scholarly journals Characterization of a Robust and pH-Stable Tannase from Mangrove-Derived Yeast Rhodosporidium diobovatum Q95

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 546
Author(s):  
Jie Pan ◽  
Ni-Na Wang ◽  
Xue-Jing Yin ◽  
Xiao-Ling Liang ◽  
Zhi-Peng Wang
Keyword(s):  
Gallic Acid ◽  
Tannic Acid ◽  
Specific Activity ◽  
Maximum Activity ◽  
Sds Page ◽  
Rhodosporidium Diobovatum ◽  
Bacteria And Fungi ◽  
Ph Range ◽  
First Time

Tannase plays a crucial role in many fields, such as the pharmaceutical industry, beverage processing, and brewing. Although many tannases derived from bacteria and fungi have been thoroughly studied, those with good pH stabilities are still less reported. In this work, a mangrove-derived yeast strain Rhodosporidium diobovatum Q95, capable of efficiently degrading tannin, was screened to induce tannase, which exhibited an activity of up to 26.4 U/mL after 48 h cultivation in the presence of 15 g/L tannic acid. The tannase coding gene TANRD was cloned and expressed in Yarrowia lipolytica. The activity of recombinant tannase (named TanRd) was as high as 27.3 U/mL. TanRd was purified by chromatography and analysed by SDS-PAGE, showing a molecular weight of 75.1 kDa. The specific activity of TanRd towards tannic acid was 676.4 U/mg. Its highest activity was obtained at 40 °C, with more than 70% of the activity observed at 25–60 °C. Furthermore, it possessed at least 60% of the activity in a broad pH range of 2.5–6.5. Notably, TanRd was excellently stable at a pH range from 3.0 to 8.0; over 65% of its maximum activity remained after incubation. Besides, the broad substrate specificity of TanRd to esters of gallic acid has attracted wide attention. In view of the above, tannase resources were developed from mangrove-derived yeasts for the first time in this study. This tannase can become a promising material in tannin biodegradation and gallic acid production.

scholarly journals Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua)

2018 ◽  
pp. 52-58
Keyword(s):  
Specific Activity ◽  
Chenopodium Quinoa ◽  
Maximum Activity ◽  
Alpha Amylase ◽  
Partial Purification ◽  
Chemical Hydrolysis ◽  
Germination Process ◽  
Sds Page ◽  
Global Population

Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua) Partial Purification and Characterization of Alpha Amylase from germinated grains from Chenopopdium quinoa (Quinua) Melissa Bedón Gómez, Oscar Nolasco Cárdenas, Carlos Santa Cruz C. y Ana I. F. Gutiérrez Román Universidad Nacional Federico Villarreal, Facultad de Ciencias Naturales y Matemática, Laboratorio de Bioquímica y Biología Molecular, Jr. Río Chepén S/N, El Agustino. Telefax: 362 - 3388 DOI: https://doi.org/10.33017/RevECIPeru2013.0007/ Resumen Las alfa amilasas son las enzimas más estudiadas e importantes en el campo biotecnológico e industrial; ya que han reemplazado por completo la hidrólisis química del almidón. Estas enzimas son imprescindibles en la elaboración de productos alimenticios, combustibles, medicamentos y detergentes con la finalidad de optimizar procesos y conservar el medio ambiente. La α-amilasa puede ser purificada de diferentes organismos como plantas, animales, hongos y bacterias; actualmente un gran número de α-amilasas bacterianas en especial del género Bacillus están disponibles comercialmente y son las más utilizadas en las industrias. Sin embargo, la producción de éstas no satisfacen los requerimientos industriales en el mundo; ya que, la demanda de esta enzima se ha incrementado en los últimos dos años y el empleo de α-amilasas bacterianas ha provocado alergias afectando al 15% de la población a nivel mundial. . En este estudio, como fuente de α-amilasa se emplearon semillas de Chenopodium quinoa (quinua) var hualhuas blanca durante el proceso de germinación; esta enzima fue parcialmente purificada por precipitación con sulfato de amonio obteniendo una actividad específica final de 35.60U/mg y un grado de purificación de 5 veces. La purificación fue confirmada por SDS-PAGE, encontrando un peso molecular de 44kDa. La actividad enzimática se evaluó mediante el método de Miller mostrando máxima actividad a pH 7 y a temperatura de 37ºC. La linealización de Lineweaver-Burk nos dio un Km de 16mg/mL y Vmax de 100µM de maltosa/min. Por lo tanto, esta caracterización reúne los pre-requisitos necesarios para la aplicación en la industria. Descriptores: Chenopodium quinoa, alfa amilasa, germinación, purificación parcial. Abstract The alpha amylases are the enzymes most studied and important in biotechnology and industry; because they have completely replaced the starch’s chemical hydrolysis. These enzymes are essential in the food production, medicines and detergents in order to optimize processes and conserve the environment. The α-amylase can be isolated from different organisms such as plants, animals, fungi and bacteria, now a large number of bacterial α-amylases especially from genus Bacillus are commercially available and they are the most used in industry. However, the production of these do not meet industry requirements in the world, because the demand for this enzyme has increased in the last two years and the use of bacterial α-amilase has caused allergies affecting the 15% of the global population. In this study, as a source of α-amylase used the seeds from Chenopodium quinoa (quinoa). Var. white hualhuas during the germination process, this enzyme was partially purified by ammonium sulfate precipitation to obtain a final specific activity of 35.60U/mg, and a grade of purification of 5 times. The purification was confirmed by SDS-PAGE, where the molecular weight was 44kDa. The enzyme activity was evaluated by Miller method showing maximum activity at pH 7 and 37ºC. The Lineweaver-Burk linearization shows a Km of 16mg/mL and Vmax of 100μM the maltose / min. Therefore, these characterizations meet the prerequisites need for industry. Keywords: Chenopodium quinoa; alpha amylase; germination; partial purification

