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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.95, #1)
Effect of two broad-spectrum antibiotics on activity and stability of continuous nitrifying system by J. Luis Campos; Juan M. Garrido; Ramón Méndez; Juan M. Lema (pp. 1-10).
The effects of two broad-spectrum antibiotics, chloramphenicol and oxytetracycline hydrochloride, on the microbial activity and biofilm stability of a mixed nitrifying culture were studied. These antibiotics are present in some wastewaters generated in cattle farms or pharmaceutical industries. A 1-L fermentor, in which nitrifiers grew both in suspension and in a biofilm, was used during the experiments. Chloramphenicol (10–250 mg/L) barely had any effect on biofilm stability and nitrification. Ammonia was fully oxidized to nitrate. However, oxytetracycline caused biofilm sloughing at concentrations of 10 mg/L, but nitrification was not inhibited at antibiotic concentrations up to 100 mg/L. When the concentration of oxytetracycline chlorohydrate was increased stepwise from 100 to 250 mg/L, nitrification was inhibited by 50%. The dissolved organic carbon measurements in both the influent and effluent showed that the antibiotics were neither mineralized by the mixed nitrifying culture nor accumulated in the system. Furthermore, the microbial tests did not reveal the presence of active antibiotics in the effluent. This fact indicates that both cloramphenicol and oxytetracycline were degraded by the nitrifying sludge but not mineralized.
Keywords: Antibiotic; chloramphenicol; oxytetracycline hydrochloride; nitrification; biofilm
New supports for enzyme immobilization based on copolymers of vinylene carbonate and acrylamide by Lunhan Ding; Yan Jiang; Lei Huang; Yonggang Li; Jiaxian Huang (pp. 11-21).
In this study, a series of beadlike and hydrophilic supports containing reactive cyclic carbonate groups for enzyme immobilization were prepared via reverse-phase suspension copolymerization of the aqueous solutions of vinylene carbonate (VCA), acrylamide (AA), and N,N′-methylene bisacrylamide in paraffin oil. The supports were used as a matrix for immobilization of trypsin and showed a considerable capacity to couple with trypsin and reasonable retention of activity for the immobilized trypsin, depending on the immobilization conditions, such as the content of VCA structural units, reaction time, and pH of the medium.
Keywords: Vinylene carbonate; trypsin; immobilization enzyme; support; acrylamide
Construction, expression, and characterization of recombinant hirudin in Escherichia coli by Qun Bi; Junshan Zhang; Yixiu Huang; Hongjun Su; Xianwan Zhou; Shenggeng Zhu (pp. 23-30).
The mutant gene of HV2-K47 was obtained by polymerase chain reaction-directed mutagenesis and expressed in Escherichia coli. Many elements that could affect its expression level were compared. The product was purified to homogeneity via three chromatographic steps—ion exchange, gel filtration, and reverse phase chromatography—on the AKTA Explorer System. The antithrombin activity of HV2-K47 is much higher than that of recombinant HV2. Some properties and expression conditions were investigated systematically, which would be useful for further studies of hirudin and other small proteins.
Keywords: Antithrombin; expression; purification; recombinant hirudin HV2-K47; secretion
Controlling electrophoretic trapping of circular DNA by addition of starch preparations to agarose gels by Kenneth D. Cole; Carlos M. Tellez; Richard Nguyen (pp. 31-43).
Starch preparations were added to agarose gels to enhance the electrophoretic trapping of circular plasmid DNA. The critical voltages required to trap the open circular (OC) and the supercoiled (SC) forms of a 13.1-kbp plasmid were measured in gels composed of agarose and added starch preparations. Modified starch preparations reduced the critical voltage required to trap the OC form of the plasmid to approximately one-third of the control value (in 1% agarose gels). Amylose (a fraction of starch with a low amount of branching) also reduced the critical voltage to trap the OC form in a similar manner. The critical voltage to trap the SC form of the plasmid was not significantly reduced by the starch preparations. The capacity to trap OC DNA was increased by the addition of higher amounts of the starch preparations added to the gels. Field inversion gel electrophoresis was used to characterize the length of the traps in the gels. The starch preparations and amylose increased the trap lengths approximately twofold. The increased trap length correlated with the decreased critical voltage required to trap the OC form of the 13.1-kbp plasmid.
Keywords: Electrophoresis; DNA; agarose gel; trapping; starch; circular DNA; plasmid; separation
Continuous cultivation of dilute-acid hydrolysates to ethanol by immobilized Saccharomyces cerevisiae by Mohammad J. Taherzadeh; Ria Millati; Claes Niklasson (pp. 45-57).
The continuous cultivation of immobilized Saccharomyces cerevisiae CBS 8066 on dilute-acid hydrolysates of forest residuals was investigated. The yeast cells were immobilized in 2–4% Ca-alginate beads. The 2% beads were not stable. However, the 3 and 4% beads were stable for at least 3 wk when an extra resource of calcium ions was available in the medium. The continuous cultivation of a dilute-acid hydrolysate by the immobilized cells at dilution rates of 0.3, 0.5, and 0.6 h−1 resulted in 86, 83, and 79% sugar consumption, respectively, and an ethanol yield between 0.45 and 0.48 g/g. The hydrolysate was fermentable at a dilution rate of 0.1 h−1 in a free-cell system but washed out at a dilution rate of 0.2 h−1. The continuous cultivation of a more inhibiting hydrolysate was not successful by either free- or immobilized-cell systems even at a low dilution rate of 0.07 h−1. However, when the hydrolysate was overlimed, it was fermentable by the immobilized cells at a dilution rate of 0.2 h−1.
Keywords: Saccharomyces cerevisiae ; dilute-acid hydrolysate; ethanol; glucose; immobilization
Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica by Leonie A. Sarubbo; Maria do Carmo Marçal; Maria Luisa C. Neves; Maria da Paz C. Silva; Lúcia F. Porto; Galba Maria Campos-Takaki (pp. 59-67).
The yeast Candida lipolytica IA 1055 produced an inducible extracellular emulsification activity while utilizing glucose at different concentrations as carbon source during batch fermentation at 27°C. In all glucose concentrations studied, maximum production of emulsification activity was detected in the stationary phase of growth, after pH reached minimal values. The bioemulsifier isolated was a complex biopolymer constituting proteins, carbohydrates, and lipids. The results obtained in this work show that the biosynthesis of a bioemulsifier is not simply a prerequisite for the degradation of extracellular hydrocarbon.
Keywords: Candida lipolytica ; biosurfactant; bioemulsifier; glucose fermentation