Cloning of Phytase Genes from Pantoea Sp. 3.5.1 and Bacillus ginsengihumi M2.11 in Pichia pastoris

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Phytases (myo-inositol hexakisphosphate hydrolase) catalyze the hydrolysis of phytate to inorganic phosphate and less phosphorylated myo-inositol derivatives and are widely used as feed additives in animal nutrition. Nevertheless, nowadays, there is a constant search for new phytases and new expression systems for better production of these important enzymes. In this study, we report cloning of two novel bacterial phytases belonging to the different enzyme classes and having different properties in the methylotrophic yeast Pichia pastoris. Sequences of agpP and phyC genes, encoding histidine acid phytase from Pantoea sp. 3.5.1 and β-propeller phytase from Bacillus ginsengihumi M2.11, respectively, were optimized and chemically synthesized according to the P. pastoris codon usage bias. The optimized genes were cloned into the yeast vectors pPINK-HC and pPINK-LC under the control of the inducible promoter AOX1 and two different signal peptides—signal sequence of α-amylase gene from Aspergillus niger and presequence of inulinase gene from Kluyveromyces maxianus. PCR analysis, restriction analysis, and DNA sequencing confirmed correct integration of agpP and phyC genes into P. pastoris genome. As a result, recombinant strains of P. pastoris with codon-optimized bacterial phytase genes (agpP and phyC), encoding histidine acid and β-propeller phytases, integrated into the genome under the alcohol oxidase promoter AOX1 and two different signal peptides, were obtained. Recombinant phytase AgpP was stably expressed and secreted into the culture medium of yeasts, whereas the expression of phyC gene was only confirmed at transcription level

Cloning and Heterologous Expression of Phytase Gene from Pantoea sp. 3.5.1

© 2016, Springer Science+Business Media New York.Phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyzes the stepwise hydrolysis of phosphate groups from phytic acid (myo-inositol hexakisphosphate) or its salt phytate. These enzymes could be potentially used for the stereospecific and efficient production of different myo-inositol phosphate isomers with therapeutic features. In the present study, we cloned the 1728 bp open reading frame encoding Pantoea sp. 3.5.1 phytase into the expression vector pET28a. The recombinant Escherichia coli BL21 pLysS pET28a/agpP strain expressing Pantoea sp. 3.5.1 AgpP phytase was obtained

Эффективность и безопасность комбинированного применения целекоксиба, диацереина и комбинации глюкозамина и хондроитина для контроля скелетно-мышечной боли, связанной с остеоартритом и неспецифической болью в спине

