The Secretion of Active Recombinant Human Gastric Lipase bySaccharomyces cerevisiae

Crabbe, Thomas; Weir, Ashley; Walton, E. F.; Brown, Michael E.; Sutton, Chris W.; N, Trétout; Bonnerjea, J.; Lowe, Peter A.; Yarranton, Geoffrey

doi:10.1006/prep.1996.0033

Cited by 20 publications

(9 citation statements)

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“…The production of recombinant gastric lipase as an alternative and sustainable source of enzyme has been explored since the 1980s. An active recombinant human gastric lipase (rHGL) was found to be produced in the yeasts Saccharomyces cerevisiae 77,140 and Schizosaccharomyces pombe, 141,142 in Cos-7 and human embryonal kidney (HEK-293) cells, 123,124 in insect cells, 120,121 as well as Nicotiana benthamiana leaves using a transient expression system. 143 Yeast and insect cell systems were not suitable however for industrial production since the lipase either remained stacked to the yeast cell wall 76,120 or was produced at low levels (<40 mg L −1 of culture).…”

Section: Production Of Gastric Lipasementioning

confidence: 99%

Relevant pH and lipase for in vitro models of gastric digestion

et al. 2016

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The development of in vitro digestion models relies on the availability of in vivo data such as digestive enzyme levels and pH values recorded in the course of meal digestion. The variations of these parameters along the GI tract are important for designing dynamic digestion models but also static models for which the choice of representative conditions of the gastric and intestinal conditions is critical. Simulating gastric digestion with a static model and a single set of parameters is particularly challenging because the variations in pH and enzyme concentration occurring in the stomach are much broader than those occurring in the small intestine. A review of the literature on this topic reveals that most models of gastric digestion use very low pH values that are not representative of the fed conditions. This is illustrated here by showing the variations in gastric pH as a function of meal gastric emptying instead of time. This representation highlights those pH values that are the most relevant for testing meal digestion in the stomach. Gastric lipolysis is still largely ignored or is performed with microbial lipases. In vivo data on gastric lipase and lipolysis have however been collected in humans and dogs during test meals. The biochemical characterization of gastric lipase has shown that this enzyme is rather unique among lipases: (i) stability and activity in the pH range 2 to 7 with an optimum at pH 4-5.4; (ii) high tensioactivity that allows resistance to bile salts and penetration into phospholipid layers covering TAG droplets; (iii) sn-3 stereospecificity for TAG hydrolysis; and (iv) resistance to pepsin. Most of these properties have been known for more than two decades and should provide a rational basis for the replacement of gastric lipase by other lipases when gastric lipase is not available.

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Section: Production Of Gastric Lipasementioning

confidence: 99%

Relevant pH and lipase for in vitro models of gastric digestion

et al. 2016

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“…Gastric lipase has hydrophobic tips exposed in the open enzyme conformation that leads to binding of the hydrophobic substrate, i.e., lipids (11), and is effective in hydrolyzing lipids after adsorbing to an oil-water interface (10). Gastric lipase has been expressed and produced in insect cell culture (12,13), yeast (14), transgenic tobacco (15), and corn seed (7,8). Different host tissues require different conditions to extract lipases.…”

Section: Introductionmentioning

confidence: 99%

“…Different host tissues require different conditions to extract lipases. The recovery of extracellular lipases from fermentation broths is simplified because lipases remain in the supernatant after centrifugation (13,14,(16)(17)(18)(19)(20). Extraction of lipases from oilseeds requires detergents (polyoxyethylene (20) monooleate (Tween 80) (21), 3-[(cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS) (22,23)) and/or a reducing agent such as dithiothreitol (24)(25)(26).…”

