scholarly journals Molecular, structural and biochemical characterization of a novel recombinant chlorophyllase from cyanobacterium Oscillatoria acuminata PCC 6304

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Sitian Gu ◽  
Xiaojun Dai ◽  
Zhengjun Xu ◽  
Qiwen Niu ◽  
Jiang Jiang ◽  
...  
Keyword(s):  
Chlorophyll A ◽  
Biochemical Characterization ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Oil Refining ◽  
Chlorophyll B ◽  
Pheophytin A ◽  
Signal Peptide Sequence ◽  
His Tag

Abstract Background Chlorophyllase catalyzes the hydrolysis of chlorophyll and produces chlorophyllide and phytol. Cyanobacterial chlorophyllases are likely to be more highly heterologously expressed than plant chlorophyllases. A novel recombinant chlorophyllase from the cyanobacterium Oscillatoria acuminata PCC 6304 was successfully expressed in Escherichia coli BL21(DE3). Results The putative N-terminal 28-amino-acid signal peptide sequence of O. acuminata chlorophyllase (OaCLH) is essential for its activity, but may confer poor solubility on OaCLH. The C-terminal fusion of a 6 × His tag caused a partial loss of activity in recombinant OaCLH, but an N-terminal 6 × His tag did not destroy its activity. The optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40 °C, respectively. Recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, but prefers chlorophyll b and chlorophyll a as substrates. The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159, Asp226, and His258. Conclusions The high-level expression and broad substrate specificity of recombinant OaCLH make it suitable for genetically engineering and a promising biocatalyst for industrial production, with applications in vegetable oil refining and laundry detergents.

Interactions between Photosynthetic Pigments Bound to Lipid and Protein Particles. Spectroscopic Properties

1979 ◽  
Vol 34 (7-8) ◽  
pp. 582-587
Author(s):  
Framçoise Techy ◽  
Monique Dinant ◽  
Jacques Aghion
Keyword(s):  
Chlorophyll A ◽  
Photosynthetic Pigments ◽  
Relative Concentration ◽  
Spectroscopic Properties ◽  
Chlorophyll B ◽  
Pheophytin A ◽  
Chlorophylls A And B ◽  
Protein Particles ◽  
Β Carotene

Abstract The spectroscopic (visible) properties of pigment-bearing lipid and protein particles extract­ ed from milk show that: 1) chlorophylls a and b bound to separate particles can form aggregates provided their relative concentration is high enough. Neither pheophytin a nor β-carotene, in the same conditions, form observable aggregates. 2) Chlorophylls a and b can co-aggregate when they are bound to the same particles. Pheophytin a as well as β-carotene seem to prevent the aggregation of chlorophyll a. β-carotene has no effect on the aggregation of chlorophyll b.

Reduction of the Formyl Group of Zinc Pheophorbide b in vitro and in vivo: a Model for the Chlorophyll b to a Transformation

1996 ◽  
Vol 51 (3-4) ◽  
pp. 185-194 ◽  
Author(s):  
Verena Scheumann ◽  
Michael Helfrich ◽  
Siegrid Schoch ◽  
Wolfhart Rüdiger
Keyword(s):  
Nmr Spectroscopy ◽  
Chlorophyll A ◽  
Chemical Reduction ◽  
Chlorophyll B ◽  
Formyl Group ◽  
Pheophorbide A ◽  
Pheophytin A ◽  
H Nmr

Abstract The chemical reduction of the formyl group of pheophorbide b with sodium cyanoborohy­ dride in methanol leads to 71-methoxy-and 71-hydroxy-pheophorbide a. The same reaction with zinc pheophorbide b yields in addition zinc pheophorbide a. This was characterized by mass and 1H -NMR spectroscopy. Infiltration of zinc pheophorbides a and b and of zinc 71-hydroxy-pheophorbide a into etiolated oat leaves yielded phytylated products. The best yield in the esterification was obtained with 71-hydroxy-pheophorbide a. Analysis of the products revealed the formation of zinc pheophytin a from all infiltrated compounds. The significance for the transformation of chlorophyll b into chlorophyll a is discussed.

scholarly journals A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study

2019 ◽  
Vol 20 (3) ◽  
pp. 780 ◽  
Author(s):  
Wei Tang ◽  
Dongming Lan ◽  
Zexin Zhao ◽  
Shuang Li ◽  
Xiuting Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Fermentation Broth ◽  
Catalytic Triad ◽  
Esterification Reaction ◽  
Monoacylglycerol Lipase ◽  
His Tag ◽  
Km Value ◽  
Mutagenesis Study ◽  
Hydrolysis Activity

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

scholarly journals Functional Characterization of a Phospholipase A2 Homolog from Rickettsia typhi

2010 ◽  
Vol 192 (13) ◽  
pp. 3294-3303 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
Nicole C. Ammerman ◽  
Khandra T. Sears ◽  
Shane M. Ceraul ◽  
Abdu F. Azad
Keyword(s):  
Functional Characterization ◽  
Gene Expression Pattern ◽  
Vero Cells ◽  
Phospholipase A ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Peptide Sequence ◽  
Transcriptional Analysis ◽  
Rickettsia Typhi ◽  
Signal Peptide Sequence

