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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.125, #1)
Bioconversion of isoeugenol into vanillin by crude enzyme extracted from soybean by Yong-Hong Li; Zhi-Hao Sun; Li-Qing Zhao; Yan Xu (pp. 1-10).
Crude enzyme extracted from soybean was used to convert isoeugenol into vanillin. The effects of several factors on the bioconversion were studied. Conversion was affected by the armount of substrate and was also improved by the addition of absorbents, among which powdered activated carbon was the best. The effect of H2O2 concentration on the conversion was also studied. The optimum concentration of H2O2 was 1% (v/v). With 10 g/L of powdered activated carbon and 0.1% H2O2 added, vanillin reached a maximum concentration of 2.46 g/L after 36 h, corresponding to a molar yield of 13.3%.
Keywords: Isoengenol; vanillin; enzyme; bioconversion; soybean
Antioxidant responses to oleic acid in two-liquid-phase suspension cultures of Taxus cuspidata by Qiu-Man Xu; Jing-Sheng Cheng; Zhi-Qiang Ge; Jing-Jin Yuan (pp. 11-26).
Two-liquid-phase plant cell cultures employ the use of a partitioning system to redirect extracellular product into a second phase. After the addition of organic solvent, in order to understand the defense system of Taxus cuspidata cells to organic solvent in two-liquid-phase suspension cultures, we investigated cells' antioxidant metabolism. The results showed that T. cuspidata cells responded to oleic acid with oxidative bursts in both intracellular H2O2 and extracellular O2 − production. Inhibition studies with diphenylene iodonium suggested that the key enzyme responsible for oxidative bursts was primarily NADPH oxidase. Investigation of the relationship between reactive oxygen species (ROS) and defense responses induced by oleic acid indicated that 4% (v/v) oleic acid increased the levels of antioxidant enzymes of superoxide dismutase, ascorbate peroxidase, and catalase and the antioxidant capacity of reduced ascorbate and glutathione. However, when oleic acid content reached a critical value (6% [v/v]), no further increase in antioxidant enzymes and antioxidant capacity was observed, indicating that the defense responses played a role in a certain range of oleic acid content, beyond which the overall ROS scavenging machinery was not induced and the peroxidation of membrane lipids emerged.
Keywords: Oleic acid; reactive oxidative species; defense response; two-liquid-phase suspension cultures; Taxus cuspidata
Hydrolysis characteristics of tissue fractions resulting from mechanical separation of corn stover by Jason A. Bootsma; Brent H. Shanks (pp. 27-39).
Corn stover has potential as a resource for both fiber and chemical needs if separation strategies can be developed to deal with its heterogeneity. Relative hydrolysis characteristics were assessed for pith (sclerenchyma and parenchyma) and fiber (collenchyma) tissue fractions derived from mechanical separation of corn stover to determine whether classification by tissue type resulted in fractions with different hydrolysis response. The physical characteristics of the tissue fractions were analyzed. The hydrolysis behavior of the fractions was evaluated under both acidic and basic conditions. The results from the hydrolysis experiments are compared with previously reported compositional analysis for the tissue fractions.
Keywords: Corn stover; hydrolysis; pith; fiber; cellulose; hemicellulose; mechanical separation
Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins by Jean-Philippe Gourdine; Pamela Greenwell; Emilie Smith-Ravin (pp. 41-52).
We used the recombinant phage display antibody system (RPAS) to obtain chimeric single-chain fragment variable (ScFv) antibodies to gill proteins of the white clam Codakia orbicularis (Linné, 1758). After three rounds of selection on immunotubes loaded with total gill protein extract, recombinant phages exhibiting antibodies to gill proteins were isolated and tested by enzyme-linked immunosorbent assay (ELISA). Clones exhibiting a high affinity for the mollusk proteins were selected for production of soluble ScFv antibodies, which were purified for subsequent analysis. ScFv antibodies exhibited a reaction specific for a protein whose molecular mass was about 15,000 Daltons and that was detected by the antigen capture technique followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.
Keywords: Antigen capture; Codakia orbicularis ; gill proteins; phage antibodies; single-chain fragment variable
An integrated process for separation of major and minor proteins from goat serum by Sulakshana Jain; M. N. Gupta (pp. 53-62).
Analysis of minor proteins in animal sera is of considerable clinical significance. To be able to detect these proteins, depletion of major proteins (albumin and immunoglobulin G [IgG]) is necessary. Many of these proteins are also required in pure form for a variety of biochemical applications. The present work uses goat serum as the system and describes the separation and purification of both major and several minor proteins. This was carried out by judicious adaptation and combination of separation technologies such as immobilized metal ion affinity chromatography (on a somewhat novel matrix), dye affinity chromatography, and lectin affinity chromatography. Albumin, IgG, α2-macroglobulin, α1-proteinase inhibitor, and transferrin were obtained from the serum. The purified preparations were found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Keywords: Albumin; immobilized metal ion affinity chromatography; IgG; α1-proteinase inhibitor; α2-macroglobulin; serum proteins
Preparation of mono- and diacylglycerols by enzymatic esterification of glycerol with conjugated linoleic acid in hexane by C. E. Martinez; J. C. Vinay; R. Brieva; C. G. Hill Jr.; H. S. Garcia (pp. 63-75).
Esterification of glycerol with conjugated linoleic acid (CLA) was carried out in hexane. Lipase from Rhizomucor miehei provided a high degree of esterification (80%) in 8 h at 50°C when used at 15% (w/w) in a system containing a 1∶2 molar ratio of glycerol to free fatty acids. Esterification levels >80% were obtained in 8 h at 40°C with 15% (w/w) lipase from Candida antarctica at the same molar ratio of reactants. The extent of esterification of CLA was >90% after 4h of reaction at 50°C with a 5% (w/w) loading of either R. miehei or C. antarctica lipase, together with a 1∶1 molar ratio of substrates. Both enzymes incorporated the original CLA as acylglycerol residues in primarily 1,3-diacylglycerol and 1-monoacylglycerol. The CLA-rich acylglycerols can be employed as emulsifiers or as substitutes for natural fats and oils.
Keywords: Conjugated linoleic acid; lipases; polyesterification; nutraceuticals; structured lipids