scholarly journals Characterization of a Novel Lutein Cleavage Dioxygenase, EhLCD, from Enterobacter hormaechei YT-3 for the Enzymatic Synthesis of 3-Hydroxy-β-ionone from Lutein

Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1257
Author(s):  
Zhangde Long ◽  
Naixin Duan ◽  
Yun Xue ◽  
Min Wang ◽  
Jigang Li ◽  
...  
Keyword(s):  
Enzymatic Synthesis ◽  
Isotope Labeling ◽  
Catalytic Efficiency ◽  
Potential Candidate ◽  
Sds Page ◽  
Cleavage Enzyme ◽  
Dependent Protein ◽  
Fragrance Compound ◽  
Enterobacter Hormaechei ◽  
Tween 40

3-Hydroxy-β-ionone, a flavor and fragrance compound with fruity violet-like characteristics, is widely applied in foodstuff and beverages, and is currently produced using synthetic chemistry. In this study, a novel lutein cleavage enzyme (EhLCD) was purified and characterized from Enterobacter hormaechei YT-3 to convert lutein to 3-hydroxy-β-ionone. Enzyme EhLCD was purified to homogeneity by ammonium sulfate precipitation, Q-Sepharose, phenyl-Sepharose, and Superdex 200 chromatography. The molecular mass of purified EhLCD, obtained by SDS-PAGE, was approximately 50 kDa. The enzyme exhibited the highest activity toward lutein, followed by zeaxanthin, β-cryptoxanthin, and β-carotene, suggesting that EhLCD exhibited higher catalytic efficiency for carotenoid substrates bearing 3-hydroxy-ionone rings. Isotope-labeling experiments showed that EhLCD incorporated oxygen from O2 into 3-hydroxy-β-ionone and followed a dioxygenase reaction mechanism for different carotenoid substrates. These results indicated that EhLCD is the first characterized bacterial lutein cleavage dioxygenase. Active EhLCD was also confirmed to be a Fe2+-dependent protein with 1 molar equivalent of non-haem Fe2+. The purified enzyme displayed optimal activity at 45 °C and pH 8.0. The optimum concentrations of the substrate, enzyme, and Tween 40 for 3-hydroxy-β-ionone production were 60 mM lutein/L, 1.5 U/mL, and 2% (w/v), respectively. Under optimum conditions, EhLCD produced 3-hydroxy-β-ionone (637.2 mg/L) in 60 min with a conversion of 87.0% (w/w), indicating that this enzyme is a potential candidate for the enzymatic synthesis of 3-hydroxy-β-ionone in biotechnological applications.

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

scholarly journals Biosynthesis, purification, characterization and immobilization of laccase from Lithothelium sp.

Chemija ◽  
2020 ◽  
Vol 31 (3) ◽  
Author(s):  
Ingrida Radveikienė ◽  
Ingrida Pilotaitė ◽  
Rimgailė Dainytė ◽  
Regina Vidžiūnaitė
Keyword(s):  
Residual Activity ◽  
Catalytic Efficiency ◽  
Hydrophobic Interaction Chromatography ◽  
Metal Salts ◽  
Biotechnological Applications ◽  
Affinity Constants ◽  
Sds Page ◽  
Wide Range ◽  
Optimum Ph ◽  
Sulphate Salts

Novel fungal laccase isoenzymes (namely L95-1 and L95-2) produced by the Ascomycete Lithothelium sp. isolated from the forest soil were purified. However, only one of them was characterized, because the other isoenzyme lost its activity during purification. Extracellular L95-1 laccase was purified 30-fold using ion-exchange and hydrophobic interaction chromatography, with an overall yield of 88%. The molecular mass of purified L95-1 was estimated to be 85 kDa by SDS-PAGE analysis. L95-1 laccase was stable at temperature 4–22°C and pH 6.0–6.5. The substrate specificity of L95-1 laccase was examined with various compounds. Determined affinity constants (KM) varied in a wide range of 3.7–2020.0 µM, whereas catalytic efficiency constants (kcat/KM) covered a range of 0.008–1.9 µM–1 s–1. The optimum pH for most substrates varied in a range from pH 5.0 to 6.0. Sodium azide and fluoride strongly inhibited L95-1 activity, whereas sulphate salts inhibited weakly. The laccase was immobilized on the Fe3O4 nanoparticles and characterized. Residual activity remained at 20% after ten cycles of ABTS oxidation reaction. The immobilized laccase showed higher tolerance to various metal salts. The properties of L95-1 laccase make it potentially useful in the biotechnological applications.

