Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming

Marchisio, Fiorela; Nardo, Luisina Di; Val, Diego Sebastián; Cerminati, Sebastián; Espariz, Martín; Rasia, Rodolfo M.; Menzella, Hugo G.; Castelli, María Eugenia

doi:10.1007/s00253-022-12081-z

Cited by 7 publications

(4 citation statements)

References 44 publications

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“…Variants of phospholipase C with a higher thermal stability can be obtained by searching the genomes of thermophilic organisms for homologous sequences. We have previously obtained and studied a natural thermostable Zn(II) PLC from Thermococcus kodakarensis (TkPLC), which is a promising candidate for industrial-scale enzymatic oil degumming at high temperatures . Alternatively, new artificial sequences can be generated by protein engineering. − In this work, we present the use of the consensus sequence approach to produce a new thermostable variant of this enzyme.…”

Section: Introductionmentioning

confidence: 99%

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design

Val,

Di Nardo,

Marchisio

et al. 2024

Biochemistry

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Proteins' extraordinary performance in recognition and catalysis has led to their use in a range of applications. However, proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here, we achieved a substantial thermal stabilization of a bacterial Zn(II)dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologues from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of the stability parameters of this stabilized mutant and other natural variants bearing similar mutations allows us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in harsh oil degumming conditions, making it suitable for industrial applications.

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Section: Introductionmentioning

confidence: 99%

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design

Val,

Di Nardo,

Marchisio

et al. 2024

Biochemistry

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“…Thus, developing thermostable and more efficient PLCs to facilitate the widespread adoption of enzymatic oil degumming has been a target of researchers in this industrial sector during the last decade. 11,14 The goal of this study was to use a novel thermostable PLC 15 (1)…”

Section: ■ Introductionmentioning

confidence: 99%

“…Avoiding capital investments and reducing operational costs will foster the adoption of enzyme-based environmentally friendly technologies. Thus, developing thermostable and more efficient PLCs to facilitate the widespread adoption of enzymatic oil degumming has been a target of researchers in this industrial sector during the last decade. , The goal of this study was to use a novel thermostable PLC for developing an enzymatic degumming process compatible with the constraints imposed by the design of the existing water-degumming plants . A brief techno-economic analysis to assess the expected impact on the oil industry and the environment is provided.…”

Section: Introductionmentioning

confidence: 99%

Sustainable Refining of Vegetable Oil Made Easy with a Designer Phospholipase C Enzyme

Val

Marchisio

Nardo

et al. 2023

J. Agric. Food Chem.

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The increasing demand pressures the vegetable oil industry to develop novel refining methods. Degumming with type C phospholipases (PLCs) is a green technology and provides extra oil. However, natural PLCs are not active under the harsh conditions used in oil refining plants, requiring additional unit operations. These upfront capital expenditures and the associated operational costs hinder the adoption of this method. Here, we present a process based on ChPLC, a synthetic PLC obtained by consensus sequence design, possessing superior thermal stability and catalytic properties. Using ChPLC, crude soybean oil degumming was completed at 80 °C in 30 min, the temperature and residence time imposed by the design of existing oil refining plants. Remarkably, an extra yield of oil of 2% was obtained using 60% of the dose recommended for PLCs marketed today, saving upfront investments and reducing the operational cost of degumming. A techno-economic analysis indicates that, for medium size plants, ChPLC reduces the overall cost of soybean oil enzymatic degumming by 58%. The process presented here facilitates the implementation of enzymatic technologies to oil producers, regardless of their processing capacity, bringing potential annual benefits in the billion-dollar range for the global economy.

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“…Variants of the phosphatidylcholine preferring Bacillus cereus phospholipase C are currently used in edible oil refining (Jiang et al,2011). We have previously worked in the engineering of this enzyme (Elena et al, 2017; Ravasi et al, 2015), and in the search for natural thermostable variants (Marchisio et al, 2022). In this work we employed the consensus sequence approach to produce a new variant of this enzyme.…”

Section: Introductionmentioning

confidence: 99%

A thermostable phospholipase C obtained by consensus design

Val¹,

Nardo²,

Marchisio³

et al. 2022

Preprint

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Proteins' extraordinary performance in recognition and catalysis have led their use in a range of applications. But proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here we achieved a substantial thermal stabilization of a bacterial Zn(II) dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologs from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of activity and stability parameters between this stabilized mutant with other natural variants bearing similar mutations allow us to pinpoint the sites most likely to be responsible for the enhancement. We show that the stabilized version of the protein retains full activity even in the harsh oil degumming conditions.

show abstract

Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming

Cited by 7 publications

References 44 publications

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design

Sustainable Refining of Vegetable Oil Made Easy with a Designer Phospholipase C Enzyme

A thermostable phospholipase C obtained by consensus design

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