Nesse trabalho avaliamos o potencial enzimático de diferentes linhagens de fungos, visando determinar a presença de mono-oxigenases capazes de oxidar cetonas aromáticas e cíclicas. Todas as linhagens empregadas apresentaram atividade de álcool desidrogenase e Baeyer-Villiger mono-oxigenases. Adicionalmente foram sintetizadas oito moléculas bi-funcionalizadas com grupos sulfeto, seleneto e carbonila (cetona). Os produtos das reações biocatalisadas foram isolados e caracterizados. / In this work, we evaluated the enzymatic potential of different Aspergillus strains, through the biotransformations of two substrates: 2- and 4-methylcyclohexanone (1a e 1b). All the strains employed showed alcohol dehydrogenase and Baeyer-Villiger monooxygenase (CPMO and CHMO) activities. These enzymes can perform ketone biorreduction and oxidation. Using the A. terreus SSP 1498 selected from the screening study, we prepared alcohols and lactones in good enantiosselectivity. In this way, other fungal strains were studied aiming to determine the presence of monooxygenase activity by means of the biotransformation of aromatic ketones. Like the Aspergillus, we observed that all strains used in this study showed alcohol dehydrogenase and Baeyer-Villiger monooxygenase (APMO) activities. We selected 1-phenyl-etanone and its para substituted derivates as substrates. Additionally, we synthesized eight examples of bi-functionalized compounds with sulfide, selenide and ketone groups. These compounds were submitted to the action of enzymatic system of different fungi which were selected from the initial screening. The products from the biotransformation were isolated and characterized.

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Chronic wounds represent an under-acknowledged socioeconomic epidemic, affecting 1.8 million new patients per year and costing the US health care system upwards of $25 billion annually. This substantial cost is rapidly growing due to a disproportionate occurrence in the ever-aging population. Key features associated with age-related impairment of wound healing include limited energy and nutrient exchange, unremitting inflammations, increased reactive oxygen species (ROS), and diminished blood flow. Most chronic wound therapies target specific molecular mechanisms; however, there are often multiple mitigating factors that prevent normal wound closure. This is likely one reason most wound therapies are minimally effective. In the standard American diet, carbohydrates are broken down for fuel (glucose). While fasting, starvation, and calorie or carbohydrate restriction, beta-oxidation of stored fats in the liver produces ketone bodies (primarily acetoacetate (AcAc) and β-hydroxybutyrate (βHB) to serve as energy metabolites for extra-hepatic tissues. In addition to enhancing metabolic physiology, ketone bodies have recently been discovered to have signaling properties that are independent of their function as energy metabolites. Here we present the evidence for a novel method of inducing therapeutic ketosis via exogenous ketone supplementation to promote enhanced ischemic wound healing in young and aged Fischer 344 rats. Preliminary mechanistic studies demonstrated that exogenous ketone supplementation enhanced wound healing via increasing proliferation and migration, decreasing lactate production, and decreasing ROS production as well as affecting inflammatory cytokines and growth factors. We conclude that exogenous ketone supplementation will be an effective, cost efficient, low toxicity therapy to promote enhancement of wound healing in an aged population.

