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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.101, #2)
Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A on BMCC and avicel by Donna L. Watson; David B. Wilson; Larry P. Walker (pp. 97-111).
In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonospora fusca E3, E4, and E5), it was observed that binding capacities for these three cellulases were 18–30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate, accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding The processive endoglucanase, Cel9A, had four times the extent of conversion of the end endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.
Keywords: Cellulases; Thermobifida fusca ; synergism; Avicel; BMCC
Recent progress on immobilization of enzymes on molecular sieves for reactions in organic solvents by Ai-Xin Yan; Xuan-Wen Li; Yun-Hua Ye (pp. 113-129).
Enzymes exhibit high selectivity and reactivity under normal conditions but are sensitive to denaturation or inactivation by pH and temperature extremes, organic solvents, and detergents. To extend the use of these biocatalysts for practical applications, the technology of immobilization of enzymes on suitable supports was developed. Recently, these immobilized biomolecules have been widely used and a variety of immobilization supports have been studied. The majority of these supports cover diverse kinds of materials such as natural or synthetic polyhydroxylic matrives, porous in organic carriers, and all kinds of functional polymers. Microporous molecular sieve, zeolite, has attracted extensive interest in research because of its distinctive physical properties and geochemistry. Recently, with the discovery of a new family of mesoporous molecular sieves, MCM-41, this series of materials shows great potential for various applications. Molecular sieves involve such a series of materials that can discriminate between molecules, particularly on the basis of size. As support materials, they offer interesting properties, such as high surface areas, hydrophobic or hydrophilic behavior, and electrostatic interaction, as well as mechanical and chemical resistance, making them attractive for enzyme immobilization. In this article, different types of molecular sieves used in different immobilization methods including physical adsorption on zeolite, entrapment in mesoporous and macroporous MCM series, as well as chemically covalent binding to functionalized molecular sieves are reviewed. Key factors affecting the application of this biotechnology are discussed systematically, and immobilization mechanisms combined with newly developed techniques to elucidate the interactions between matrixes and enzyme molecules are also introduced.
Keywords: Zeolite; molecular sieve; enzyme; immobilization; MCM-41; matrix
Optimization of glycerol fed-batch fermentation in different reactor states by Dongming Xie; Dehua Liu; Haoli Zhu; Jianan Zhang (pp. 131-151).
To optimize the fed-batch processes of glycerol fermentation in different reactor states, typical bioreactors including 500-mL shaking flask, 600-mL and 15-L airlift loop reactor, and 5-L stirred vessel were investigated. It was found that by reestimating the values of only two variable kinetic parameters associated with physical transport phenomena in a reactor, the macrokinetic model of glycerol fermentation proposed in previous work could describe well the batch processes in different reactor states. This variable kinetic parameter (VKP) approach was further applied to model-based optimization of discrete-pulse feed (DPF) strategies of both glucose and corn steep slurry for glycerol fed-batch fermentation. The experimental results showed that, compared with the feed strategies determined just by limited experimental optimization in previous work, the DPF strategies with VKPs adjusted could improve glycerol productivity at least by 27% in the scale-down and scale-up reactor states. The approach proposed appeared promising for further modeling and optimization of glycerol fermentation or the similar bioprocesses in larger scales.
Keywords: Macrokinetic model; variable kinetic parameter; glycerol production; fed-batch fermentation; discrete-pulse feed strategy; reactor state
Optimization of β-carotene production from synthetic medium by Blakeslea trispora by Fani Mantzouridou; Triantafyllos Roukasa; Parthena Kotzekidoua; Maria Liakopoulou (pp. 153-175).
The effect of inoculum, pH, carbon and nitrogen source, natural oils, fatty acids, antioxidant, and precursors on β-carotene production by Blakeslea trispora in shake-flask culture was investigated. The highest concentration of β-carotene was obtained in the medium (pH 7.0) inoculated with one loop of each culture. Sucrose, glycerol, cornmeal, soy protein acid hydrolysate, and distiller's solubles did not improve the production of β-carotene. By contrast, glucose, corn steep liquor, antioxidant, olive oil, soybean oil, cottonseed oil, oleic and linoleic acids, and kerosene significantly increased the β-carotene production. A central composite design was employed to determine the maximum β-carotene production at optimum values for the process variables (linoleic acid, kerosene, and antioxidant). The fit of the model was found to be good. Linoleic acid, kerosene, and antioxidant had a strong linear effect on β-carotene production. The concentration of β-carotene was significantly affected by linoleic acid-kerosene and linoleic acid-antioxidant interactions as well as by the negative quadratic effects of these variables. The interaction between kerosene and antioxidant had no significant linear effect. The maximum β-carotene concentration (2.88 g/L) was obtained at concentrations of 17.15 g/L of linoleic acid, 39.25 g/L of kerosene, and 9.04 g/L of antioxidant.
Keywords: β-Carotene; synthetic medium; Blakeslea trispora ; shake flask; mathematical modeling