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

scholarly journals Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1800 ◽  
Author(s):  
Nurfadhilah Eko Sukohidayat ◽  
Mohammad Zarei ◽  
Badlishah Baharin ◽  
Mohd Manap
Keyword(s):  
Phase System ◽  
Two Phase ◽  
Aqueous Two Phase System ◽  
Process Characteristics ◽  
Sds Page ◽  
Ph Range ◽  
Lipase Purification ◽  
Triton X ◽  
First Time

Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30–60 °C and pH range of 6–11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na+, Mg2+, K+ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

scholarly journals Purification, Characterization of L-Methioninase from Candida tropicalis, and Its Application as an Anticancer

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Mohsen Helmy Selim ◽  
El-Zahraa Karm Eldin ◽  
Moataza Mahmoud Saad ◽  
El-Sayed Eliwa Mostafa ◽  
Yosrea Hassan Shetia ◽  
...  
Keyword(s):  
Candida Tropicalis ◽  
Anticancer Agent ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumor Cell Lines ◽  
Sds Page ◽  
Optimum Ph ◽  
Ph Range

The aim of the present study is to purify L-methioninase from Candida tropicalis 34.19-fold with 27.98% recovery after ion exchange chromatography followed by gel filtration. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular weight of 46 KDa. Its optimum temperature was 45 to 55 and thermal stability was 55°C for 15 min. The enzyme had optimum pH at 6.5 and stability at a pH range of 5.5 to 7.0 for 24 hr. The maximum activity was observed with substrate concentration of 30 µM and Km was 0.5 mM. The enzyme was strongly inhibited by Cd+2 and Cu+2 while it was enhanced by Na+, Ni+2, and Mg+2 at 10 mM while Ca+2 had slight activation at 20 mM. In addition, the potential application of the L-methioninase as an anticancer agent against various types of tumor cell lines is discussed.

scholarly journals (458) pH Optima for Myrosinase Activity in Crude Extracts from Cruciferous Crops

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1037B-1037
Author(s):  
Craig S. Charron ◽  
Carl E. Sams
Keyword(s):  
Plant Pathogens ◽  
Specific Activity ◽  
Degradation Products ◽  
Maximum Activity ◽  
Brussels Sprouts ◽  
Significant Interest ◽  
Ph Range ◽  
Citrate Phosphate ◽  
Myrosinase Activity

