Production, Purification and Characterization of Extracellular Tannase from a Newly Isolated Yeast, Geotrichum cucujoidarum

With an aim to isolate a tannase positive organism, the microbial mat growing on the stored areca extract leachate surface was screened. Once the tannase positive organism was isolated, it was identified by ITS/18S rRNA gene sequencing. Further, the enzyme was purified and examined for its biochemical properties. A potent extracellular tannase-producing yeast was isolated and was identified as Geotrichum cucujoidarum. After the shake flask studies, the enzyme activity of 4.42 U/ml and specific activity of 29.86 U/mg were achieved in a medium with tannic acid as an inducer. Later, ethanol (70%) precipitation followed by purification through FPLC using SEC 650 column resulted in 166.37 U/mg specific activity and a recovery of 50.54%. The purified enzyme was a monomer with a molecular weight of 63 kDa. The optimum pH and the temperature of the enzyme were found to be 5.0 and 30°C, respectively. The Michaelis-Menten constant (Km) was found to be 2.9 mM, and the turn over number (kcat) and catalytic efficiency (kcat/km) of the purified tannase were 102 S-1 and 35.17 mM-1S-1 respectively. Temperature and pH stability profiles of the enzyme, influence of various metal ions, chelators and surfactants on enzyme activity and kinetic constants of enzyme shows that the tannase produced from Geotrichum cucujoidarum is unique and is a potential candidate for further studies

Иммуномодулирующие свойства пробиотика на основе молочнокислых бактерий и растительного компонента

Визначено імуномодулювальну дію базової пробіотичної композиції молочнокислих бактерій на основі роду Lactobacillus, рослинного компонента карбюлози та комплексного пробіотика з карбюлозою на експериментальній моделі інтактних мишей. Встановлено, що введення до складу пробіотика карбюлози підвищувало функціональну активність клітин фагоцитарної системи, а саме поглинальну активність макрофагів (за показником фагоцитозу). Після введення інтактним мишам комплексного препарату на основі композиції молочнокислих бактерій з карбюлозою виявлено тенденцію до підвищення кількості CD19+ В-лімфоцитів, кількості CD25+-клітин у селезінці, до яких належать активовані Т- та В-лімфоцити, а також активовані макрофаги. Під впливом чистої карбюлози у селезінці дослідних мишей також підвищувалась кількість CD4+-клітин (на дев’яту добу) та CD25+-клітин (на третю та шосту добу). Отримані дані свідчать про потенційну здатність як базової композиції пробіотика, так і комплексного препарату з карбюлозою, направляти розвиток імунної відповіді по клітинному типу, важливому при захисті як від бактеріальних, так і вірусних патогенів.Purpose of this work was determination of the immunomodulatory effects of base probiotic composition of lactic acid bacteria based on genus Lactobacillus and the complex probiotic with carbyuloza on an experimental model of intact mice. It was found, that injection of carbyuloza in the probiotic composition increased functional activity of phagocytic system — namely, absorbing activity of macrophages. After injection of complex composition to intact mice, there was a tendency to increase the number of CD19+ B-lymphocytes, CD25+ cells in the spleen including activated T- and B-lymphocytes and activated macrophages. In addition, under the influence of net carbyuloza in spleens of mice the number of CD4+ cells (on 9th day), and CD25+ cells (at the third day and 6th) where up. The received data indicate the potential ability of a base composition of the probiotic and complex preparation with carbyuloza guide to the development of the immune response to cell type which is important in protecting against bacterial and viral pathogens.Определено иммуномодулирующее действие препаратов: базовой пробиотической композиции молочнокислых бактерий на основе рода Lactobacillus, растительного компонента карбюлозы и комплексного пробиотика с карбюлозой на экспериментальной модели интактных мышей. Установлено, что введение в состав пробиотика карбюлозы повышало функциональную активность клеток фагоцитарной системы, а именно поглощающую активность макрофагов (по показателю фагоцитоза). После введения интактным мышам комплексного препарата на основе композиции молочнокислых бактерий с карбюлозой выявленатенденция к увеличению количества CD19+ В-лимфоцитов, количества CD25+-клеток в селезенке, к которым относятся активированные Т- и В-лимфоциты, а также активированные макрофаги. Под влиянием чистой карбюлозы в селезенке мышей также повышалось количество CD4+-клеток (на девятые сутки)и CD25+-клеток (на третьи и шестые сутки). Полученные данные свидетельствуют о потенциальной способности как базовой композиции пробиотика, так и комплексного препарата с карбюлозой, направлять развитие иммунного ответа по клеточному типу, важном при защите как от бактериальных, так и вирусных патогенов

