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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.120, #1)
Expression of human β-defensin-2 with multiple joined genes in Escherichia coli by Zhinan Xu; Fang Wang; Li Peng; Xiangming Fang; Peilin Cen (pp. 1-13).
Human β-defensin (HBD)-2 is a small cationic peptide with a broad range of antimicrobial activity. In this study, multiple copies of the hBD2 gene were linked in tandem, and a number of different Escherichia coli expression vectors were evaluated, including pQE-30, pBV220, pET-28a(+), and pGEX-4T-2. No expression of multiple joined genes was detectable in the pQE-30 expression system, whereas in pBV220 with one or two joined hBD2 genes and in pET-28a(+) with one, two, or four copies, target proteins were expressed at a low level. Only when pGEX-4T-2 was applied as expression plasmid with one or two joined hBD2 genes were target proteins expressed in high level, and the expressed fusion proteins account for 26 and 16% of the total insoluble proteins, respectively. In the pGEX-4T-2 and pET-28a(+) expression systems, the effects of multiple joined genes on the growth of host strains and plasmid stability were examined. Host cells containing plasmid carrying fewer copies of hBD2 genes were faster in cell growth. Plasmid stability decreased with an increase in multiple joined genes, which was especially noticeable in the pET-28a(+) system. Furthermore, the presence of glucose in culture medium brought about a positive effect on plasmid stability when using pET28-nhBD2 as expression vectors.
Keywords: Antimicrobial peptide; human β-defensin-2; tandem repeated genes; fusion expression; plasmid stability; T7 expression system
Role of pyruvate and ascorbate production in regulation of antioxidant enzymes and membrane LPO levels in Fusarium Acuminatum by Hulya Ayar Kayali; Leman Tarhan (pp. 15-27).
The role of pyruvate and ascorbate in the regulation of superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase enzymes; and, therefore, membrane lipid peroxidation (LPO) levels in Fusarium acuminatum was investigated in media containing either glycerin or glucose as a carbon source, depending on the incubation period, in the range of 5–25 g/L. Increasing SOD activity between d 9 and 16 of the incubation period showed a positive correlation with a significant increase in pyruvate production up to 15 g/L of glycerin and glucose. In addition, maximum ascorbate production was observed at 15 g/L of glycerin as 82.5 ± 2.1 and 20 g/L of glucose as 54±1.51, whereas CAT activity decreased with an increased concentration of both carbon sources. When compared with the LPO levels determined in media supplemented with glycerin and glucose, the minimum LPO level was 1.88±0.028 nmol of malondialdehyde/g wet wt at 15 g/L of glycerin on d 16, at which it was also observed to have a maximum pyruvate and ascorbate production and SOD, CAT, and GSH-Px activities of 75±1.42 µg/mL, 82.5±2.1 µg/mL, 32.5±0.634 µg/mL, 86.8±2.58 IU/mg, and 1.867 IU/mg, respectively. These results indicate that the biosynthesis of pyruvate and ascorbate may be involved in the regulation of antioxidant enzymes, depending on the glycerin and glucose concentrations, and also this defense network was effective in preventing membrane damage from oxidative stress.
Keywords: Antioxidant enzymes; ascorbate; carbon sources; Fusarium sp.; pyruvate
Efficiency control in large-scale genotyping using analysis of variance by Geert T. Spijker; Marcel Bruinenberg; Gerard J. te Meerman (pp. 29-36).
The efficiency of the genotyping process is determined by many simultaneous factors. In actual genotyping, a production run is often preceded by small-scale experiments to find optimal conditions. We propose to use statistical analysis of production run data as well, to gain insight into factors important for the outcome of genotyping. As an example, we show that analysis of variance (ANOVA) applied to the first-pass results of a genetic study reveals important determinants of genotyping success. The largest factor limiting genotyping appeared to be interindividual variation among DNA samples, explaining 20% of the variance, and a smaller reaction volume, sizing failure, and differences among markers all explained ∼10%. Other potentially important factors, such as sample position within the plate and reusing electrophoresis matrix, appeared to be of minor influence. About 55% of the total variance could be explained by systematic factors. These results show that ANOVA can provide valuable feedback to improve genotyping efficiency. We propose to adjust genotype production runs using principles of experimental design in order to maximize genotyping efficiency at little additional cost.
Keywords: Laboratory procedure; polymerase chain reaction; genotyping; efficiency; analysis of variance
Enhancing profitability of dry mill ethanol plants by Srinivasan Rajagopalan; Elankovan Ponnampalam; Darold McCalla; Mark Stowers (pp. 37-50).
An Aspen Plus™ modeling platform was developed to evaluate the performance of the conversion process of degermed defibered corn (DDC) to ethanol in 15- and 40-million gallons per year (MGPY) dry mill ethanol plants. Upstream corn milling equipment in conventional dry mill ethanol plants was replaced with germ and fiber separation equipment. DDC with higher starch content was fed to the existing saccharification and fermentation units, resulting in higher ethanol productivity than with regular corn. The results of the DDC models were compared with those of conventional dry mill ethanol process models. A simple financial analysis that included capital and operating costs, revenues, earnings, and return on investment was created to evaluate each model comparatively. Case studies were performed on 15- and 40-MGPY base case models with two DDC process designs and DDC with a mechanical oil extraction process.
Keywords: Dry mill; degermed defibered corn; ethanol; modeling; economics
Innovative kinetic and thermodynamic analysis of a purified superactive xylanase from Scopulariopsis sp. by Ahmed Jawaad Afzal; Sikander Ali; Farooq Latif; Muhammad Ibrahim Rajoka; Khawar Sohail Siddiqui (pp. 51-70).
Two isoenzymes of endo-1,4-β-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The k cat values of LAX and HAX for oat-spelt xylan at 40°C, pH 6.5, were 95,000 and 9900 min−1 and the K m values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared with HAX was not owing to a reduction in ΔH # but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50°C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (ΔH #=306 kJ/mol) compared with HAX (ΔH #=264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (ΔS*=−232 J/[mol·K]) was more thermostable than HAX (ΔS*=490 J/[mol·K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that k cat/K m does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.
Keywords: Charge isomers; enzyme purification; energy-activity diagram; fungus; kinetic characterization; kinetic constant; Scopulariopsis ; stability-function relationship; superactive xylanase
Characteriztion of a novel feather-degrading Bacillus sp. strain by Paricia Orsoo Werlang; Adriano Brandelli (pp. 71-79).
Feather waste is generated in large amounts as a byproduct of commercial poultry processing. This residue is almost pure keratin, which is not easily degradable by common proteolytic enzymes. A feather-degrading bacterium was isolated from poultry feathers in decomposition. The strain identified as kr16 showed important feather-degrading activity when grown on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. The isolate was characterized according to the phenotypical characteristics and biochemical profiling that belong to the Bacillus genus. Keratinolytic activity of this isolate was monitored during cultivation of the bacterium on raw feathers at different temperatures. Maximum growth and feather-degrading activity were observed at 30–37°C. The keratinolytic enzyme had a pH optimum ranging from 8.0 to 11.0 and a temperature optimum of 45–65°C. The keratinase was strongly inhibited by EDTA and the metal ions Hg2+ and Sn2+.
Keywords: Bacillus ; feather digestion; keratinase; poultry waste; protease