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Subject: search.filters.subject.Mutation

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    The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2)
    (Elsevier, 2015) Prins, Alaric; Kleinsmidt, L.; Khan, Nuraan; Kirby, Bronwyn; Kudanga, Tukayi; Vollmer, Jannik; Pleiss, Juergen; Burton, Stephanie; Le Roes-Hill, Marilize
    tBacterial laccases show low activities but can be of biotechnological interest due to industrially suit-able characteristics such as thermostability and tolerance to alkaline pH. In this study, three separatemutations (M298F, V290N and V290A) were introduced at or near the T1 copper site of the small lac-case (SLAC) from Streptomyces coelicolor A3(2) and biochemical properties were assessed in comparisonwith the native enzyme. The mutation, V290N showed approximately double the activity of SLAC whenABTS was used as substrate while the specific activity of SLAC-M298F was 4–5 times higher than that ofSLAC when the assays were performed at ≥70◦C. There was no significant difference in activity with 2,6-dimethoxyphenol (2,6-DMP); however, there was a significant shift in the optimal pH from pH 9.5 (SLAC)to 7.5 (SLAC-V290N). Optimal temperature for activity was not significantly altered but thermostabilitywas reduced in all three mutants. The substrate range of the mutant variants remained largely unchanged,with the exception of SLAC-M298F which was unable to oxidise veratryl alcohol. Interestingly, the “typ-ical” laccase inhibitor, sodium azide, had no significant inhibitory effect on the activity of SLAC-M298F,which also exhibited increased resistance to inhibition by sulfhydryl compounds. SLAC-V290N showedhigher catalytic efficiency for 2,6-DMP (kcat/Km= 2.226 mM−1s−1) and ABTS (kcat/Km= 1.874 mM−1s−1)compared to SLAC (kcat/Km= 1.615 mM−1s−1for 2,6-DMP and kcat/Km= 1.611 mM−1s−1for ABTS). Thisstudy has shown that three ligands that are closely associated with the T1 copper in SLAC play a key rolein maintaining enzymatic activity. Whilst the introduction of mutations at these sites negated favourablecharacteristics such as thermostability, several favourable effects were observed. This study has alsoextended the knowledge base on the biochemical characteristics of SLAC, and its suitability as a templatefor engineering with the aim of widening its potential range of industrial applications.
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    Mutation of Eremothecium gossypii and statistical media optimization to increase riboflavin production
    (2011) Govender, Sharon; Swalaha, Feroz Mahomed; Permaul, Kugen
    Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.