Elevated ketone production and utilization results in a host of health benefits. The aim of this study was to assess the rate of ketone production during a prolonged fast and to evaluate how an initial bout of exercise influences this production. Mood and hunger, along with plasma insulin and glucagon, were also assessed. In this crossover study, 20 adult subjects (11 Male, 9 Female) completed two 36-hour fasts, with one protocol requiring the subject to complete an intense treadmill exercise session at the beginning of the fast. Ketone levels were assessed via blood ketone meter and recorded every two hours. Subjective mood and hunger ratings were also recorded every two hours. Venipuncture was performed every 12 hours to assess plasma insulin and glucagon. The mean area under the ketone production curve for the nonexercise intervention was 19.19 ± 2.59 mmol/L and 27.49 ± 2.59 mmol/L for the exercise intervention, resulting in a significant 8.30 mmol/L difference between conditions (95% probability interval was 1.94 to 14.82 mmol/L). The mean time to nutritional ketosis was 21.07 ± 2.95 hours with fasting alone, and 17.5 ± 1.69 hours when combined with exercise (posterior probability = 0.89). There was a significant decrease in insulin over time (F(3,133) = 61.75, p < 0.0001). There was also a significant increase in glucagon over time (F(3,133) = 21.10, p < 0.0001). Hunger and stomach discomfort did not differ between conditions. Anger (F(10,394) = 2.74, p = 0.0028), depression (F(10,394) = 2.91, p = 0.0016), tension (F(10,394) = 2.29, p = 0.0128), vigor (F(10,394) = 11.65, p < 0.0001), and fatigue (F(10,394) = 10.60, p = 0.0001) increased over the course of the fast, but did not differ between conditions. Completing aerobic exercise at the beginning of a 36-hour fast results in significantly more ketone production. The impact of exercise on ketone production comes at little or no impact on hunger, stomach discomfort and negative moods. A difference in time to achieving nutritional ketosis between conditions may exist, but this was not observed in this study.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18301号 / 工博第3893号 / 新制||工||1597(附属図書館) / 31159 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 村上 正浩, 教授 杉野目 道紀, 教授 大江 浩一 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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Introduction: Acute ketone monoester (KE) supplementation affects exercise responses but there are equivocal data regarding the effects on exercise efficiency. We examined the effect of ketone monoester ingestion on exercise efficiency during cycling and probed further the influence of supplement dose and exercise intensity. This study was registered prior to data collection at ClinicalTrials.org (NCT05665855). Methods: Twenty-eight trained adults were recruited [16 males, 12 females; peak oxygen uptake (V̇O2peak): 59±11 ml·kg-1·min-1]. Participants completed three experimental trials in a randomized, crossover, and double-blinded manner, each separated by ~1 week. Participants ingested either a 0.3 (KE-LO) or 0.6 g/kg (KE-HI) body mass dose of KE or a flavour-matched placebo (PLAC) ~30 min prior to exercise. The incremental cycling protocol involved a 3-minute warm-up, three 5-minute stages at 75%, 100%, and 125% of individual ventilatory threshold, and a ramp increase to volitional exhaustion. Expired gases and heart rate were measured continually during exercise. Results: Venous blood [ß-hydroxybutyrate], the major circulating ketone body, was higher in both KE conditions compared to PLAC and also different between conditions (3.0±1.1 and 2.3±0.6 vs 0.2±0.1 mM; all p<0.05). There were no differences in submaximal exercise V̇O2, exercise economy, gross efficiency, or delta efficiency between conditions (all p>0.05). Submaximal exercise heart rate and ventilation were higher in both KE conditions compared to PLAC (141±11 and 141±12 vs 137±12 beats/min; 63±14 and 62±13 vs 60±13 L/min, respectively; all p<0.05). Peak power output at V̇O2peak was lower in KE-HI compared to both KE-LO and PLAC (329±60 vs 339±62 and 341±61 W; both p<0.05). Conclusion: KE supplementation did not alter exercise efficiency during submaximal cycling. KE ingestion increased cardiorespiratory stress during submaximal exercise and the higher dose reduced peak aerobic power output. Future studies should investigate the mechanisms by which KE ingestion alters exercise responses. / Thesis / Master of Science (MSc) / Endurance exercise performance is determined by many variables including the efficiency of the individual. This can be measured during cycling by calculating the ratio of oxygen uptake relative to power output. Ketone supplements have been suggested to alter exercise efficiency. We investigated this issue by having trained adults complete an incremental cycling protocol on three occasions. Before exercise the participants ingested either a small or large dose of a ketone supplement or a taste-matched placebo drink. Exercise efficiency was not different between the conditions but ventilation rate and heart rate were higher during the ketone supplemented trials compared to the placebo. The power output that the participants could achieve at maximal exercise was reduced in the high dose ketone condition. Our study does not support the use of ketone supplements as a strategy to enhance endurance exercise performance. Future studies should investigate the mechanisms by which ketones affect exercise responses.

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