico Chemical ◽

And Function ◽

Better Than

AbstractUDP-dependent glycosyltransferases (UGTs) are enzymes that glycosylate a wide variety of natural products, thereby modifying their physico-chemical properties, i.e. solubility, stability, reactivity, and function. To successfully leverage the UGTs in biocatalytic processes, we need to be able to screen and characterise them in vitro, which requires efficient heterologous expression in amenable hosts, preferably Escherichia coli. However, many UGTs are insoluble when expressed in standard and attempted optimised E. coli conditions, resulting in many unproductive and costly experiments. To overcome this limitation, we have investigated the performance of 11 existing solubility predictors on a dataset of 57 UGTs expressed in E. coli. We show that SoluProt outperforms other methods in terms of both threshold-independent and threshold-dependent measures. Among the benchmarked methods, only SoluProt is significantly better than random predictors using both measures. Moreover, we show that SoluProt uses a threshold for separating soluble and insoluble proteins that is optimal for our dataset. Hence, we conclude that using SoluProt to select UGT sequences for in vitro investigation will significantly increase the success rate of soluble expression, thereby minimising cost and enabling efficient characterisation efforts for biocatalysis research.

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Polymer Particles Bearing Recombinant LEL CD81 as Trapping Systems for Hepatitis C Virus

Pharmaceutics ◽

10.3390/pharmaceutics13050672 ◽

2021 ◽

Vol 13 (5) ◽

pp. 672

Author(s):

Dmitry Polyakov ◽

Ekaterina Sinitsyna ◽

Natalia Grudinina ◽

Mariia Antipchik ◽

Rodion Sakhabeev ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Fusion Protein ◽

Core Protein ◽

Chemical Properties ◽

Extracellular Loop ◽

E Coli ◽

Physico Chemical ◽

Urgent Task ◽

Large Extracellular Loop

Hepatitis C is one of the most common social diseases in the world. The improvements in both the early diagnostics of the hepatitis C and the treatment of acute viremia caused by hepatitis C virus are undoubtedly an urgent task. In present work, we offered the micro- and nanotraps for the capturing of HCV. As a capturing moiety, we designed and synthesized in E. coli a fusion protein consisting of large extracellular loop of CD81 receptor and streptavidin as spacing part. The obtained protein has been immobilized on the surface of PLA-based micro- and nanoparticles. The developed trapping systems were characterized in terms of their physico-chemical properties. In order to illustrate the ability of developed micro- and nanotraps to bind HCV, E2 core protein of HCV was synthesized as a fusion protein with GFP. Interaction of E2 protein and hepatitis C virus-mimicking particles with the developed trapping systems were testified by several methods.

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Decoration of specific sites on freeze-fractured membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081565 ◽

1978 ◽

Vol 36 (2) ◽

pp. 140-141

Author(s):

H. Gross ◽

H. Moor

Keyword(s):

Pure Water ◽

Freeze Fracture ◽

Chemical Properties ◽

Surface Forces ◽

Sulfate Pentahydrate ◽

Copper Sulfate Pentahydrate ◽

Physico Chemical ◽

Water Of Hydration ◽

Small Container ◽

Binding Forces

Fracturing under ultrahigh vacuum (UHV, p ≤ 10-9 Torr) produces membrane fracture faces devoid of contamination. Such clean surfaces are a prerequisite foe studies of interactions between condensing molecules is possible and surface forces are unequally distributed, the condensate will accumulate at places with high binding forces; crystallites will arise which may be useful a probes for surface sites with specific physico-chemical properties. Specific “decoration” with crystallites can be achieved nby exposing membrane fracture faces to water vopour. A device was developed which enables the production of pure water vapour and the controlled variation of its partial pressure in an UHV freeze-fracture apparatus (Fig.1a). Under vaccum (≤ 10-3 Torr), small container filled with copper-sulfate-pentahydrate is heated with a heating coil, with the temperature controlled by means of a thermocouple. The water of hydration thereby released enters a storage vessel.

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PHYSICO CHEMICAL PROPERTIES OF NICKEL OXIDE AT HIGH TEMPERATURE

Le Journal de Physique Colloques ◽

10.1051/jphyscol:1976798 ◽

1976 ◽

Vol 37 (C7) ◽

pp. C7-438-C7-442 ◽

Cited By ~ 2

Author(s):

R. FARHI ◽

G. PETOT-ERVAS

Keyword(s):

High Temperature ◽

Nickel Oxide ◽

Chemical Properties ◽

Physico Chemical

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Physico-Chemical Properties of Recombinant Desulphatohirudin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645073 ◽

1990 ◽

Vol 63 (03) ◽

pp. 499-504 ◽

Cited By ~ 4

Author(s):

A Electricwala ◽

L Irons ◽

R Wait ◽

R J G Carr ◽

R J Ling ◽

...

Keyword(s):

Molecular Weight ◽

Gel Filtration ◽

Chemical Properties ◽

Alpha Helix ◽

Apparent Molecular Weight ◽

Correlation Spectroscopy ◽

Nmr Studies ◽

Recombinant Hirudin ◽

Physico Chemical ◽

Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

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