Co-oxydation of linoleic acid to volatile compounds by lipoxygenase isoenzymes from soya beans (author's transl)]

Lipoxygenase isoenzymes L-1 (optimum pH 9.0) and L-2 (pH 6.5) were incubated with linoleic acid. The extracted volatile compounds were separated by gas-chromatography and analysed by mass spectrometry. The relative amounts of volatile carbonyl compounds, which were formed during catalysis were determined (mole percent). L-1 yielded hexanal (greater than 90% at pH 7 and 70% at pH 8.5). L-2 at pH 7 yielded hexanal (31), two geometric isomers of 2,4-decadienal (40), 2-trans-heptenal (12) and 2-trans-octenal (10).     

Co-oxidation of carotene and crocin by soyabean lipoxygenase isoenzymes

Summary Three lipoxygenases that occur in soya beans were separated chromatographically. L-1 (optimum pH = 9.0), L-2 (pH 6.5), L-3 (pH 6.5). The velocities with which these enzymes co-oxidise-carotene or Crocin in the presence of linoleic acid or linoleyl sulphate weremeasured. The carotenoid turnover was related to each lipoxygenase activity.-carotene/linoleic acid = 55% (L-2), 43% (L-3), 6% (L-1), Crocin/linoleic acid = 17,8% (L-2), 14,3% (L-3), 3.3% (L-1), crocin/linoleyl sulphate = 24% (L-3), 4,2% (L-1).The relationship between the reaction rate of the Crocin bleaching and the concentrations of the enzyme, Crocin and linoleic acid was determined. To explain the differences between the pH-6.5 (L-2, L-3) and the alkaline (L-1) lipoxygenases it is supposed that L-2 and L-3 form specially active radicals that are able to co-oxidise polyenes. Both enzymes possess a hydrophobic bonding position, in the neighbourhood of the active site, for-carotene.

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Co-oxidation von Carotin und Crocin durch Lipoxygenase-Isoenzyme aus der Sojabohne

Zusammenfassung Drei in Sojabohnen vorkommende Lipoxygenasen wurden chromatographisch getrennt: L-1 (pH-Optimum 9,0), L-2 (pH 6,5), L-3 (pH 6,5). Gemessen wurden die Geschwindigkeiten mit denen diese Enzyme-Carotin oder Crocin in Gegenwart von Linolsäure oder Linoleylsulfat co-oxydieren. Die Carotinoid-Umsätze wurden auf die jeweilige Lipoxygenase-Aktivität bezogen.-Carotin/Linolsäure: 55% (L-2), 43% (L-3), 6% (L-1), Crocin/Linolsäure: 17,8% (L-2),14,3% (L-3),3,3% (L-1), Crocin/Linoleylsulfat: 24% (L-3), 4,2% (L-1).Für die Crocin-Bleichung wurde die Abhängigkeit der Reaktionsgeschwindigkeit von der Enzym-, Crocin- und Linolsäure-Konzentration bestimmt.Zur Erklärung der Unterschiede zwischen den pH 6,5- (L-2, L-3) und der alkalischen Lipoxygenase (L-1) wird angenommen: L-2 und L-3 bilden besonders aktiv Radikale, welche die Polyene co-oxydieren können. Beide Enzyme besitzen in der Nähe des aktiven Zentrums eine hydrophobe Bindungsstelle für das-Carotin.

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We are grateful to the Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg for supporting this work.     

Effects of ingredients on the oxidation of linoleic acid by lipoxygenase in bread doughs