   The combined use of drugs with different mechanisms of action is the main principle of musculoskeletal pain control in rheumatic diseases. However, there are few studies evaluating the efficacy of this approach in real practice.Objective: to determine the efficacy and safety of the combined use of celecoxib, diacerein, and the combination of glucosamine + chondroitin in osteoarthritis (OA) and chronic nonspecific low back pain (NSLBP).   Material and methods. Statistical analysis of data obtained during a 3-month open observational study was performed. We included 1569 patients (63.6 % women and 36.4 % men, mean age 58.7 ± 11.0 years) with knee OA (kOA), hip OA (hOA), generalized OA (gOA), and chronic NSLBP with moderate/severe pain (≥ 4 on a numeric rating scale, NRS 0–10) who required nonsteroidal anti-inflammatory drugs. Celecoxib 200 mg twice daily was prescribed, with the dose reduced to 200 mg per day or taken “as needed" after significant pain relief; diacerein 50 mg twice daily; and a medication of glucosamine 250 mg and chondroitin 200 mg, 2 capsules 2–3 times daily. Outcomes were assessed after 3 months using the dynamics of pain, fatigue, dysfunction (according to NRS), and the “Patient Acceptable Symptom State” (PASS) indicator.   Results and discussion. 80.2 % of patients completed the 3 month course of treatment, 4.4 % discontinued treatment due to adverse events (AEs), and for 15.4 % of patients there was no follow-up. After 3 months of treatment ≥ 50 % decrease (from baseline) in the severity of symptoms was noted in 83.4 % of patients for pain on movement, in 83.7 % for pain at rest, in 78.6 % for pain at night, in 80.8 % for dysfunction, and in 83.4 % for fatigue. 87.7 % of patients reported PASS. There were no significant differences in treatment outcomes for different localizations of OA and NSLBP: a ≥ 50 % pain reduction in kOA was achieved in 81.6 % of patients, in hOA – in 82.2 %, in gOA – in 85.0 %, in NSLBP – in 88.1 %. AEs were registered in 350 (22.4 %) patients, the most frequent was dyspepsia (n = 280, 17.8 %), diarrhea was recorded in 37 (2.4 %) cases. No serious AEs requiring hospitalization were registered.   Conclusion. Combination therapy with celecoxib, diacerein, and a combination of glucosamine and chondroitin significantly reduces the severity of symptoms of OA and NSLBS.   Комплексное применение препаратов с различным механизмом  действия – основной принцип контроля скелетно-мышечной боли при ревматических заболеваниях. Однако имеется лишь небольшое число работ, в которых оценивается эффективность такого подхода в реальной практике.   Цель исследования – определение эффективности и безопасности сочетанного применения целекоксиба, диацереина и комбинации глюкозамин + хондроитин при остеоартрите (ОА) и хронической неспецифической боли в спине (НБС).   Материал и методы. Выполнен статистический анализ данных, полученных в ходе 3-месячного открытого наблюдательного исследования. Было включено 1569 пациентов (63,6 % женщин и 36,4 % мужчин, средний возраст – 58,7 ± 11,0 года) с ОА коленного (КС), тазобедренного (ТБС) суставов, генерализованным ОА (ГОА) и хронической НБС, испытываваших умеренную/выраженную боль (≥ 4 по числовой рейтинговой шкале, ЧРШ 0–10) и нуждавшихся в приеме нестероидных противовоспалительных препаратов. Назначались целекоксиб 200 мг 2 раза в сутки со снижением дозы до 200 мг/сут и использованием «по требованию» после значительного уменьшения боли; диацереин 50 мг 2 раза в сутки и препарат глюкозамина 250 мг и хондроитина 200 мг по 2 капсулы 2–3 раза в день. Оценка результатов проводилась через 3 мес по динамике боли, усталости, нарушения функции (по ЧРШ), а также показателю «состояние симптомов, приемлемое для пациента» (ССПП).   Результаты и обсуждение. 3-месячный курс лечения закончили 80,2 % больных, 4,4 % прервали лечение из-за нежелательных явлений (НЯ), 15,4 % выпали из-под наблюдения. Через 3 мес уменьшение выраженности ≥ 50 % по сравнению с исходным уровнем для боли при движении отмечено у 83,4 % пациентов, боли в покое – у 83,7 %, боли ночью – у 78,6 %, нарушения функции – у 80,8 %, усталости – у 83,4 %; 87,7 % пациентов указали на ССПП. Не выявлено значимых различий результатов лечения при различной локализации ОА и НБС: ≥ 50 % уменьшение боли при ОА КС достигнуто у 81,6 % больных, при ОА ТБС – у 82,2 %, при ГОА – у 85,0 %, при НБС – у 88,1 %. НЯ зарегистрированы у 350 (22,4 %) пациентов, наиболее часто встречалась диспепсия (n = 280, 17,8 %), диарея отмечалась в 37 (2,4 %) случаях. Серьезные НЯ, потребовавшие госпитализации, не зафиксированы.   Заключение. Комбинированная терапия с использованием целекоксиба, диацереина и комбинации глюкозамина и хондроитина обеспечивает существенное уменьшение выраженности симптомов ОА и НБС

Phytate-hydrolyzing rhizobacteria: abiotic stress tolerance and antimicrobial activity

© The Authors 2020. Phytate-hydrolyzing bacteria Pantoea sp. 3.1, 3.2, 3.5.2, 3.6.1 and Bacillus ginsengihumi M2.11 were previously isolated from the soil samples of the Republic of Tatarstan. The effect of cultivation conditions on the growth dynamics as well as antimicrobial activity was determined. All four Pantoea strains showed optimum growth at 26 °C and 28 °C and pH 6.0-7.0. The optimum conditions for the growth of B. ginsengihumi M2.11 strain was determined to be 26 °C, 28 °C and 37 °C and alkaline pH 7 and 8. Salt concentration in the range of 0 to 1000 mM did not significantly affect the growth of the strains. Antagonistic activity of Pantoea sp. 3.5.2 was studied against phytopathogenic micromycetes, identified as Alternaria alternata and Bipolaris sorokiniana. In the presence of bacterial isolate growth of A. alternata was inhibited by 57% and growth of B. sorokiniana - by 85%. Minor growth inhibition by Pantoea sp. 3.5.2 of gram-negative bacteria from Enterobacteriaceae family was observed. The presence of fungicidal activity in the Pantoea strain together with its ability to hydrolyze soil phytates and overcome abiotic stress factors in soil can possibly serve as the basis for the new fungicide of microbial origin

Cloning of Phytase Genes from Pantoea Sp. 3.5.1 and Bacillus ginsengihumi M2.11 in Pichia pastoris

