High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.

Three branches of phospholipase C signaling pathway promote hepatocyte growth in rat liver regeneration.

In general, the phospholipase C (PLC) signaling pathway is involved in many physiological activities, including cell growth. However, little is known regarding how the PLC signaling pathway participates in regulating hepatocyte (HC) growth during liver regeneration (LR). To further explore the influence of the PLC signaling pathway on HCs at the cellular level, HCs of high purity and vitality were isolated using Percoll density-gradient centrifugation after partial hepatectomy. The genes of the PLC signaling pathway and target genes of transcription factors in the pathway were obtained by searching the pathways and transcription factor databases, and changes in gene expression of isolated HCs were examined using the Rat Genome 230 2.0 Microarray. The results suggested that various genes involved in the pathway (including 151 known genes and 39 homologous genes) and cell growth (including 262 known genes and 37 homologous genes) were associated with LR. Subsequently, the synergetic effect of these genes in LR was analyzed using a mathematical model (Et) according to their expression profiles. The results showed that the Et values of G protein-coupled receptor/PLC, integrin/PLC, and growth factor receptor/PLC branches of the PLC pathway were all significantly strengthened during the progression and termination phases of LR. The synergetic effect of target genes, in parallel with target gene-related cell growth, was also enhanced during whole rat LR, suggesting the potential positive effect of PLC on HC growth. The present data indicate that the PLC signaling pathway may promote HC growth through 3 mechanisms during rat LR after partial hepatectomy.

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A.

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity.

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta-galactosidase activity. beta-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Paracoccidioides brasiliensis expresses both glycosylphosphatidylinositol-anchored proteins and a potent phospholipase C.

This study reports, for the first time, the detection of glycosylphosphatidylinositol (GPI) membrane anchors in proteins of a pathogenic fungus, Paracoccidioides brasiliensis. Taking into account that fungal antigens are found in the sera of paracoccidioidiomycosis patients and that cleavage of this glycolipid by phospholipases is a means of selective protein release, the presence of an enzyme with this property has also been investigated. Using a methodological approach in which the proteins were immobilized on nitrocellulose, treated with phospholipase C of Trypanosoma brucei and then probed with antibodies which recognize the 1,2-cyclic-phosphate inositol moiety formed as a reaction product in proteins bearing the glycolipid anchor, it was possible to detect a major glycoprotein in the 80- to 90-kDa range, as well as two other minor species of 66 and 43 kDa. All of them bind to Concanavalin-A and are also substrates of a very potent fungal phospholipase C which is inhibited by p-chloromercuri-phenylsulfonic acid and is insensitive to EDTA. The integrity of glycosylphosphatidylinositol anchors in proteins of P. brasiliensis is impaired by 0.1 M NaOH, a finding indicative of a diacyl glycerolipid moiety which is quite surprising since it is, with the exception of African trypanosomes surface proteins and Torpedo acetylcholinesterase, an uncommon feature among GPIs in general. The present findings may have implications in the pathology of paracoccidiodomycosis.

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline.

1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-Pi basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite. 2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate. 3. The peak containing the PLC activity revealed a single protein after SDS-PAGE. 4. The method could also be applied to purify PLC produced in a low-Pi complex medium. The resultant preparation was not homogeneous. 5. The molecular weight for both PLC preparations was about 70 kDa. 6. Both PLC used phosphatidylcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent KM of 25 mM for p-nitrophenylphosphorylcholine. 7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100. 8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP1-BSM plus choline but not the enzyme from the LP1-CM.