Flour and water doughs containing 1-¹⁴C-linoleic acid (18:2) and various ingredients were prepared to study the oxidation of linoleic acid by lipoxygenase in bread doughs. Lipids were extracted, treated with diazomethane, and ¹⁴C-labelled fatty acid methyl esters separated by thin-layer chromatography. Radioactivity was determined in silica gel bands containing unoxidised 18:2, hydroperoxy acids (L₁), hydroxy acids (L₂), hydroxyepoxy acids (L₃) and trihydroxy acids (L₄). Minor components detected by autoradiography were present mainly in L₃ and L₄. Recoveries of total radioactivity were always > 95%. Untreated flour-water dough was mixed aerobically for ?4 min, rested, and the lipids extracted after 10 min total dough time. Yields of ¹⁴C products were unoxidised 18:2 = 28.6 μmol, L₄ = 93.9 μmol/100 g dry flour. Similar yields were obtained from ClO₂-treated flour, both after 10 min and 60 min dough time. Salt, salt + yeast, or salt + yeast + ascorbic acid in the dough did not reduce 18:2 oxidation significantly, but increased L₃ at the expense of L₄. Soya flour preparations inhibited linoleic acid oxidation by 25–44%, but pure soya lipoxygenase had no effect at all. Heat treatment reduced the inhibitory effect of soya flour. Accessible thiol groups were not essential for lipoxygenase activity or for the reduction of L₁ to L₂ since adding cysteine or N-ethyl maleimide had negligible effects on the 18:2 oxidation products. Most of the flour carotenoids (xanthophylls) were bleached by wheat enzymes in non supplemented doughs, and all were bleached in doughs supplemented with soya flour. ¹⁴C-labelled triglyceride was not oxidised except in doughs containing soya flour mixed in air (1.5% oxidation) or oxygen (3 % oxidation). Soya flour contains lipoxygenase isoenzymes (principally lipoxygenase-2) which oxidise linoleate in triglycerides. This isoenzyme is evidently not present in wheat.     

On lipoxygenase and enzymes which decompose linoleic acid hydroperoxides in rye (author's transl)]

An enzyme fraction from rye containing lipoxygenase activity was investigated. The molecular weight of lipoxygenase was found to be about 102000. Two bands groups with isoelectric points between 5.1-5.5 and 5.8-6.4 were obtained by isoelectric focusing. Three isoenzymes could be separated by ion exchange chromatography. Lipoxygenase has optimum activity at pH 7.3-7.5 and predominantly forms 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (13-LHPO). In rye the 13-LHPO is converted to alpha-ketols by a high molecular protein fraction. This isomerase converts the LHPO formed by rye lipoxygenase predominantly to 12,13-ketohydroxy acids. The Michaelis Constant of isomerase is 3-5 X 10(-5), using LHPO as substrate. At low protein concentrations the reaction velocity of LHPO-conversion increases linearly with protein concentration.     

Purification and some properties of wheat germ lipoxygenase

Lipoxygenase from the germ of bread wheat was purified to near homogeneity by a classical scheme. After extraction at pH 4.5 from defatted germ, lipoxygenase activity was precipitated by 40% saturation (NH₄)₂SO₄ from a 25% saturation supernatant. After dissolution in a phosphate buffer at pH 7 and extensive dialysis against this buffer, the extract was submitted to gel filtration on Ultrogel AcA 34. The final step of DEAE Sephadex A50 chromatography gave three peaks (L₁, L₂ and L₃) with lipoxygenase activity. The total yield of the purification procedure was close to 30% and the degree of purification varied from 200 to 300 depending on the fraction considered. The three isoenzymes were also detected by disc electrophoresis using a specific staining method and were isolated by isoelectric focusing in a liquid medium. The molecular weight for each isoenzyme was determined to be 90 000–95 000 by gel filtration and 110 000 by electrophoresis in a gradient of acrylamide concentration. The stability, pH optimum and calcium effect have been studied. L₂ and L₃ were less stable than L₁. Optimum pH ranged between 6–6.5 and neither isoenzyme activity was affected by calcium ions. L₁ was twice as active towards β-carotene as L₂ and L₃ for the same level of oxygen uptake, but in comparison to lipoxygenase from horsebean the co-oxidising power of wheat lipoxygenase was very poor.     

Calcium Interaction with Substrates of Type-2 Pea Lipoxygenases

The calcium activation of two purified type-2 pea lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.11.12) isoenzymes was investigated. With ethanolic linoleate or linolenate substrates, oxygen uptake rates were stimulated by calcium while with Tween 20-dispersed substrates, oxygen uptake was inhibited by calcium. With methyl linoleate or trilinolein substrates, oxygen uptake rates were not affected by the addition of calcium. Results indicate that calcium activation of lipoxygenase cannot be used to differentiate between lipoxygenase isoenzymes because the effect is more related to substrate state and concentration, as well as time of addition of calcium to the reaction mixture, rather than to the isoenzyme tested.     

