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

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.

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

The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.

Cloning and Production of Thermostable Enzymes for the Hydrolysis of Steryl Glucosides in Biodiesel.

Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.

Industrial uses of phospholipases: current state and future applications.

Phospholipids play a central role in all living organisms. Phospholipases, the enzymes aimed at modifying phospholipids, are consequently widespread in nature and play diverse roles, from lipid metabolism and cellular signaling in eukaryotes to virulence and nutrient acquisition in microbes. Phospholipases catalyze the hydrolysis of one or more ester or phosphodiester bonds of glycerophospholipids. The use of phospholipases with industrial purposes has constantly increased over the last 30 years. This demand is rapidly growing given the ongoing improvements in protein engineering and the reduction of enzymes manufacturing costs, making them suitable for industrial use. Here, a general overview of phopholipases A, B, C, and D and their industrial application is presented along with potential new uses for these enzymes. We draw attention to commercial phospholipases used to improve the emulsifying properties of products in the baking, egg, and dairy industries. On the other hand, the improvement of oil degumming by phospholipases is thoroughly analyzed. Moreover, recent developments in enzymatic biodiesel production and the use of phospholipases for the synthesis of phospholipids with pharmaceutical or nutritional value are reviewed.

The ßγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability.

ßγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca2+-binding proteins with a large diversity and variable properties in Ca2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal ßγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal ßγ-crystallin domain was deleted (LS-PIPLCΔCRY) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLCΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.

The production, properties, and applications of thermostable steryl glucosidases.

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable ß-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.

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.

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis.

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.

PhoP can activate its target genes in a PhoQ-independent manner.

The PhoP/PhoQ two-component system controls the extracellular magnesium depletion response in Salmonella enterica. Previous studies have shown that PhoP is unable to up-regulate its target genes in the absence of PhoQ function. In this work, we demonstrate that PhoP overexpression can substitute for PhoQ- and phosphorylation-dependent activation. Either a high concentration of PhoP or activation via phosphorylation stimulates PhoP self-association.