Characterization of metabolic pathway of linoleic acid 9-hydroperoxide in cytosolic fraction of potato tubers and identification of reaction products

Potato tubers are shown to contain a unique lipoxygenase pathway to form 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) from linoleic acid. Here, we report the metabolic pathway of 9-HPODE in the cytosolic fraction and the characterization of enzymes involved in the conversion of metabolites. The analysis of enzymatic reaction products at pH 5.5 revealed the formation of 9-keto-10,12-octadecadienoic acid, 9-hydroxy-10,12-octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid. The cytosolic enzymes were separated by anion-exchange chromatography into two fractions E1 and E2, having molecular masses of 66 and 54 kDa, respectively. The enzyme fraction E1 only produced 9-keto-10,12-octadecadienoic acid, whereas E2 formed other products. The enzyme E1 showed higher reactivity with 13- and 9-hydroperoxide of α-linolenic acid than 9-HPODE, but no reaction with hydroxy fatty acids. In contrast, the enzyme E2 showed the highest reactivity with 9-HPODE, followed by hydroperoxides of α-linolenic acid and arachidonic acid. We also evaluated the antibacterial activity of hydroxy fatty acids against Erwinia carotovora T-29, a bacterium infecting potato tubers. Growth of the bacteria was suppressed more potently with 9- or 13-hydroxy fatty acids than dihydroxy or trihydroxy fatty acids, suggesting a role for the metabolites in the resistance of bacterial infection.     

镧对软腐欧文氏菌生长及其胞外酶活性的影响

研究了镧对软腐细菌（Ｅｒｗｉｎｉａｃｈｒｙｓａｎｔｈｅｍｉ，Ｅｃｈ）生长及胞外酶活性的影响。结果表明，Ｌａ３＋浓度为５０、１００、１５０、２００、２５０、３００和３５０ｍｇ／Ｌ时，固体培养中对Ｅｃｈ生长均呈抑制作用，且随浓度提高，抑制作用加强，表现为菌落出现时间比对照延长，７ｄ后的菌落直径比对照减小。液体培养中，浓度＜２００ｍｇ／Ｌ，Ｌａ在２４ｈ内对Ｅｃｈ生长有轻微刺激作用；但随着浓度提高和培养时间的延长，Ｅｃｈ生长明显受到抑制。当Ｌａ３＋浓度＞３５０ｍｇ／Ｌ，Ｅｃｈ生长基本处于停止状态；浓度为２００ｍｇ／Ｌ，对Ｅｃｈ产生胞外酶能力有一定影响，其中对纤维素酶活性增强最明显，其次是蛋白酶，而对果胶酶活性有减弱作用。经稀土处理的无细胞滤液对马铃薯块茎组织的浸离力降低。     

Large-scale recovery and purification ofl-asparaginase fromErwinia carotovora

A large-scale process was developed to purify gram quantities of a therapeutic enzyme,L-asparaginase, from submerged cultures ofErwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column ofl-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. the final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis,N-terminal sequencing, peptide mapping, and immunological comparison.     

Molecular Weight and Aggregation of Erwinia Gum in Aqueous Solutions

Erwinia(E) gum is composed of glucose, fucose, galactose and glucuronic acid. The weight-average molecular weights Mw, number-average molecular weights Mn and intrinsic viscosities[η] of the four fractions and the unfractionated E gum in aqueous solutions at desired temperatures were studied by light scattering, membrane osmometry, size exclusion chromatography(SEC) and viscometry. The experimental results prove that E gum formed aggregates in the aqueous solution at 25 ℃ and the aggregates were broken gradually with increasing temperature. The dissociation of the aggregates of E gum in the aqueous solution started at 36 ℃, and was completed at around 90 ℃. The [η] values of E gum and its fractions are much higher than those of the conventional polymers with the similar molecular weights, and decrease with increasing NaCl concentration.