Substrate Report for: Tween-20

Degradation of the surfactant polysorbate (PS) by enzyme impurities has been previously suggested as a mechanism for the formation of visible and subvisible particles that affect product quality. Although chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously characterized, enzymatic degradation of PS remains poorly understood. In this report, enzyme-mediated hydrolysis of the major components of PS was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. PS20 and PS80 tested contained 99% of laurate and 98% oleate esters, respectively, were heterogeneous with respect to head group, and contained a distribution of ester types. Carboxylester hydrolases tested included those from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. PS hydrolysis was monitored by observing the change in the peak area of major PS components over time and quantified using a parameter called t50, which was defined as the time required for each peak to reach 50% of its initial value. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), and the identity of the fatty acid ester tail (C12 vs C18:1). In addition, the pattern of PS hydrolysis was unique to the type of enzyme used. Importantly, we observed that no PS component was completely resistant to the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes. LAY ABSTRACT: Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously studied, but enzymatic degradation of PS remains poorly understood. In this study, enzyme-mediated hydrolysis of the major components in a heterogeneous mixture of PS20 or PS80 was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. Carboxylester hydrolases from a broad range of organisms were tested, including enzymes from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), the identity of the fatty acid ester tail (C12 vs C18:1), and the identity of the enzyme. Importantly, no PS component was completely resistant to all the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying or identifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.

CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.

Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.

Degradation of the surfactant polysorbate (PS) by enzyme impurities has been previously suggested as a mechanism for the formation of visible and subvisible particles that affect product quality. Although chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously characterized, enzymatic degradation of PS remains poorly understood. In this report, enzyme-mediated hydrolysis of the major components of PS was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. PS20 and PS80 tested contained 99% of laurate and 98% oleate esters, respectively, were heterogeneous with respect to head group, and contained a distribution of ester types. Carboxylester hydrolases tested included those from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. PS hydrolysis was monitored by observing the change in the peak area of major PS components over time and quantified using a parameter called t50, which was defined as the time required for each peak to reach 50% of its initial value. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), and the identity of the fatty acid ester tail (C12 vs C18:1). In addition, the pattern of PS hydrolysis was unique to the type of enzyme used. Importantly, we observed that no PS component was completely resistant to the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes. LAY ABSTRACT: Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously studied, but enzymatic degradation of PS remains poorly understood. In this study, enzyme-mediated hydrolysis of the major components in a heterogeneous mixture of PS20 or PS80 was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. Carboxylester hydrolases from a broad range of organisms were tested, including enzymes from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), the identity of the fatty acid ester tail (C12 vs C18:1), and the identity of the enzyme. Importantly, no PS component was completely resistant to all the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying or identifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.

CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.

Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.

Ten clinical isolates of Pseudomonas cepacia from the sputum of cystic fibrosis patients were examined for the ability to produce lipase. Lipase substrates used included egg yolk agar, four different polyoxyethylene sorbitans (Tweens), and p-nitrophenylphosphorylcholine, a chromogenic substrate used to assay for phospholipase C. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in either tryptose minimal medium or chemically defined medium. Lipase activity increased in the filtrates if the cultures were allowed to proceed into the stationary phase. None of the isolates produced phospholipase C. Lipase activity on Tween 20 ranged from 41.6 X 10(-3) to 640.0 X 10(-3) U/micrograms of protein. The activity was similar or slightly lower when Tween 40, 60, or 80 was used as the substrate. There was no correlation between lipase activity on Tween and that demonstrated on egg yolk agar. Lipase activity increased as pH increased from 7.0 to 9.0. Boiling for 5 min resulted in 66% loss of enzyme activity. The remaining activity continued to decrease with increasing boiling time. The enzyme was purified by gel filtration on Sephadex G-200, and the resultant preparation, when subjected to polyacrylamide gel electrophoresis, resulted in a single protein band (molecular weight, approximately 25,000) from which lipase activity could be eluted. The purified lipase was not cytotoxic to HeLa cells, nor was it toxic when injected intravenously into mice.