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Recently, heavy-ion beams have been used widely to generate mutants of higher plants, because, in contrast to other mutagenic techniques, they induce mutations with high frequency at a relatively low dose, and various phenotypes may be obtained without other plant characteristics being affected9,10,11.
To date, uricase catalytic mechanisms are unknown and evolution biotechnology is practical to obtain uricase mutants of higher catalytic capacity.
The approaches were evaluated in terms of the ability to create mutants of higher order resulting in higher efficacy and less effort when compared with first order mutants.
For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed.
The practice of evolution biotechnology requires an efficient and cost-effective analytical method for reliable assay of catalytic capacities of mutants, and a suitable decision method for recognizing mutants of higher catalytic capacity as positive candidates or potential hits.
The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells.
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Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity.
Studies on mutant of higher plants showed that CYP97A3 and CYP97C1 are the enzymes primarily responsible for catalyzing hydroxylation of β- and ϵ-ring of α-carotene, respectively, producing α-branch xanthophylls (lutein), the most abundant carotenoids in light-harvesting complexes (LHCs) which is key structural and functional components of light harvesting [ 6, 8, 26, 33, 37, 39, 59, 72].
The ratio of docked versus moving mitochondria increases in tam3/tam9 mutants because of higher number of motile mitochondria.
However, paralogous mutants of the high-B hypersensitive RP subunit mutants, including rpn8b, rpn2b, rpt2b-1, and rpt5b-3, were not hypersensitive to high-B stress (Supplementary Fig. 4a e).
Their transcript levels are strongly increased in deletion mutants of the "high glucose" group and decreased in deletion mutants of the "low glucose" group.
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