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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.110, #1)
Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane by P. R. S. Leirião; L. J. P. Fonseca; M. A. Taipa; J. M. S. Cabral; M. Mateus (pp. 1-10).
A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 μmol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 μmol of H2O2 reduced/(h·Uimmob HRP).
Keywords: Horseradish peroxidase activity; stability; immobilized enzyme productivity; polyacrylonitrile membrane
l-Asparaginase release from Escherichia coli cells with aqueous two-phase micellar systems by Minjun Qin; Fengsheng Zhao (pp. 11-21).
A method was proposed to release and separate l-asparaginase (EC 3.5.1.1) from Escherichia coli ATCC 11303 cells with aqueous two-phase micellar systems. The systems were composed of K2HPO4 and Triton X-100. The method combines enzyme release with enzyme purification. The influence of Triton X-100 concentration, K2HPO4 concentration, and pH on the release and partition of l-asparaginase was investigated. Experimental results showed that E. coli cells treated with 9.4% (w/v) K2HPO4 and 15% (w/v) Triton X-100 at 25°C for 15–20 h released nearly 80% of the enzyme. Most of the released enzyme was partitioned to the bottom phase (phosphate-rich phase). The effects of Triton X-100 concentration, K2HPO4 concentration, and pH on cloud point were also studied. Electron micrography indicated that the chemical treatment altered the inner structure of E. coli cells significantly.
Keywords: l-Asparaginase; enzyme release; Escherichia coli ; aqueous two-phase micellar system; dipotassium hydrogen phosphate; Triton X-100
Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins by Valeria Grazú; Karen Ovsejevi; Karina Cuadra; Lorena Betancor; Carmen Manta; Francisco Batista-Viera (pp. 23-32).
Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae β-galactosidases and β-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 μmol of SH groups/g of dried gel). Both reductants allowed an increase of three- (for K. lactis β-galactosidase) and fourfold (for A. oryzae β-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis β-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae β-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in β-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.
Keywords: Protein reduction; reducing agents; immobilized reductants; disulfide reductants; sulfhydryl residues; disulfide reduction; cystine bonds
Effect of tributylphenyltetraethoxylate on enzyme production of Pleurotus Ostreatus by Cristina Ramos; Helena Morais; Esther Forgács; Tibor Cserháti (pp. 33-44).
The effect of the addition of the nonionic surfactant tributylphenyl-tetraethoxylate to culture media on pH and extracellular protein content, and on production of β-glucosidase, xylanase, laccase, and manganese-dependent and-independent peroxidases by the edible fungus Pleurotus ostreatus was determined. The relationship between fermentation parameters and concentration of surfactant was assessed by multiple linear regression analysis, and the similarities and differences among the fermentation parameters were elucidated by principal component analysis. Calculations proved that except for xylanase all other cultivation parameters were significantly influenced by the surfactant, with the effect higher at higher surfactant concentrations. Surfactant increased the production of β-glucosidase and inhibited laccase and manganese-dependent and-independent peroxidase activities.
Keywords: Enzyme production; Pleurotus ostreatus ; nonionic surfactant; principal component analysis
Effect of enzymatic interesterification on melting point of palm olein by Atif A. A. Yassin; Ibrahim O. Mohamed; Mohd N. Ibrahim; Mohd S. A. Yusoff (pp. 45-52).
Immobilized PS-C ‘Amano’ II lipase was used to catalyze the interesterification of palm olein (POo) with 30, 50, and 70% stearic acid in n-hexane at 60°C. The catalytic performance of the immobilized lipase was evaluated by determining the composition change of fatty acyl groups and triacylglycerol (TAG) by gas liquid chromatography and high-performance liquid chromatography, respectively. The interesterification process resulted in the formation of new TAGs, mainly tripalmitin and dipalmitostearin, both of which were absent in the original oil. These changes in TAG composition resulted in an increase in slip melting point, from the original 25.5°C to 36.3, 37.0, and 40.0°C in the modified POo with 30, 50, and 70% stearic acid, respectively. All the reactions attained steady state in about 6 h. This type of work will find great applications in food industries, such as confectionery.
Keywords: Interesterification; PS-C ‘Amano’ II lipase; palm olein; stearic acid; slip melting point
Calcium alginate gel as encapsulation matrix for coimmobilized enzyme systems by A. Blandino; M. Macías; D. Cantero (pp. 53-60).
Encapsulation within calcium alginate gel capsules was used to produce acoimmobilized enzyme system. Glucose oxidase (GOD) and catalase (CAT) were chosen as model enzymes. The same values of V max and K mapp for the GOD encapsulated system and for the GOD-CAT coencapsulated system were calculated. When gel beads and capsules were compared, the same catalyst deactivation sequence for the two enzymes was observed. However, when capsules were employed as immobilization support, GOD efficiencies were higher than for the gel beads. These results were explained in terms of the structure of the capsules.
Keywords: Calcium alginate; gel capsules; gel beads; coimmobilization; glucose oxidase; catalase