B. cereus phospholipase C engineering for efficient degumming of vegetable oil

Elena, Claudia; Cerminati, Sebastián; Ravasi, Pablo; Rasia, Rodolfo M.; Peirú, Salvador; Menzella, Hugo G.; Castelli, María Eugenia

doi:10.1016/j.procbio.2017.01.011

Cited by 24 publications

(20 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…After showing the principle of CopySwitch with the easy-to-monitor GFP reporter protein, we applied the system to the expression of a secreted and industrially relevant enzyme. The phospholipase mPLC from B. cereus SBUG 516 has been chosen, which can be applied for the degumming of oils (Elena et al, 2017;Jiang et al, 2015) . Furthermore, it already has been shown to be expressed in and secreted from B. subtilis and its activity can be assayed photometrically (Durban et al, 2007) .…”

Section: Application Of Copyswitch To Change Mplc Expression Levelsmentioning

confidence: 99%

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

Nadler

Bracharz

Kabisch

2018

Preprint

View full text Add to dashboard Cite

The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis , we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.

show abstract

Section: Application Of Copyswitch To Change Mplc Expression Levelsmentioning

confidence: 99%

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

Nadler

Bracharz

Kabisch

2018

Preprint

View full text Add to dashboard Cite

show abstract

“…The mPLC is a monomeric, phosphatidylcholine-preferring PLC (E.C.3.1.4.3, UniProt-ID P09598), containing three catalytically relevant zinc ions. It is used in the industrial degumming of oils (Jiang et al, 2015; Elena et al, 2017). Its sequence comprises of the amino acids 39–283 (mature form) and is preceded by the first 33 amino acids of the B. subtilis amyE gene, which function as a signal peptide for secretion (Durban et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

Nadler

Bracharz

Kabisch

2019

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. While there are numerous ways of doing so on protein or mRNA level, our approach aims for the underlying number of coding sequences. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. Strains are transformed with a linearized, low copy number plasmid containing an antibiotic resistance marker and homology regions up- and downstream of the gene of interest. Said gene is copied onto the vector, rendering it circular and replicative and thus selectable. We could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful transformation. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher. A major benefit of this method is the ability to work with very large regions of interest, since all relevant steps are carried out in vivo and are thus far less prone to mechanical DNA damage.

show abstract

“…ese need to be removed in the degumming process. e purpose of degumming removes colloidal impurities in crude oil improving the quality of it, building a good foundation for the subsequent processes [1,2]. Phospholipids in soybean crude oil are divided into hydratable phospholipids (HPs) and nonhydratable phospholipids (NHPs).…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Application of Nanomagnetic Immobilized Phospholipase C

Jiang

Tang

et al. 2019

Journal of Chemistry

View full text Add to dashboard Cite

The nanomagnetic carrier (Fe3O4@SiO2@p(GMA)) was prepared by atom transfer radical polymerization, and then, the free phospholipase C (PLC) was immobilized on it proved by the results of FT-IR analysis. The enzyme loading was 135.64 mg/g, the enzyme activity was 8560.7 U/g, the average particle size was 99.86 ± 0.80 nm, and the specific saturation magnetization was 16.00 ± 0.20 emu/g. PLC-Fe3O4@SiO2@p(GMA) showed the highest activities at the pH of 7.5, and tolerance temperature raised to 65°C in soybean lecithin emulsion. Enzymatic degumming with PLC-Fe3O4@SiO2@p(GMA) under the conditions of the enzyme dosage of 110 mg/kg, reaction temperature of 65°C, pH of 7.5, and reaction time of 2.5 h resulted in residual phosphorus of 64.7 mg/kg, 1,2-diacylglycerol (1,2-DAG) contents of 1.07%, and oil yield of 98.1%. Moreover, PLC-Fe3O4@SiO2@p(GMA) still possessed more than 80% of its initial activity after 5 cycles.

show abstract

B. cereus phospholipase C engineering for efficient degumming of vegetable oil

Cited by 24 publications

References 21 publications

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

Synthesis and Application of Nanomagnetic Immobilized Phospholipase C

Contact Info

Product

Resources

About