Purification of phospholipase-C from Bacillus cereus by affinity chromatography on 2-(4-aminophenylsulphonyl)ethyl-cellulose

Kamberov, E.; Ivanov, Alexander E.

doi:10.1016/s0378-4347(00)83407-6

Cited by 10 publications

(4 citation statements)

References 22 publications

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“…The maximum PC-PLC secretion at the end of the exponential growth phase corroborates the literature findings on phospholipase enzyme production during the late logarithmic and early stationary phases in Gram-positive bacteria [32]. Similar results regarding the secretion of phospholipase C were noted in C. perfringens and B. cereus [26,[33][34][35]. In Gram-negative bacteria, maximal enzymatic secretion occurs at a later stage in culture development, influenced by their physiological characteristics [36][37][38][39].…”

Section: Resultssupporting

confidence: 87%

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer

Baev,

Iliev,

Stefanov

et al. 2024

CIMB

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Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine–cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC.

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Section: Resultssupporting

confidence: 87%

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer

Baev,

Iliev,

Stefanov

et al. 2024

CIMB

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“…In contrast, the phosphatidylcholine-specific PLC [E.C.3.1.4.3] prefers phosphatidylcholine [3]. Due to its high structural and catalytical similarity to mammalian PLC the enzyme from Bacillus cereus is also subject of medical research [4]. This could be the explanation why PLC are the best-investigated bacterial enzymes of this class.…”

Section: Introductionmentioning

confidence: 99%

An improved assay for the determination of phospholipase C activity

Durban

Bornscheuer

2007

Euro J Lipid Sci & Tech

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Phospholipases C (PLC, EC 3.1.4.3) are enzymes that specifically hydrolyze the C-O-P bond in phospholipids, yielding sn-1,2(2,3)-diacylglycerides and the phosphate residue bearing the corresponding headgroup. The biochemical characterization of PLC requires methods for the reliable determination of their activity. Here, an assay is described in which the phosphate residue released by the PLC is cleaved with an alkaline phosphatase. The phosphate formed is then extracted with n-butanol and quantified as phosphomolybate complex. The applicability of this method is demonstrated for a concentration range from 10 nM to 10 mM for a range of phospholipids bearing different headgroups in an aqueous and a two-phase system. The method has the additional advantage that the crude enzyme can be used without the need for purification.

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“…Some PLCs are more substrate specific than others. The inositol-specific PLC [E.C.3.1.4.10] for example only cleaves phosphatidylinositol-4,5-bisphosphate into 1,2-diacylglycerides and inositol- (1,4,5)-triphosphate, which is an important second messenger in calcium metabolism [2]. In contrast, phosphatidyl choline specific phospholipase C (PC-PLC) [E.C.3.1.4.3] prefers phosphatidyl choline [3].…”

Section: Introductionmentioning

confidence: 99%

“…Due to its high structural and catalytic similarity to mammalian PLCs, the enzyme from B. cereus is also subject to medical research. Additionally, the enzyme is used to research lipid metabolism, lipid chemistry, blood coagulation and eucaryotic cell membranes [4]. For this reason PC-PLCs are the best investigated bacterial enzymes of this class.…”

Section: Introductionmentioning

confidence: 99%

An assay system for the detection of phospholipase C activity

Durban

Bornscheuer

2003

Euro J Lipid Sci & Tech

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Phospholipase C (PLC, EC 3.1.4.3) enzymes specifically hydrolyze the C-O-P-bond in phospholipids, yielding sn-1,2(2,3)-diglycerides and a phosphate residue bearing the corresponding head group. Biochemical characterization of PLC requires methods for determination of activity. During characterization and purification, proteins are separated by polyacrylamide gel electrophoresis (PAGE). For direct identification and visualization of PLC, a new assay for activity staining in native and renatured SDS-PAGE is described. Incubation of a gel containing an active PLC in the presence of a-naphthylphosphorylcholine leads to a-naphthol formation. This reacts with the diazonium salt Fast Red, forming a red dye which allows clear determination of PLC purity, molecular weight and substrate specificity. The assay was verified using commercially available PC-PLC and new PC-PLC-producing Bacillus cereus strains. The substrate a-NPC was prepared by chemical synthesis at an overall yield of 12%.

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Purification of phospholipase-C from Bacillus cereus by affinity chromatography on 2-(4-aminophenylsulphonyl)ethyl-cellulose

Cited by 10 publications

References 22 publications

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer

An improved assay for the determination of phospholipase C activity

An assay system for the detection of phospholipase C activity

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