Characterization of purified green bell pepper hydroperoxide lyase expressed by Yarrowia lipolytica: Radicals detection during catalysis

Santiago-Gómez, Mirna P.; Vergely, Catherine; Policar, Clotilde; Nicaud, Jean‐Marc; Belin, Jean‐Marc; Rochette, Luc; Husson, Florence

doi:10.1016/j.enzmictec.2006.11.017

Cited by 19 publications

(17 citation statements)

References 21 publications

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“…The enzymes were mainly characterized after purification through extraction from plant tissues [2,9,[24][25][26]. Recently, researchers have been trying to express the plant HPL gene in E. coli and P. pastoris and characterize the recombinant enzyme [13][14][15][16][17][18]. Herein, a novel cDNA encoding HPL from S. tuberosum leaves has been cloned, sequenced, and expressed in E. coli M15.…”

Section: Discussionmentioning

confidence: 99%

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Molecular cloning, expression, and enzymatic characterization of Solanum tuberosum hydroperoxide lyase

Xue

Jiang

et al. 2012

Eur Food Res Technol

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A cDNA encoding hydroperoxide lyase (HPL) was isolated from Solanum tuberosum, cloned into pQE-30 vector, and expressed in E. coli. The recombinant protein was purified by nickel affinity chromatography and showed an approximate molecular weight of 54 kDa by SDS-PAGE analysis, which was similar to the predicted value based on the putative amino acid sequences (53.9 kDa). 13-Hydroperoxy-linolenic acid (13-HPOT) was the preferred substrate for the enzyme compared with 13-hydroperoxy-linoleic acid (13-HPOD). The corresponding volatile products were 2(E)-hexenal and n-hexanal tested by headspace-gas chromatography, respectively. The enzyme was optimally active at 25°C and pH 6.5. The K m , V max , and the catalytic efficiency (V max /K m ) for 13-HPOT were 56.6 lM, 71.3 units/mg, and 1.26 units/mg Á lM, respectively. Activity of the recombinant potato HPL increased when Triton X-100, sodium chloride, or potassium chloride was added in the reaction mixture, while calcium chloride decreased activity of the recombinant enzyme.

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Section: Discussionmentioning

confidence: 99%

“…HPL can be extracted from plant tissues or expressed in E. coli, Yarrowia lipolytica, and Pichia pastoris as heterologous protein [8,[13][14][15][16][17][18]. Unlike plant, E. coli, Y. lipolytica, and P. pastoris could produce only one hydroperoxide-metabolizing enzyme leading to the expected product.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning, expression, and enzymatic characterization of Solanum tuberosum hydroperoxide lyase

Xue

Jiang

et al. 2012

Eur Food Res Technol

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“…-dependent glucose dehydrogenase from Bacillus megaterium provided a functioning cofactor regeneration system and demonstrated the feasibility of such coupled reactions in an engineered host (Schewe et al 2008). Intermediate radical species, shown to occur, for example, during the biotransformation of a-farnesene (Krings et al 2008a, b) and fatty acid hydroperoxides (Santiago-Gomez et al 2007) (''green'' 2-(E)-hexenal; Table 1), are supposed to account for the frequently observed multitude of reaction products.…”

Section: Engineering the Catalystmentioning

confidence: 99%

Biotechnology of flavours—the next generation

2009

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Volatile organic chemicals (flavours, aromas) are the sensory principles of many consumer products and govern their acceptance and market success. Flavours from microorganisms compete with the traditional agricultural sources. Screening for overproducers, elucidation of metabolic pathways and precursors and application of conventional bioengineering has resulted in a set of more than 100 commercial aroma chemicals derived via biotechnology. Various routes may lead to volatile metabolites: De novo synthesis from elementary biochemical units, degradation of larger substrates such as lipids, and functionalization of immediate flavour precursor molecules. More recently, the field was stimulated by the increasing preference of alienated consumers for products bearing the label "natural", and by the vivid discussion on healthy and "functional" food ingredients. The unmistakable call for sustainable sources and environmentally friendly production is forcing the industry to move towards a greener chemistry. Progress is expected from the toolbox of genetic engineering which is expected to help in identifying metabolic bottlenecks and in creating novel high-yielding strains. Bioengineering, in a complementary way, provides promising technical options, such as improved substrate dosage, gas-phase or two-phase reactions and in situ product recovery.

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“…The tomato enzyme cleaved only 13-hydroperoxides from linoleic acid and ␣-linolenic acid, whereas HPO-lyase from alfalfa (Medico sativa L.) also accepted 9-hydroperoxides as substrates to form the respective volatile C 9 -aldehydes (Noordermeer et al, 1999). Classical tools of molecular biology, such as His-tagging of proteins and immobilised metal affinity chromatography of the tagged species, support the search for flavour enzymes (Santiago-Gomez et al, 2007). The short-term aim is to express both active lipoxygenase and HPO-lyase in a recombinant micro-organism.…”

Section: Genetically Engineered Plant Enzymesmentioning

confidence: 99%