Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.
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There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process. (+info)
Chromosomal assignment of RFLP linkage groups harboring important QTLs on an intraspecific cotton (Gossypium hirsutum L.) Joinmap.
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Chromosome identities were assigned to 15 linkage groups of the RFLP joinmap developed from four intraspecific cotton (Gossypium hirsutum L.) populations with different genetic backgrounds (Acala, Delta, and Texas Plains). The linkage groups were assigned to chromosomes by deficiency analysis of probes in the previously published joinmap, based on genomic DNA from hypoaneuploid chromosome substitution lines. These findings were integrated with QTL identification for multiple fiber and yield traits. Overall results revealed the presence of 63 QTLs on five different chromosomes of the A subgenome (chromosomes-03, -07, -09, -10, and -12) and 29 QTLs on the three different D subgenome (chromosomes-14 Lo, -20, and the long arm of -26). Linkage group-1 (chromosome-03) harbored 26 QTLs, covering 117 cM with 54 RFLP loci. Linkage group-2, (the long arm of chromosome-26) harbored 19 QTLs, covering 77.6 cM with 27 RFLP loci. Approximately 49% of the putative 92 QTLs for agronomic and fiber quality traits were placed on the above two major joinmap linkage groups, which correspond to just two different chromosomes, indicating that cotton chromosomes may have islands of high and low meiotic recombination like some other eukaryotic organisms. In addition, it reveals highly recombined and putative gene abundant regions in the cotton genome. QTLs for fiber quality traits in certain regions are located between two RFLP markers with an average of less than one cM (approximately 0.4-0.6 Mb) and possibly represent targets for map-based cloning. Identification of chromosomal location of RFLP markers common to different intra- and interspecific-populations will facilitate development of portable framework markers, as well as genetic and physical mapping of the cotton genome. (+info)
Mode of action of cellulases on dyed cotton with a reactive dye.
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Cotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases. (+info)
The delayed initiation and slow elongation of fuzz-like short fibre cells in relation to altered patterns of sucrose synthase expression and plasmodesmata gating in a lintless mutant of cotton.
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Cotton (Gossypium hirsutum L.) seed develops single-celled long fibres (lint) from the seed-coat epidermis at anthesis. Previous studies have shown that the initiation and rapid elongation of these fibres requires the expression of sucrose synthase (Sus) and, potentially, a transient closure of plasmodesmata. This study extends the previous work to examine the patterns of Sus expression and plasmodesmata gating in fuzz-like short fibres of a mutant that shows delayed initiation and much slower and reduced elongation of the fibre cells. Immunolocalization studies revealed delayed expression of Sus in the mutant seed-coat epidermis that correlates temporally and spatially with the initiation of the fibre cells. Anatomically, these short fibres differed from the normal lint in that their basal ends enlarged immediately after initiation, while the majority of the normal lint on wild-type seed did not show this enlargement until the end of elongation. Suppression of Sus expression in the seed-coat epidermis of the transgenic plants reduced the length of both lint and short fuzz fibres at maturity, suggesting that the growth of short fibres also requires high levels of Sus expression. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed no closure of plasmodesmata during the entire elongation period of short fibres from the mutant seed. These results show (i) the delayed initiation of fuzz-like short fibres from the mutant seed correlates with delayed or insufficient expression of Sus in a subset of seed-coat epidermal cells destined to become fibres and (ii) the much shortened elongation of the fibres from the mutant may be related to their inability to close plasmodesmata. (+info)
Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers.
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Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues, respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159wD mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159wD cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p. (+info)
Use and abuse of cotton buds.
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Ear injuries caused by cotton buds are commonly seen in ear, nose and throat (ENT) practice. We asked 1000 patients attending an ENT referral clinic whether they used cotton buds to clean the ear canal. Of the 325 who responded, 171 said they did. The frequency of use was no higher in those with ear complaints than in those with nose and other complaints. 15-20% of respondents disagreed with the statements that cotton buds can cause infections, wax impaction or perforations. On the evidence of this survey, manufacturers' warnings need to be fortified. (+info)
Cotton bud and ear cleaning--a loose tip cotton bud?
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Complications that arise from self-cleaning the external ear canal are common. This is a prospective study using standard questionnaire on the practice of ear cleaning in 50 subjects. A loose tip cotton bud was also shown to them to assess if it was acceptable to replace their current cotton bud. Thirty six percent of them clean their ears by introducing an object into the ear canal once or more a day. The commonest reason to clean the ears was the presence of earwax. Only 6% used a cotton bud to mop fluid from the ear canal. Almost all (92%) the subjects used a cotton bud to clean the ear. A complication rate of 2% was noted. A loose tip cotton swab was shown to the subjects. Despite explaining that it is safer and better, only 24% of the subject were willing to change to this loose tip cotton bud. Seventy four percent of the subjects cleaned their ear regularly because of earwax. The misconception of needing to clean the ear canal by introducing an object into the ear is rampant. This practice should be avoided and health care providers can play an important role in this. (+info)
A 20-year follow-up study on chronic respiratory effects of exposure to cotton dust.
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In order to evaluate chronic effects of long-term exposure to cotton dust on respiratory health, and the role of dust and endotoxin, longitudinal changes in lung function and respiratory symptoms were observed prospectively from 1981 to 2001 in 447 cotton textile workers, along with 472 silk textile controls. The results from five surveys conducted over the 20-yr period are reported, including standardised questionnaires, pre- and post-shift spirometric measurements, work-area inhalable dust sample collections and airborne Gram-bacterial endotoxin analysis. Cotton workers had more persistent respiratory symptoms and greater annual declines in forced expiratory volume in one second (FEV1) and forced vital capacity as compared with silk workers. After exposure cessation, in the final 5-yr period, the rate of FEV1 decline tended to slow in nonsmoking males, but not in nonsmoking females. Workers who reported byssinotic symptoms more persistently suffered greater declines in FEV1. Chronic loss in lung function was more strongly associated with exposure to endotoxin than to dust. In conclusion, the current study suggests that long-term exposure to cotton dust, in which airborne endotoxin appears to play an important role, results in substantial adverse chronic respiratory effects. (+info)