Canola Phytase: Isolation and Characterization

Two phytase isoenzymes were isolated from 8-day germinated canola cv Regent. Gel filtration chromatography of an ammonium sulfate fractionated extract on Sephadex G-100 produced one peak with phytase activity. The phytase fraction was separated into two isoenzymes by DEAE-cellulose chromatography. The optimum pH was 4.5–5.0 and 5.0 for the phytase isoenzymes 1 and 2, respectively. Both isoenzymes exhibited maximum activity at 50°C. Km values at pH 5.0 were 0.36 and 0.25 mM for phytase 1 and 2 isoenzymes, respectively, while molecular weight determination showed both fraction were identical with a molecular weight of 70,100 ± 4,000 daltons.     

不同培养条件对红曲霉产红曲色素的研究

本研究主要针对发酵液培养基中不同碳源、氮源、pH以及装液量分别进行单因素五水平的试验,确定三个较好的水平,再进行四因素三水平正交试验以确定红曲霉产红曲色素的最佳条件.在单因素试验中,碳源以玉米粉、淀粉、麦芽糖较好;氮源以硝酸钠、大豆粉、氯化铵较好;pH值以pH3.8、5.4、7.0较好;装液量以250ml三角瓶装100、125、150ml较好.通过四因素三水平正交试验,确定出红曲霉产红曲色素的最佳培养条件为A3BtC1D3,即麦芽糖、大豆粉、pH3.8、装液量150ml/250ml组合最佳.     

Winged bean lipoxygenase—Part 2: Physicochemical properties

Winged bean lipoxygenase (linoleate: oxygen oxidoreductase EC 1.13.11.12) isoenzymes FI and FII were isolated and purified according to the method of Truong et al. (1980).FI and FII were both highly specific for linoleic acid. They exhibited optimal activity at pH 6·0 and 5·8, respectively at 30°C. An activation energy of 4·5 kcal mol^?1 was calculated for this lipoxygenase within the temperature range of 30–50°C.At 0·075% Tween 20, FI and FII had K_m values for linoleic acid of 0·44 and 0·37 × 10^?3M, respectively, compared to 0·4 × 10^?3M for the crude enzyme. Maximal activity was obtained at 1·6 × 10^?3M. Higher levels of Tween 20 inhibited the lipoxygenase activity.Both isoenzymes had identical average molecular weight of 80 000 daltons by gel filtration and SDS gel electrophoresis.FI and FII isoenzymes were strongly inhibited by Hg⁺⁺, Mn⁺⁺, Mg⁺⁺ and Fe⁺⁺⁺ and activated by Zn⁺⁺, Co⁺⁺ and Fe⁺⁺. A difference in the degree of inhibition or activation was observed between FI and FII response. Ca⁺⁺ inhibited both FI and FII but the former was more sensitive to Ca⁺⁺. KCN also inhibited the two isoenzymes.Among the antioxidants tested, butylated hydroxytoluene and butylated hydroxyanisole most effectively inhibited both FI and FII at only 10^?6M. Sulphydryl reagents such as iodoacetamide and dithiothreitol have little effect on the lipoxygenase isoenzyme activity.The lipoxygenase isoenzymes were more stable at neutral pH. The enzyme in the crude extract and especially in situ was more stable to heat treatment.     

Oxidation of free and esterified linoleic and linolenic acids in bread doughs by wheat and soya lipoxygenases

Lipids containing ¹⁴C-labelled linoleic and linolenic acids were added to flour which was then made into bread doughs with or without 2% enzyme-active soya flour. The ¹⁴C-labelled lipids and their oxidation products were analysed as intact lipids or as ¹⁴C fatty acid methyl esters by thin-layer chromatography. In the control doughs wheat lipoxygenase oxidised free fatty acids and monoglycerides. In doughs containing soya flour, steryl esters, triglycerides, diglycerides, mono-galactosyl diglycerides, digalactosyl diglycerides and phosphatidyl choline were also oxidised, and the oxidation of these lipids is attributed to soya lipoxygenase.     

