In Search of Engineered Prokaryotic Chlorophyllases: A Bioinformatics Approach

Sharafi, Ebrahim; Farmani, Jamshid; Parizi, Ali Pakdin; Dehestani, Ali

doi:10.1007/s12257-018-0143-6

Cited by 8 publications

(8 citation statements)

References 34 publications

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“…1. As reported by Sharafi et al (2017) and Sharafi et al (2018), all the chlorophyllase proteins shared the specific lipase motif GXSXG (indicated by a red line in Fig. 1).…”

Section: Resultssupporting

confidence: 77%

“…The active site of OscChlase and CitChlase were predicted to be at 159 (Ser), 226 (Asp), 258 (His), and 122 (Leu), 195 (Pro), respectively. As documented previously (Sharafi et al, 2017;Sharafi et al, 2018), plant, bacterial, and cyanobacterial chlorophyllases share a catalytic triad composed of serine, arginine, and histidine residues, which was also detected in the predicted OscChlase tertiary model. However, this catalytic triad was not detected in the predicted tertiary model for CitChlase.…”

Section: Chlorophyll Removal From Olive and Canola Oil Using Recombinant Enzymessupporting

confidence: 78%

“…In search of potential chlorophyllases for recombinant production, several bioinformatics analyses have been performed on chlorophyllase genes isolated from plants (Sharafi et al, 2017) and bacterial (Sharafi et al, 2018) sources. Based on those bioinformatics analyses, Sharafi et al (2018) reported the chlorophyllase from Oscillatoria acuminata as a good candidate for the recombinant production of a high‐activity enzyme. On the other hand, one of the most important sources of chlorophyllase enzymes is citrus plants, which are grown all over the world in more than 140 countries (Liu et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Chlorophyllase‐Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

Sharafi

Dehestani

Farmani

et al. 2021

J Americ Oil Chem Soc

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Chlorophyllase converts chlorophyll and pheophytin into their colorless derivatives (chlorophyllide/ pheophorbide and phytol). This activity can be used in chlorophyll removal from vegetable oils. Chlorophyllase genes from Oscillatoria acuminata (OscChlase) and Citrus aurantium (CitChlase) were isolated, cloned, and expressed in E. coli. Bioinformatics analysis showed that both chlorophyllases shared a conserved GHSXG lipase motif responsible for their catalytic activity. SDS-PAGE and immunoblot assays revealed that both enzymes had a molecular weight of 35 kDa. The purified chlorophyllases were stable at a broad range of temperatures and showed the highest activity at 40 C. OscChlase and CitChlase exhibited the highest activity at pH 6.0 and 7.0, respectively. Enzyme kinetics analysis revealed that OscChlase was able to hydrolyze bacteriochlorophyll-a more efficiently than the recombinant CitChlase (Vmax/Km of 0.38 for OscChlase vs. 0.01 min −1 mg protein for CitChlase). Instead, CitChlase hydrolyzed chlorophyll-b more efficiently than OscChlase. Both enzymes were able to reduce the chlorophyll content of olive (from 623.1 to as low as 87.2 mg per kg oil) and canola oil (from 537.2 to as low as 101.1 mg per kg oil). The ratio of oil to the aqueous reaction media affected chlorophyll hydrolysis (P < 0.05). The lower the oil ratio was (10%), the higher the chlorophyll removal was (75-86%). The efficiency of CitChlase in chlorophyll removal was higher than that of OscChlase at oil ratios of 10 and 20, but lower at 30% ratio (P < 0.05). This is the first report on the application of recombinant OscChlase and CitChlase in chlorophyll removal (up to 86%) from vegetable oils.

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“…1. As reported by Sharafi et al (2017) and Sharafi et al (2018), all the chlorophyllase proteins shared the specific lipase motif GXSXG (indicated by a red line in Fig. 1).…”

Section: Resultssupporting

confidence: 77%

Section: Chlorophyll Removal From Olive and Canola Oil Using Recombinant Enzymessupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

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Chlorophyllase‐Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

Sharafi

Dehestani

Farmani

et al. 2021

J Americ Oil Chem Soc

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“…ATCC 51472 preferentially hydrolyzes BChl a to produce BChlide a [ 23 ]. Sharafi et al also predicted that all bacterial and cyanobacterial chlorophyllases prefer BChl a as their substrate [ 24 , 30 ]. Therefore, this is the first report of a cyanobacterial chlorophyllase with a substrate preference for Chl a and Chl b rather than BChl a.…”

Section: Resultsmentioning

confidence: 99%

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

Dai

et al. 2021

Microb Cell Fact

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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.

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“…Also, the aliphatic index is dependent on the volume occupied by aliphatic side chains (alanine, valine, leucine, and isoleucine) [ 15 ]. In addition, the instability index indicates the degree of protein instability, and the GRAVY index determines the hydrophilicity/hydrophobicity of the protein [ 16 , 17 ].…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of inulinases by bioinformatics analysis of bacterial glycoside hydrolases family 32 (GH32)

Khosravi

Fard

Hosseininezhad³

et al. 2023

Engineering in Life Sciences

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The glycoside hydrolase family contains enzymes that break the glycosidic bonds of carbohydrates by hydrolysis. Inulinase is one of the most important industrial enzymes in the family of Glycoside Hydrolases 32 (GH32). In this study, to identify and classify bacterial inulinases initially, 16,002 protein sequences belonging to the GH32 family were obtained using various databases. The inulin-effective enzymes (endoinulinase and exoinulinase) were identified. Eight endoinulinases (EC 3.2.1.7) and 4318 exoinulinases (EC 3.2.1.80) were found. Then, the localization of endoinulinase and exoinulinase enzymes in the cell was predicted. Among them, two extracellular endoinulinases and 1232 extracellular exoinulinases were found. The biochemical properties of 363 enzymes of the genus Arthrobacter, Bacillus, and Streptomyces (most abundant) showed that exoinulinases have an acid isoelectric point up to the neutral range due to their amino acid length. That is, the smaller the protein (336 aa), the more acidic the pI (4.39), and the larger the protein (1207 aa), the pI is in the neutral range (8.84). Also, a negative gravitational index indicates the hydrophilicity of exoinulinases. Finally, considering the biochemical properties affecting protein stability and post-translational changes studies, one enzyme for endoinulinase and 40 enzymes with desirable characteristics were selected to identify their enzyme production sources. To screen and isolate enzyme-containing strains, now with the expansion of databases and the development of bioinformatics tools, it is possible to classify, review and analyze a lot of data related to different enzymeproducing strains. Although, in laboratory studies, a maximum of 20 to 30 strains can be examined. Therefore, when more strains are examined, finally, strains with more stable and efficient enzymes were selected and introduced for laboratory activities. The findings of this study can help researchers to select the Abbreviation: GH32, Glycoside Hydrolase 32.

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In Search of Engineered Prokaryotic Chlorophyllases: A Bioinformatics Approach

Cited by 8 publications

References 34 publications

Chlorophyllase‐Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

Chlorophyllase‐Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

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

Identification and characterization of inulinases by bioinformatics analysis of bacterial glycoside hydrolases family 32 (GH32)

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