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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.87, #1)
Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica by Cheon Seok Park; Ching Chuan Chang; Dewey D. Y. Ryu (pp. 1-15).
The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.
Keywords: Gene expression; secretion; cellulase; endoglucanase I; high-cell density culture; fed-batch fermentation; Yarrowia lipolytica
Isolation and characterization of a novel feather-degrading bacterial strain by Sidnei Sangali; Adriano Brandelli (pp. 17-24).
Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common proteolytic enzymes. Feather-degrading bacteria were isolated from a Brazilian poultry industrial waste. Among these isolates, a strain identified as kr2 was the best feather-degrading organism when grown on basal medium containing 10 g/L of native feather as a source of energy, carbon, and nitrogen. The isolate was characterized according to morphological characteristics and biochemical tests belonging to the Vibrionaceae family. Keratinolytic activity of this isolate was monitored throughout the cultivation of the bacterium on raw feather at different temperatures. The optimum temperature for growth was about 30°C, at which maximum enzyme and soluble protein production were achieved. The enzyme had a pH and temperature optima of 8.0 and 55°C, respectively.
Keywords: Vibrioneceae ; feather digestion; keratinase; poultry waste; protease
A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine by Mohyee E. Eldefrawi; Nehad L. Azer; Nidhi Nath; Nabil A. Anis; Madan S. Bangalore; Kevin P. O’Connell; Robert P. Schwartz; Jeremy Wright (pp. 25-35).
An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 µg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).
Keywords: Morphine; urinalysis; monoclonal antibody; polymethyl methacrylate; beads; KinExA™
Continuous production of antibiotics in an airlift fermentor utilizing a transverse magnetic field by Zakaria Al-Qodah (pp. 37-55).
In this study, a series of experiments was conducted to demonstrate the feasibility of continuous production of penicillin antibiotic using a three-phase magneto airlift fermentor with immobilized Penicillium chrysogenum. The fermentation processes were carried out in a 2.4-L external loop airlift utilizing a transverse magnetic field. It was found that the application of the magnetic field to a bed of ferromagnetic beads affects both the hydrodynamics of the reactor and the rate of the bioconversion process occurring inside it. One hundred hours after startup, the maximum penicillin concentration increased 48% as the magnetic field intensity increased from 0 to 35 mT, owing to the increased residence time of the substrate in the riser and the positive effect of the magnetic field on the effective fluid-solid interfacial area. In addition, the detached biomass concentration in the liquid phase was found to be only 5% of the immobilized biomass, owing to low shear levels and the absence of friction among the solid-phase particles.
Keywords: Magnetic stabilization; magneto airlift reactors; magnetic field; penicillin production; penicillium chrysogenum
Cellular association of glucosyltransferases in Leuconostoc mesenteroides and effects of detergent on cell association by James C. Zahnley; Michael R. Smith (pp. 57-70).
Most glucosyltransferase (GTF) activity in sucrose-grown cultures of some strains of Leuconostoc mesenteroides is found with the cell pellet after centrifugation. GTFs are known to bind to dextrans, and it was traditionally assumed that cell-associated GTFs were bound to those dextrans that cosedimented with the cells. We used a mutant strain (LC-17), derived from strain NRRL B-1355, which produced dextransucrase in the absence of dextrans, to investigate the extent to which GTFs were bound to cells or dextrans. Much of the GTF activity in glucose-grown cultures of strain LC-17, which do not produce dextran, was located in the cell pellets. Soluble enzyme activity increased when cell suspensions from glucose- or sucrose-grown cultures were incubated with mild nonionic detergents or zwitterionic reagents. Alternansucrase produced by the parent strain B-1355 was almost entirely associated with cells under conditions in which dextrans were or were not produced. Alternansucrase, but not dextransucrase, tended to be enriched in the particulate fraction of B-1355 cells that had been broken in a French press. The distribution of alternansucrase and the effects of detergents on the distribution of GTFs suggest that soluble GTFs sequestered in the cytoplasm, and GTFs bound or adsorbed to the cell membrane are probably the major contributors to the cell-associated GTF activity.
Keywords: Dextransucrase; glucosyltransferase; Leuconostoc mesenteroides ; solubilization; cell-associated enzyme; alternansucrase