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Phytases (myo-inositol hexakisphosphate hydrolase) catalyze the hydrolysis of phytate to inorganic phosphate and less phosphorylated myo-inositol derivatives and are widely used as feed additives in animal nutrition. Nevertheless, nowadays, there is a constant search for new phytases and new expression systems for better production of these important enzymes. In this study, we report cloning of two novel bacterial phytases belonging to the different enzyme classes and having different properties in the methylotrophic yeast Pichia pastoris. Sequences of agpP and phyC genes, encoding histidine acid phytase from Pantoea sp. 3.5.1 and β-propeller phytase from Bacillus ginsengihumi M2.11, respectively, were optimized and chemically synthesized according to the P. pastoris codon usage bias. The optimized genes were cloned into the yeast vectors pPINK-HC and pPINK-LC under the control of the inducible promoter AOX1 and two different signal peptides—signal sequence of α-amylase gene from Aspergillus niger and presequence of inulinase gene from Kluyveromyces maxianus. PCR analysis, restriction analysis, and DNA sequencing confirmed correct integration of agpP and phyC genes into P. pastoris genome. As a result, recombinant strains of P. pastoris with codon-optimized bacterial phytase genes (agpP and phyC), encoding histidine acid and β-propeller phytases, integrated into the genome under the alcohol oxidase promoter AOX1 and two different signal peptides, were obtained. Recombinant phytase AgpP was stably expressed and secreted into the culture medium of yeasts, whereas the expression of phyC gene was only confirmed at transcription level

Expression of

Phytic acid is the main storage form of organic phosphorus. Due to its structural features, phosphorus in phytate is inaccessible for assimilation by animals. Moreover, remaining inaccessible reservoir of phosphorus for animal nutrition, phytic acid is capable of forming insoluble complex salts, which lead to soil and water pollution. Мicrobial enzymes - phytases, capable of decomposing phytic acid to organic phosphorus are being used as feed additives in animal nutrition to solve this problem. Thus, search and development of technologies for the production of enzymes on an industrial scale are the most urgent. Methylotrophic yeast P. pastoris are widely used in biotechnology, as an efficient system for the recombinant proteins expression. They have many advantages, including rapid growth on inexpensive media, a wide range of molecular tools for genetic manipulation in optimizing production processes, they are safe for humans and animals, carry-out many post-translational modifications and produce recombinant proteins intracellularly or extracellularly within a short period of time. It was found that the recombinant P. pastoris strains pPINK-LC-α-MF -phyC, pPINK-HC-α-amyl -phyC, pPINK-LC-α-amyl -phyC, pPINK-HC-α-MF -phyC are able to produce and to secrete B. ginsengihumi bacterial phytase M 2.11 phyC. The maximum activity was observed in the pPINK-LC-α-MF strain – 2.6 (U / mg). Recombinant B. ginsengihumi M 2.11 phytases exhibited high activity in a wide pH range from 2.5 to 9.0. The MF-phyC-HC construction is pH stable. The temperature optimum of all recombinant phytases corresponds to 37 ° C; recombinant phytases retain their activity in the range from -80 to 90C

Cloning and Heterologous Expression of Phytase Gene from Pantoea sp. 3.5.1

© 2016, Springer Science+Business Media New York.Phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyzes the stepwise hydrolysis of phosphate groups from phytic acid (myo-inositol hexakisphosphate) or its salt phytate. These enzymes could be potentially used for the stereospecific and efficient production of different myo-inositol phosphate isomers with therapeutic features. In the present study, we cloned the 1728 bp open reading frame encoding Pantoea sp. 3.5.1 phytase into the expression vector pET28a. The recombinant Escherichia coli BL21 pLysS pET28a/agpP strain expressing Pantoea sp. 3.5.1 AgpP phytase was obtained

Cloning and Heterologous Expression of Phytase Gene from Pantoea sp. 3.5.1

© 2016, Springer Science+Business Media New York.Phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyzes the stepwise hydrolysis of phosphate groups from phytic acid (myo-inositol hexakisphosphate) or its salt phytate. These enzymes could be potentially used for the stereospecific and efficient production of different myo-inositol phosphate isomers with therapeutic features. In the present study, we cloned the 1728 bp open reading frame encoding Pantoea sp. 3.5.1 phytase into the expression vector pET28a. The recombinant Escherichia coli BL21 pLysS pET28a/agpP strain expressing Pantoea sp. 3.5.1 AgpP phytase was obtained

Cloning and Heterologous Expression of Phytase Gene from Pantoea sp. 3.5.1

© 2016, Springer Science+Business Media New York.Phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyzes the stepwise hydrolysis of phosphate groups from phytic acid (myo-inositol hexakisphosphate) or its salt phytate. These enzymes could be potentially used for the stereospecific and efficient production of different myo-inositol phosphate isomers with therapeutic features. In the present study, we cloned the 1728 bp open reading frame encoding Pantoea sp. 3.5.1 phytase into the expression vector pET28a. The recombinant Escherichia coli BL21 pLysS pET28a/agpP strain expressing Pantoea sp. 3.5.1 AgpP phytase was obtained