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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.119, #3)
Denitrification in presence of benzene, toluene, and m-xylene by A. Peña-Calva; A. Olmos-Dichara; G. Viniegra-González; F. M. Cuervo-López; J. Gómez (pp. 195-208).
Denitrification of the electron donors toluene-C(15–100 mg/L), m-xylene-C (15–70 mg/L), benzene-C (5–25 mg/L), and acetate-C as experimental reference (50–140 mg/L) was carried out in batch culture. An initial concentration of 1.1±0.15 g of volatile suspended solids/L of denitrifying sludge without previous exposure to aromatic compounds was used as inoculum. The results showed toluene and nitrate consumption efficiency (E T and E N′ respectively) of 100%. Toluene was completely mineralized (oxidized) to CO2. In all cases, the N2 (γN2) and HCO 3 − yields (γHCO3) were 0.97±0.01 and 0.8±0.05, respectively. The consumption efficiency (E x ) of m-xylene (53±5.7%) was partial. The γN2 and γHCO3 were 0.96±0.01 and 0.86±0.02, respectively. Benzene was not consumed under denitrifying conditions. The specific consumption rates of toluene (q T ) and m-xylene (q X ) were lower than that of acetate (q A ). The differences in specific consumption rates were probably owing to the negative effect of benzene, toluene, and isomers of xylene on the cell membrane.
Keywords: Toluene; m-xylene; benzene; denitrification; kinetics
RNA synthetic activity of glutamate dehydrogenase by Godson O. Osuji; Jonas Konan; Gitonga M’Mbijjewe (pp. 209-228).
The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology forassay of the enzyme’s activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.
Keywords: Glutamate dehydrogenase; α-ketoglutarate; differential display polymerase chain reaction; nucleotide chromatography; deamination; amination
Purification and characterization of an extracellular β-glucosidase with high transglucosylation activity and stability from Aspergillus niger No. 5.1 by Yu Xie; Yunhua Gao; Zhiffeng Chen (pp. 229-240).
An extracellular β-glucosidase was extracted from the culture filtrate of Aspergillus niger No. 5.1 and purified to homogeneity by using ammonium sulfate precipitation, Chitopearl-DEAE chromatography, and Sephadex G-100 chromatography. The specific activity of the enzyme was enriched 6.33-fold, with a recovery of 11.67%. The enzyme was a monomer and the molecular mass was 67.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 66.5 kDa by gel-filtration chromatography. The enzyme had optimum activity at pH 6.0 and 60°C and was stable over the pH range of 3.0–9.0. It showed specificity of hydrolysis for p-nitrophenyl-β-d-glucoside and cellobiose. The K m and V max values of the enzyme for cellobiose and salicin were 5.34 mM, 2.57 µmol/(mL·s), and 3.09 mM, 1.34 µmol/(mL·s), respectively. Both amino acid composition and N-terminal amino acid sequence of the enzyme were determined, which provides useful information for cloning of this enzyme.
Keywords: β-Glucosidase; Aspergillus niger ; purification; characterization; kinetic parameters; amino acid
Early phase process scale-up challenges for fungal and filamentous bacterial cultures by B. H. Junker; M. Hesse; B. Burgess; P. Masurekar; N. Connors; A. Seeley (pp. 241-277).
Culture pelleting and morphology has a strong influence on process productivity and success for fungal and filamentous bacterial cultures. This impact is particularly evident with early phase secondary metabolite processes with limited process definition. A compilation of factors affecting filamentous or pelleting morphology described in the literature indicates potential leads for developing process-specific control methodologies. An evaluation of the factors mediating citric acid production is one example of an industrially important application of these techniques. For five model fungal and filamentous bacterial processes in an industrial fermentation pilot plant, process development strategies were developed and effectively implemented with the goal of achieving reasonable fermentation titers early in the process development cycle. Examples of approaches included the use of additives to minimize pelleting in inoculum shake flasks, the use of large-volume frozen bagged inoculum obtained from agitated seed fermentors, and variations in production medium composition and fermentor operating conditions. Results were evaluated with respect to productivity of desired secondary metabolites as well as process scalability. On-line measurements were utilized to indirectly evaluate the cultivation impact of changes in medium and process development. Key laboratory to pilot plant scale-up issues also were identified and often addressed in subsequent cultivations.
Keywords: Morphology; pellet; fermentation; scale-up; Aspergillus ; Sordaria ; Streptomyces ; Chalara