Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

Atroshenko, D. L.; Shelomov, M. D.; Zarubina, S. A.; Negru, Nikita Y.; Golubev, I. V.; Савин, С. С.; Тишков, В. И.

doi:10.3390/ijms20184412

Cited by 5 publications

(4 citation statements)

References 29 publications

(44 reference statements)

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“…В отсутствие экзогенного FAD понижение концентрации TvDAAO вызывает снижение температурной стабильности, но добавление 100-кратного избытка FAD приводит к тому, что скорость инактивации перестает зависеть от концентрации фермента. Ранее в нашей лаборатории был подробно изучен процесс инактивации рекомбинантной TvDAAO дикого типа и ее различных мутантных форм при повышенных значениях температуры [12][13][14]. Установлено, что в диапазоне температур от 50 до 60 °C процесс термоинактивации протекает в две стадии в соответствии с диссоциативным механизмом.…”

Section: Kinetics Of Thermoinactivation Of D-amino Acid Oxidaseunclassified

“…Плазмида была получена клонированием гена opadaao1 в плазмиду pET24a(+) по сайтам рестрикции NdeI и XhoI. Условия культивирования и очистки были идентичны описанным для TvDAAO [13,14]. Согласно данным аналитического электрофореза в полиакриламидном геле в присутствии додецилсудьфата натрия, чистота полученных препаратов составляла не менее 98%.…”

Section: получение высокоочищенных препаратов Opadaao1unclassified

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Kinetics of Thermoinactivation of D-Amino Acid Oxidase Opadaao1 From the Ogataea Parapolymorpha Dl-1 Yeast

Koshkina

SERGEYEV

FEDOROV

et al. 2023

Lomonosov Chemistry Journal

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Our earlier annotation of genome of the yeast Ogataea parapolymorpha DL-1 made it possible to identify ve genes of potential D-amino acids oxidases. All opadaao1 - opadaao5 genes were cloned and expressed in E. coli. Four OpaDAAO1- OpaDAAO4 enzymes were obtained in highly puri ed form and their catalytic properties were studied. It was found that among all DAAOs described in the literature so far, the enzyme OpaDAAO1 has the highest catalytic constant kcat with D-Ala, which makes it promising for practical applications. However, in addition to good catalytic parameters, effective application of the enzyme in practice requires high stability and knowledge of the inactivation mechanism, including at elevated temperatures. In this work we studied the effect of elevated temperatures on the stability of OpaDAAO1. The enzyme was shown to have high thermal stability in comparison with majority of other D-amino acid oxidases. The kinetics of OpaDAAO1 inactivation at different temperatures, initial concentrations of the enzyme, and in the presence of exogenous FAD were studied. A possible kinetic scheme of inactivation was proposed based on the data obtained.

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Section: Kinetics Of Thermoinactivation Of D-amino Acid Oxidaseunclassified

Section: получение высокоочищенных препаратов Opadaao1unclassified

Kinetics of Thermoinactivation of D-Amino Acid Oxidase Opadaao1 From the Ogataea Parapolymorpha Dl-1 Yeast

Koshkina

SERGEYEV

FEDOROV

et al. 2023

Lomonosov Chemistry Journal

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“…D-amino acid oxidase is also widely used in practice [ 6 , 7 , 8 , 9 ]. For example, DAAO from the yeast Trigonopsis variabilis is used in the two-enzyme biocatalytic process for the production of 7-aminocephalosporonic acid (7-ACA) from cephalosporin C [ 10 , 11 ]. This process reduces the consumption of organic solvents by 400 times compared to the previously used purely chemical method.…”

Section: Introductionmentioning

confidence: 99%

“…Оксидаза D-аминокислот также широко используется на практике [6][7][8][9]. Например, DAAO из дрожжей Trigonopsis variabilis используется ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ в двухферментном биокаталитическом процессе получения 7-аминоцефалоспорановой кислоты (7-АЦК) из цефалоспорина С [10,11]. Это позволяет сократить расход органических растворителей в 400 раз по сравнению с ранее используемым чисто химическим процессом.…”

Section: Introductionunclassified

Bioinformatics-Structural Approach to the Search for New D-Amino Acid Oxidases

et al. 2023

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D-amino acid oxidase (DAAO, EC 1.2.1.2) plays an important role in the functioning of prokaryotes as well as of lower (yeast and fungi) and higher eukaryotes (mammals). DAAO genes have not yet been found in archaean genomes. D-amino acid oxidase is increasingly used in various fields, which requires the development of new variants of the enzyme with specific properties. However, even within one related group (bacteria, yeasts and fungi, mammals), DAAOs show very low homology between amino acid sequences. In particular, this fact is clearly observed in the case of DAAO from bacteria. The high variability in the primary structures of DAAO severely limits the search for new enzymes in known genomes. As a result, many (if not most) DAAO genes remain either unannotated or incorrectly annotated. We propose an approach that uses bioinformatic methods in combination with general 3D structure and active center structure analysis to confirm that the gene found encodes D-amino acid oxidase and to predict the possible type of its substrate specificity. Using a homology search, we obtained a set of candidate sequences, modelled the tertiary structure of the selected enzymes, and compared them with experimental and model structures of known DAAOs. The effectiveness of the proposed approach for discrimination of DAAOs and glycine oxidases is shown. Using this approach, new DAAO genes were found in the genomes of six strains of extremophilic bacteria, and for the first time in the world, one gene was identified in the genome of halophilic archaea. Preliminary experiments confirmed the predicted specificity of DAAO from Natronosporangium hydrolyticum ACPA39 with D-Leu and D-Phe.

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