Section: Introductionmentioning

confidence: 99%

Extraction of Recombinant Dog Gastric Lipase from Transgenic Corn Seed

Zhong

Glatz

2006

J. Agric. Food Chem.

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Several approaches were examined for extracting the relatively hydrophobic protein recombinant dog gastric lipase (rDGL) expressed in the endosperm of transgenic corn seed. The first approach used minimal processing of the seed before extraction (i.e. simple grinding of whole seed) followed by selective extraction to eliminate 72% of contaminant proteins without compromising rDGL recovery from the meal of whole grain. The second approach added defatting of the whole grain meal to reduce the amount of detergent in the subsequent step for extracting rDGL. The third approach incorporated dry-milling of the corn to recover an endosperm rich fraction, followed by extraction of this fraction. The dry milling strategy was most effective, resulting in recovery of 35 U rDGL/g of corn seed (50 U/g of endosperm) with a specific activity of 9 U/mg compared to 22 U and 3 U/mg for the first strategy and 36 U and 3.7 U/mg for the second. The reductions in host protein contamination and lower detergent levels of the endosperm route should simplify downstream purification steps. Several approaches were examined for extracting the relatively hydrophobic protein recombinant dog gastric lipase (rDGL) expressed in the endosperm of transgenic corn seed. The first approach used minimal processing of the seed before extraction (i.e. simple grinding of whole seed) followed by selective extraction to eliminate 72% of contaminant proteins without compromising rDGL recovery from the meal of whole grain. The second approach added defatting of the whole grain meal to reduce the amount of detergent in the subsequent step for extracting rDGL. The third approach incorporated dry-milling of the corn to recover an endosperm rich fraction, followed by extraction of this fraction. The dry milling strategy was most effective, resulting in recovery of 35 U rDGL/g of corn seed (50 U/g of endosperm) with a specific activity of 9 U/mg compared to 22 U and 3 U/mg for the first strategy and 36 U and 3.7 U/mg for the second. The reductions in host protein contamination and lower detergent levels of the endosperm route should simplify downstream purification steps.

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“…Modern biotechnology has developed various lipases for applications in foods, detergents, and pharmaceuticals (9). As a therapeutic protein, recombinant human gastric lipase has been expressed and produced in insects (10,11), yeast (12), and recombinant dog gastric lipase in transgenic tobacco (13). Transgenic corn has been used as a host to express and produce recombinant dog gastric lipase in seeds (14).…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Assay Method for Evaluating the Lipase Activity in Complex Extracts from Transgenic Corn Seed

Zhong

Glatz

2006

J. Agric. Food Chem.

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A colorimetric method was established to determine the activity of recombinant lipase in extracts from transgenic corn seed. The system was an oil-in-water emulsion that was stabilized by a surfactant to accommodate the organic phase substrate and aqueous phase enzyme. The lipase activity was measured by monitoring the release of nitrophenol at 346 nm from the substrate, 4-nitrophenyl butyrate. Emulsions prepared with various surfactant types and concentrations were tested. For each surfactant, the measured activity was greatest when the surfactant concentration was close to the critical micelle concentration, consistent with the changing trend of oil droplet size as a function of surfactant concentration. The optimal system, with 0.01% (w/w) Tween 80, demonstrated good reproducibility, high sensitivity, robustness, and a linear response to lipase concentration. A colorimetric method was established to determine the activity of recombinant lipase in extracts from transgenic corn seed. The system was an oil-in-water emulsion that was stabilized by a surfactant to accommodate the organic phase substrate and aqueous phase enzyme. The lipase activity was measured by monitoring the release of nitrophenol at 346 nm from the substrate, 4-nitrophenyl butyrate.Emulsions prepared with various surfactant types and concentrations were tested. For each surfactant, the measured activity was greatest when the surfactant concentration was close to the critical micelle concentration, consistent with the changing trend of oil droplet size as a function of surfactant concentration. The optimal system, with 0.01% (w/w) Tween 80, demonstrated good reproducibility, high sensitivity, robustness, and a linear response to lipase concentration.

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The Secretion of Active Recombinant Human Gastric Lipase bySaccharomyces cerevisiae

Cited by 20 publications

References 0 publications

Relevant pH and lipase for in vitro models of gastric digestion

Relevant pH and lipase for in vitro models of gastric digestion

Extraction of Recombinant Dog Gastric Lipase from Transgenic Corn Seed

Enzymatic Assay Method for Evaluating the Lipase Activity in Complex Extracts from Transgenic Corn Seed

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