scholarly journals ATP citrate-lyase and glycogen synthase kinase-3β in 3T3-L1 cells during differentiation into adipocytes

1994 ◽  
Vol 300 (2) ◽  
pp. 477-482 ◽  
Author(s):  
W B Benjamin ◽  
S N Pentyala ◽  
J R Woodgett ◽  
Y Hod ◽  
D Marshak
Keyword(s):  
Glycogen Synthase ◽  
Protein Band ◽  
Glycogen Synthase Kinase ◽  
Fat Cells ◽  
Apparent Mass ◽  
Atp Citrate Lyase ◽  
Protein Levels ◽  
Citrate Lyase ◽  
Sds Page ◽  
Dependent Protein

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these ‘down-regulated’ cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

Possible Roles for Protein Phosphorylation in Platelet Responses

1979 ◽  
Author(s):  
R.J. Haslam ◽  
J.E.B. Fox ◽  
S.E. Salama ◽  
J.A. Lynham
Keyword(s):  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Platelet Function ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Type I ◽  
Sds Page ◽  
Dependent Protein ◽  
32P Incorporation

The relationships between the phosphorylation of specific platelet polypeptides and platelet function were studied using washed human platelets labelled by preincubation with [32p] Pi. Platelet polypeptides were separated by SDS-PAGE and 32P incorporation into them determined by autoradiography. Whereas induction of platelet aggregation alone did not affect protein phosphorylation, induction of the release reaction increased 3P incorporation into several polypeptides (P75,P47,P40,P27,P20,P19), including the P-light chain of platelet myosin (P20). These changes were inhibited by drugs that blocked Ca2 movements and may be due to activation of Ca2+-dependent protein kinases. Compounds that inhibited platelet function by increasing cyclic AMP (e.g. PCE1) also suppressed these reactions but, in addition, increased phosphorylation of other polypeptides (P50,P49,P36,P24,P22). Type I and Type II cyclic AMP-dependent protein kinases were present in platelets and may mediate Che latter effects of cyclic AMP. Subcellular fractionation of 32p-labelled platelets that had been exposed to PCE1 showed that P24 was present in membranes that could take up Ca2+ by an ATP-dependent mechanism. Membranes from PCE1-treated platelets took up Ca2+ more rapidly than control membranes. Thus, the cyclic AMP-dependent phosphorylation of P24 may stimulate the removal of Ca2+ from platelet cytosol and suppress Ca2+-dependent phosphorylation reactions necessary for release of granule constituents.

scholarly journals Rapid purification and identification of calcyphosine, a Ca2+-binding protein phosphorylated by protein kinase A

1995 ◽  
Vol 306 (1) ◽  
pp. 147-151 ◽  
Author(s):  
R Lecocq ◽  
F Lamy ◽  
C Erneux ◽  
J E Dumont
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Conformational Changes ◽  
Hydrophobic Interaction Chromatography ◽  
Peptide Sequencing ◽  
Native Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Rapid Purification ◽  
Kinase A

A method is presented for the rapid purification of dog thyroid calcyphosine, a protein previously identified as a major substrate for cyclic AMP-dependent protein kinase in dog thyroid slices stimulated by thyrotropin [Lecocq, Lamy and Dumont (1979) Eur. J. Biochem. 102, 147-152]. The protein was previously identified as a spot on two-dimensional gels and is now purified in its native form by a procedure involving three chromatographic steps. Homogeneous calcyphosine identified by SDS/PAGE, immunoblotting and peptide sequencing can be obtained within 7 h. As for calmodulin, Ca(2+)-dependent conformational changes can be shown by Ca(2+)-dependent hydrophobic interaction chromatography using phenyl-Sepharose. Unlike calmodulin, calcyphosine is a substrate for protein kinase A.

scholarly journals Production of the 13C/15N single-labeled insecticidal protein Cry1Ab/Ac for the assessment of metabolic fate using recombinant Escherichia coli

2020 ◽  
Author(s):  
Zibo Wang ◽  
Cong Hu ◽  
Yu Sun ◽  
Wei Jiang ◽  
Guogan Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stable Isotope ◽  
Inclusion Bodies ◽  
Isotope Labeling ◽  
Recombinant Expression ◽  
Insecticidal Protein ◽  
Cry Protein ◽  
Environmental Behaviors ◽  
Cry Proteins ◽  
Sds Page