ABSTRACT Phospholipase A2 (PLA2) has long been proposed to be involved in rickettsial entry into host cells, escape from the phagosome to evade destruction by lysosomal exposure, and lysis of the host cells. However, the corresponding rickettsial gene(s) encoding a protein with PLA2 activity has not been identified or functionally characterized. Here, we report that the Rickettsia typhi genome possesses two genes encoding patatin-like PLA2 proteins, RT0590 and RT0522. Sequence analysis of RT0522 and RT0590 reveals the presence of the conserved motifs essential for PLA2 activity. Transcriptional analysis indicates that RT0522, but not RT0590, is transcribed at all stages of intracellular growth of R. typhi in Vero cells. The differential gene expression pattern of RT0522 at various stages of growth suggests its potential role during R. typhi infection of host cells. In silico, RT0522 is predicted to be noncytoplasmic and its gene does not encode a recognizable signal peptide sequence. However, our data indicate that RT0522 is secreted into the host cytoplasm. In addition, we observe that RT0522 protein expression is cytotoxic to both yeast and Vero cells. Importantly, we demonstrate that recombinant RT0522 possesses phospholipase A activity that requires a eukaryotic host cofactor for activation. Both cytotoxicity and phospholipase A activity associated with RT0522 were reduced by PLA2 inhibitors. Site-directed mutagenesis of predicted catalytic Ser/Asp residues of RT0522 also eliminates cytotoxicity and phospholipase A activity. To our knowledge, RT0522 is the first protein identified from Rickettsia typhi with functional phospholipase A activity.

Absorption Coefficients of Chlorophyll Derivatives

1968 ◽  
Vol 25 (3) ◽  
pp. 523-540 ◽  
Author(s):  
Seward R. Brown
Keyword(s):  
Uniform Distribution ◽  
Chlorophyll A ◽  
Chlorophyll B ◽  
Pheophorbide A ◽  
Absorption Coefficients ◽  
Pheophytin A ◽  
Carbon 14 ◽  
Chlorophylls A And B ◽  
Laboratory Cultures ◽  
Chlorophyll Derivatives

Methods are described for the isolation of chlorophylls a and b, and for the preparation of 12 chlorophyll derivatives, including crystalline pheophorbide a, from these starting compounds. Laboratory cultures of Chlorella were fed C14O2. After 9 days growth the cells were heat-killed, their pigments extracted, and chlorophylls a and b isolated chromatographically. The following C14-labelled compounds were prepared: chlorophyll a, allomerized chlorophyll a, chlorophyllide a, pheophytin a, allomerized pheophytin a, pheophorbide a, methyl pheophorbide a, chlorophyll b, chlorophyllide b, methyl chlorophyllide b, allomerized chlorophyllide b, allomerized methyl chlorophyllide b, pheophorbide b, and allomerized pheophorbide b. Assuming uniform distribution of carbon-14 label in the parent chlorophyll molecules, specific and molar absorption coefficients were calculated from the specific radioactivities of these derivatives.

EFFECT OF MAGNESIUM CARBONATE AND SODIUM PHOSPHATE ON THE EXTRACTION OF CHLOROPHYLL-LIKE PIGMENTS AFTER THERMAL PROCESSING OF SPINACH PUREE

1970 ◽  
Vol 33 (10) ◽  
pp. 456-459 ◽  
Author(s):  
D. L. Fleischman ◽  
F. M. Clydesdale ◽  
F. J. Francis
Keyword(s):  
Chlorophyll A ◽  
Thermal Processing ◽  
Sodium Phosphate ◽  
Magnesium Carbonate ◽  
Chlorophyll B ◽  
Pheophorbide A ◽  
Pheophytin A ◽  
Green Color

Because buffers are important in maintaining green color in vegetable purees, a study was undertaken to determine if sodium phosphate and/or magnesium carbonate would effect extraction of some or all of the chlorophyll-like pigments in spinach puree and thus interfere with their analysis. Samples were prepared with and without addition of the above additives and pigment analyses were carried out for chlorophylls, chlorophyllides, pheophytins, and pheophorbides. It was found that addition of sodium phosphate and/or magnesium carbonate prior to blanching and processing caused chlorophyll b, pheophytin a, pheophorbide a, and pheophorbide b to be bound to some extent. Pheophytin b was unaffected Chlorophyll a, chlorophyllide a, and chlorophyllide b were present in such small quantities that no conclusions could be drawn about their extractability. This phenomenon is an important consideration in the analysis of chlorophyll-like pigments in buffered systems and also might supply a suitable means for removal of chlorophyll-like pigments from foods and beverages where the color they impart is undesirable.

Etude sur la teneur en pigment de frênes qui croissent dans des conditions différentes

1973 ◽  
Vol 37 ◽  
Author(s):  
N. Lust
Keyword(s):  
Chlorophyll A ◽  
Strong Influence ◽  
The Other ◽  
Pigment Content ◽  
Chlorophyll B ◽  
Light Quantity ◽  
Yellow Pigments ◽  
The One ◽  
Green Pigments

Pigment content of ashes grown up under different circumstances - The pigment content (chlorophyll a, chlorophyll b,  xanthophyll and carotene) has been researched with ashes grown up under  different light circumstances and varying in age and height.     The results prove that the general laws concerning the influence of light  on the pigment content, don’t always work.     The phenomen is very complex. The light quantity is very important in some  cases, but insignificant in others. It seems origin and height of plants have  a strong influence. The results prove also the influence of the environment  is much higher on small plants as on big ones.     The research indicates finally the correlation between the green pigments,  the yellow pigments, and between the green pigments on the one side and the  yellow ones on the other side.

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