Occurrence and distribution of lipoxygenase in Camellia sinensis (L) O Kuntze and their changes during CTC black tea manufacture under southern Indian conditions

The optimal activity of tea lipoxygenase was found to be at pH 7·5 and 9·0, indicating the presence of two types of isoenzymes. Tea lipoxygenase was inhibited by cyanide and a small fraction appeared to be quite heat stable. Clonal and seasonal variations in lipoxygenase activity was significant. An increase in lipoxygenase activity was observed with leaf maturity. The lipoxygenase activity decreases with age from pruning and increases with an increase in plucking interval. The changes of lipoxygenase activity during CTC manufacture revealed that it increased with the degree of withering and upon rolling. However, a gradual decline was recorded during the fermentation and drying processes. The residual lipoxygenase activity in black tea is expected to affect its quality on storage. © 1998 Society of Chemical Industry.     

Quantitative immunoassay for soya protein in raw and sterilized meat products

An enzyme-linked immunosorbent assay (ELISA) is described for the quantitative analysis of soya protein in meat products. The assay is applicable to raw and sterilized products and is not dependent on soya variety and type of soya ingredient (concentrate, isolate, etc.). Therefore, the assay can be applied without knowledge of product composition, heat pretreatment and other processing conditions. The ELISA is specific for soya; no interference of other product components has been found. The detection limit of the ELISA is 0.5% soya protein. Qualitative analysis by immunoblotting is possible at much lower concentrations. The antibodies used are specific for sodium dodecyl sulphate (SDS) denatured soya protein subunits. The products are extracted with SDS and 2-mercaptoethanol, which guarantees optimum recovery of the protein components. Product extracts can be analysed directly by ELISA without removal of the SDS by using nitrocellulose as a solid phase.     

Sensory evaluation and flavour analysis of soymilk produced from lipoxygenase‐free soya beans after modified processes and pulsed light treatment

Conventional static pulsed light (PL) treatment completely inactivated lipoxygenase (LOX) in whole soya beans to prevent off‐flavours produced by LOX. However, the produced soymilk had inadequate values for total solid content. The fully soaked treatment (FST) and shaking treatment (SHT) were best in terms of increasing total solid content of soymilk from 3.7% for control soymilk to 4.6% and 4.3%, respectively; therefore, these treatments were chosen for soymilk sensory evaluation. No significant statistical differences in sensory evaluation scores existed between the control and soymilk produced from FST. The SHT for 130 s had higher overall liking (4.8) and flavour (4.6) scores compared with the control (4.1 and 3.9, respectively). Flavour analysis (purge and trap GC‐MS) of the soymilk revealed that the ethanol peak area was the biggest difference between the treatments. This study demonstrated soya beans treated with PL have no negative sensory effect in general. Producing lipoxygenase‐free soya bean is an important achievement to enhance soyfood products because lipoxygenase catalyses lipid oxidation, which takes place when lipoxygenase is released during soy milling.     

Total inactivation of lipoxygenase in whole soya bean by pulsed light and the effect of pulsed light on the chemical properties of soya milk produced from the treated soya beans

The inactivation of lipoxygenase (LOX) in the whole soya bean prevents lipid oxidation that produces an off‐flavour of soya food. The inactivation of lipoxygenase in the whole soya bean by pulsed light (PL) was examined with three distances (5, 7 and 9 cm) from the PL strobe and for different durations. Soya bean was treated with PL with and without ice surrounding the soya bean sample tray for limiting the rise in sample temperature. Results show that without ice surrounding the sample tray, the lowest LOX residual activity was 4.7%, 0.4% and 0.0% for 80‐s duration at 5 cm distance from the PL strobe, 110 s at 7 cm from the strobe and 150 s at 9 cm from the strobe, respectively; the soya bean temperature after treatment was 109.6, 116.3 and 114.8 °C, respectively. The instantaneous temperatures of the soya bean core measured during PL operating were above 100 °C. The lipoxygenase band was disappeared after longest PL treatments of each distance compared with the LOX band control as assessed by electrophoresis. The pulsed light had no negative effect on peroxide value of produced soya milk. However, PL reduced significantly the total solid amount and changed the colour of the produced soya milk. The residual activity with sample cooling by ice during treatment was 79.0%, 98.8% and 95.7%, with sample temperatures of 81.7, 91.2 and 66.9 °C, respectively. This study indicates that PL illumination could fully inactivate LOX in whole soya beans, with the photo‐thermal effect of PL as the main factor responsible for the inactivation of LOX.     