Abstract Stable isotope-labeled Cry1Ab/Ac protein is necessary for the metabolic study of exogenous insecticidal protein in soil using the stable isotope labeling technique, but no recombinant expression protocols for this protein have been reported. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which obtained the safety certificate in China, was subcloned into pUC57 in this study, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM IPTG was added to these cells and cultured at 37°C for 4 h to induce the expressed protein to form inclusion bodies in the presence of M9 medium containing either [U-13C] glucose (5% 13C-enriched) or [15N] ammonium chloride (5% 15N-enriched). Then Cry inclusion bodies were dissolved in urea and purified by Ni column affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by SDS-PAGE and Western blot. The purities of 13C/15N-labeled Cry proteins were each 99%, the amounts of which were 12.6 mg/L and 8.8 mg/L. The δ 13C and δ 15N values of 13C-labeled Cry protein and 15N-labeled Cry protein were 3268.68‰ and 2854.28‰, respectively. An insecticidal test showed that the prokaryotic expression of Cry1Ab/Ac protein had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies of Cry protein in soil and the characteristics of nitrogen (N) and carbon (C) transformation. The findings will also provide a reference and basis for elucidating the environmental behaviors and ecological effects of Cry plants and expressed products.

scholarly journals The Potential of Candida Biofilm Protein as Bioreceptor for Candidiasis Immunoassay

2018 ◽  
Vol 14 (1) ◽  
pp. 47 ◽  
Author(s):  
Masfufatun Masfufatun ◽  
Loo Haryanto ◽  
Harsono Harsono
Keyword(s):  
Molecular Weight ◽  
Candida Albicans ◽  
Polyclonal Antibody ◽  
Control Group ◽  
Potential Candidate ◽  
Dot Blot ◽  
Protein Extract ◽  
Treatment Groups ◽  
Sds Page ◽  
Mechanical Methods

Abstract: Candidiasis or infection that is caused by Candida has become a new list of the therapeutical problems recently. The difficulties in diagnosing are the main cause of the unsatisfactory results from common therapies and diagnosis methods. This has urged researchers to find alternative ways in candidiasis diagnosis such as serology-based detection using antigen or antibody development. The aim of this study was to evaluate the potential of protein derived from Candida albicans biofilm as bioreceptor on candidiasis immunoassay through Dot Blot method. The research method used descriptive method with the following stages: (1) preparation of Candida albicans biofilm (2) extraction of Candida albicans protein through enzymatic and mechanical methods, (3) determination of protein molecular weight with SDS-PAGE (4) production of polyclonal anti- candida and (5) analysis of protein extract as bioreeceptor on dot blot. Profile of biofilm proteins on SDS-PAGE analysis were shown on molecular weight 27,42; 29,89; 38,10; 44,90; 48,75; 52,92; 55,14; 59,86; 70,56; 87,36; 102,54;115,05; 130,14;143,14;181,53 kD. There were differences in the intensity of dots in the control group (44070) and treatment groups (63170.5). It is noticeable that biofilm protein extract of C. albicans can be used for induction of anti-Candida polyclonal antibody production as the potential candidate of bioreceptor in candidiasis immunoassay. Keywords: SDS-PAGE, polyclonal antibody, immunoassay, dot blot, biofilm

Monoclonal Antibodies Against the Heparin- Dependent Protein C Inhibitor Suitable for Inhibitor Purification and Assay of Inhibitor Complexes

1988 ◽  
Vol 60 (02) ◽  
pp. 334-339 ◽  
Author(s):  
Martin Laurell ◽  
Timothy H Carlson ◽  
Johan Stenflo
Keyword(s):  
Monoclonal Antibodies ◽  
Protein C ◽  
Activated Protein C ◽  
Average Yield ◽  
Amidolytic Activity ◽  
Specific Antibodies ◽  
Sds Page ◽  
Dependent Protein ◽  
Fresh Plasma ◽  
Formation Of Complexes

summaryTwo different monoclonal antibodies against the heparin- dependent inhibitor of human activated protein C were produced, using cleaved modified inhibitor for immunization and partially purified inhibitor for screening of the hybridomas, One of the antibodies recognized free and complexed forms of the inhibitor in immunoblotting experiments. The other antibody was used to develop an assay for A PC -PCI inhibitor complexes. Using the assay the formation of complexes was studied in plasma, both in the presence and absence of heparin. The rate of complex formation was similar to that reported previously for the loss of activated protein C amidolytic activity in plasma. The same antibody was also immobilized on Sepharose and used to purify the inhibitor from fresh human plasma. The purified material appeared as two narrowly spaced bands with Mr about 57,000 in SDS-PAGE. The average yield from 1 liter of fresh plasma was 1 mg of inhibitor, The purified inhibitor formed SDS stable complexes with activated protein G and urokinase that could be identified in immunoblots using specific antibodies.

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