Production of Oxalate Oxidase from Endophytic Ochrobactrum intermedium CL6

Four oxalate degrading endophytic bacteria were isolated from oxalate rich Colocasia esculenta tubers. Based upon the oxalate oxidase (EC 1.2.3.4) activity produced in nutrient medium, one bacterium was selected and identified as Ochrobactrum intermedium by 16S rDNA sequencing. Studies on effect of nutritional and non-nutritional parameters showed that oxalate oxidase production is inducible, requires Manganese ions in the medium, and very low fill-up volume is beneficial. Shake flask fermentation carried out with medium comprising Sucrose, Ammonium chloride, Sodium oxalate along with basal salts gave 0.5 UmL-1 oxalate oxidase activity and 0.454 Umg-1specific activity after 65h of fermentation

L-Asparaginase Production using Solid-state Fermentation by an Endophytic Talaromyces pinophilus Isolated from Rhizomes of Curcuma amada

In recent times, exploration of endophytes for L-asparaginase production is gradually gaining momentum. This work deals with studies on the production of L-asparaginase from Talaromyces pinophilus, an endophytic fungus isolated from the rhizomes of Curcuma amada. L-asparaginase production was carried out by Submerged Fermentation (SmF) followed by Solid-state Fermentation (SSF). A liquid medium was designed and optimized using Plackett-Burman Design and Response Surface Methodology (RSM), under SmF. Additionally, optimal concentrations of various metal salts were incorporated in the optimized liquid medium, by one-factor-at-a-time experiments. To further enhance L-asparaginase production, SSF was carried out using Polyurethane Foam (PUF) as inert support impregnated with the optimized liquid medium. Effects of PUF cube volume, mass of PUF, moisture content, initial medium pH, and incubation temperature on the enzyme production in SSF were optimized by one-factor-at-a-time approach.L-asparaginase production enhanced from 80.8 U/mL in the unoptimized medium to 94.4 U/mL in the optimized medium under SmF. Enzyme production further increased to 120.3 U/mL under SSF by using PUF soaked in the optimized liquid medium. This study highlights the benefits of carrying out SSF with PUF, using the same liquid medium optimized for SmF - a novel approach to enhance the enzyme yield (in our case an increase of about 27% was observed). To the best of our knowledge, this is the first report on the production of L-asparaginase by both SmF and SSF, from an endophyte Talaromyces pinophilus isolated from the rhizomes of Curcuma amada

Methods available to assess therapeutic potential of fibrinolytic enzymes of microbial origin: a review

Abstract Fibrinolytic enzymes are agents administered for the treatment of myocardial infarctions, strokes, cardiac and respiratory failure. Although several microorganisms are known to produce these fibrinolytic enzymes, only a few of such enzymes, along with the age-old oral anticoagulants, have been employed in the clinical and therapeutic applications in humans. The use of these agents is associated with drawbacks such as allergic reactions and bleeding complications; therefore, it necessitates frequent monitoring of drug levels in the blood. Due to this, there is an impetus on the current effort to identify newer potential candidates from the novel microbial sources which show longer half-life, higher fibrin specificity, higher therapeutic index and lesser allergic reactions. Various methods are available for the preliminary evaluation of a potential drug candidate for the therapeutic use. Choosing the right combination of in vitro and in vivo methods would give crucial insight on the therapeutic potential of the chosen test compound. This article discusses various assay techniques, in vitro trails and in vivo models available, to help researchers in choosing right biological methods and its combinations to evaluate efficacy of potential drug candidate

Isolation and screening of endophytes from the rhizomes of some Zingiberaceae plants for L-asparaginase production

<p>Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from <i>Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium,</i> and <i>Zingiber officinale</i>; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from <i>Curcuma amada</i>, identified as <i>Talaromyces pinophilus</i>, was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. <i>Talaromyces pinophilus</i> initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family.</p

Isolation and screening of endophytes from the rhizomes of some Zingiberaceae plants for L-asparaginase production

<p>Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from <i>Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium,</i> and <i>Zingiber officinale</i>; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from <i>Curcuma amada</i>, identified as <i>Talaromyces pinophilus</i>, was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. <i>Talaromyces pinophilus</i> initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family.</p

Optimization of oxalate-free starch production from taro flour by oxalate oxidase assisted process

Taro (Colocasia esculenta) starch is known to possess unique physical and functional properties such as low amylose content, A-crystalline form, small granules, higher swelling power, etc. Due to the presence of significant amount of calcium oxalate crystals, the food industry is reluctant to explore this unique and cheap starch source for various food applications. Traditional processes utilizing various physical and chemical methods to remove oxalate content of starch inevitably change its physical and functional properties. However, using oxalate oxidase can effectively remove oxalates without altering the unique properties of starch. Hence, an attempt was made to optimize oxalate oxidase assisted starch extraction process from taro flour using response surface methodology. A central composite design comprising 20 experimental trials with 10 cube points augmented with six axial points and four replicates at the center point was applied. A mathematical model was developed to show the effect of taro flour concentration, enzyme load and incubation time on the oxalate removal. Validity of the model was experimentally verified and found that 98.3% of total oxalates can be removed under optimal conditions. This is the first report of optimization of the production of starch from taro flour using microbial oxalate oxidase. © 2020 Taylor & Francis Group, LLC