Identification and characterization of lipoxygenase in fresh culinary herbs

The aim of this project was to study the biochemical characterization of lipoxygenase extracted from basil (Ocimum basilicum L.), rosemary (Rosmarinus officinalis L.), and sage (Salvia officinalis L.) leaves. The lipoxygenase extracted from these culinary herbs was frozen with liquid N₂ and ground into powder before adding ice-cold phosphate buffer (pH 7). The crude extracts from the three aromatic plants showed various specific activities and the highest specific activity and the lowest protein content were observed for the basil extracts. The optimum pH values for lipoxygenase extracted from basil and sage ranged between 5 and 6, while pH 6 was the optimum value for lipoxygenase activity for rosemary. The optimum temperature was 40°C for all of the crude extracts analyzed. Considering the biochemical characteristics evaluated for lipoxygenase from each culinary herb, suitable strategies for the inactivation of the enzyme can be proposed in order to reduce its detrimental effects on fresh aromatic herbs.     

Some products of winged bean lipoxygenase-catalysed reaction: A note

Purified isoenzymes of lipoxygenase from winged bean were shown to catalyse the formation of hydroperoxides as well as carbonyl compounds. Preliminary experiments indicate formation of two volatile compounds by lipoxygenase in a model purified lipoxygenase-linoleic acid system which are similar to two of the volatile components of raw winged bean homogenate.     

Two Lipoxygenases from Germinated Barley-Heat and Kilning Stability

Using ammonium sulfate precipitation followed by hydrophobic and ion exchange chromatography, two lipoxygenase isoenzymes, LOX 1 and LOX 2, were 18.3- and 44.5-fold purified from germinated barley, with 18 and 24% recovery of activity respectively. LOX 1 and LOX 2 were characterized by isoelectric points 4.9 and 6.4, and molecular weights of 90 kd and 110 kd, respectively. Apparent Km values for linoleic acid were 0.06 mM for LOX 1 and 0.18 mM for LOX 2. LOX 1 converted linoleic acid to 9 and 13 hydroperoxides at about 4:1, whereas the 13 hydroperoxide was the major product formed by LOX 2 (ratio 3:7). For both isoforms, thermal inactivation data indicated first order kinetics with activation energies influenced by ionic strength and pH. Isoenzymes composition was analyzed for three kilning schemes: the 1:3 ratio between LOX 1 and LOX 2 observed in germinated barley increased during the course of kilning.     

The effect of lipoxygenase action on the mechanical development of wheat flour doughs

The mechanical development of dough has been followed by measurement of the relaxation time of dough samples after mixing to various levels of work input. Parallel determinations of free and bound lipid have also been made. When doughs were mixed in air, addition of full-fat, enzyme-active soya bean flour (subsequently referred to as “soya flour”) resulted in an increase in relaxation time, particularly at higher work levels. The magnitude of this improvement increased with increasing work input and was dependent on the rate of work input. Addition of soya flour also enabled a higher level of mechanical work to be introduced before dough breakdown occurred. When doughs were mixed under nitrogen, or when the soya flour was heat denatured, no change in the rheological properties compared with the respective control doughs was found. The release of bound lipid, which occurred during dough development in air in the presence of soya flour, could also be induced by adding purified lipoxygenase to the dough, together with linoleic acid as a substrate. This resulted in rheological changes similar to those observed using soya flour. However addition of enzymically pre-peroxidised lipid to doughs mixed in nitrogen was without effect on relaxation times. These findings suggest that lipid release is linked with structural changes in dough protein and provide further support for a mechanism of coupled oxidation of protein -SH groups by lipoxygenase.     

DOGR1 and DOGR2: Two genes from Saccharomyces cerevisiae that confer 2-deoxyglucose resistance when overexpressed

Saccharomyces cerevisiae contains two genes (DOG^R1 and DOG^R2) that are able to confer 2-deoxyglucose resistance when they are overexpressed. These genes are very similar, sharing 92% identity at the protein level. They code for two isoenzymes with 2-deoxyglucose-6 phosphate (2-DOG-6P) phosphatase activity. These enzymes have been purified and characterized. Dog^R1p shows an optimum pH of 6, an optimum temperature of 30°C and a K_M on 2-DOG-6P of 17 mM. Dog^R2p shows a similar optimum pH, but the optimum temperature is 40°C and it exhibits a K_M on 2-DOG-6P of 41 mM. Both enzymes require 10 mM-MgCl₂ for maximal activity and they are inhibited by inorganic phosphate.