There has been significant interest in the glucosinolate-myrosinase system in plants of the Brassicaceae due to accumulating evidence that some glucosinolate degradation products are anticarcinogenic and/or suppressive to plant pathogens. Because glucosinolate hydrolysis is catalyzed by endogenous myrosinase, characterization of myrosinase activity is important for elucidating the potential bioactivity of crop glucosinolates. We measured the specific activity in citrate-phosphate buffer extracts across the pH range 4.5–6.5 of two cultivars each of five Brassica groups grown during two fall and two spring seasons. Specific activity in two kale cultivars was highly variable, but tended to have highest activity from pH 5.0–6.0. In both cauliflower cultivars from Fall 2000, Fall 2001, and Spring 2002, optimal pH was around pH 6.0. In Spring 2000, however, specific activity was highest at pH 5.0. Maximum specific activity in both cabbage cultivars occurred in the pH range 5.5–6.0 in Fall 2000, Fall 2001, and Spring 2002. In Spring 2000, specific activity in `Red Acre' cabbage was uniform across the range pH 4.5–5.5 and maximum specific activity was at pH 5.0 for `Early Round Dutch' cabbage. Both brussels sprouts cultivars had pH maxima around pH 5.5–6.0 and significantly lower activity at pH 4.5. Specific activity in broccoli was much like that of cauliflower in that highest activity occurred around pH 5.5–6.0 in Fall 2000, Fall 2001, and Spring 2002, but in Spring 2000, maximum activity was at pH 5.0. These results indicate that in most cases, pH optima were in the range 5.5–6.0, but varied somewhat with season and genotype.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

scholarly journals Biochemical Isolation and Characterization of Hyaluronidase Enzyme from Venom of Egyptian Honey Bee Apis Mellifera Lamarckii

2020 ◽  
Vol 64 (1) ◽  
pp. 153-164
Author(s):  
Mohammed M. Abdel-Monsef ◽  
Hind A. Zidan ◽  
Doaa A. Darwish ◽  
Hassan M. Masoud ◽  
Mohamed S. Helmy ◽  
...  
Keyword(s):  
Specific Activity ◽  
Deae Cellulose ◽  
Bee Venom ◽  
Native Form ◽  
Native Page ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Wide Range ◽  
First Time

AbstractThe hyaluronidase enzyme has been used in many such fields of medicine as ophthalmology, orthopaedia, internal medicine, gynecology, surgery, oncology and dermatology. In this study, the hyaluronidase enzyme was purified and characterized for the first time from Egyptian bee venom homogeneously using DEAE-cellulose and Sephacryl S-300 columns. Bee venom hyaluronidase specific activity was 411.7 units/mg protein with 49.9% yield and 3.23-fold purification. The molecular weight of the purified bee venom hyaluronidase native form was 37 kDa. The purified enzyme was found homogeneous on native PAGE and SDS-PAGE, with two congruent subunits of 18.4 kDa and isoelectric point (pI) of 8.6–8.8. The enzyme was found to be stable over a wide range of temperature (20–60°C) and pH (4.5–6.5), and its optimum activity at 37°C, pH 5.4 and 0.15 M NaCl. Km for bee venom hyaluronidase was 0.029 mg/ml hyaluronic acid and its activity was elevated in presence of MgCl2 and ZnCl2 and lowered in presence of FeCl2. Heparin inhibited the hyaluronidase enzyme noncompetitively with a Ki value of 2.9 units heparin and one binding site on the enzyme molecule.

scholarly journals Purification and characterization of GlcNAc-6-P 2-epimerase from Escherichia coli K92.

2007 ◽  
Vol 54 (2) ◽  
pp. 387-399 ◽  
Author(s):  
Miguel Angel Ferrero ◽  
Honorina Martínez-Blanco ◽  
Federico Felino Lopez-Velasco ◽  
Carlos Ezquerro-Sáenz ◽  
Nicolas Navasa ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Metabolic Flux ◽  
Maximum Activity ◽  
Logarithmic Phase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Inducible Protein ◽  
First Time

N-Acetylmannosamine (ManNAc) is the first committed intermediate in sialic acid metabolism. Thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. In prokaryotic organisms, UDP-N-acetylglucosamine (GlcNAc) 2-epimerase and GlcNAc-6-P 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc-6-P, respectively. We have purified for the first time native GlcNAc-6-P 2-epimerase from bacterial source to apparent homogeneity (1 200 fold) using Butyl-agarose, DEAE-FPLC and Mannose-6-P-agarose chromatography. By SDS/PAGE the pure enzyme showed a molecular mass of 38.4 +/- 0.2 kDa. The maximum activity was achieved at pH 7.8 and 37 degrees C. Under these conditions, the K(m) calculated for GlcNAc-6-P was 1.5 mM. The 2-epimerase activity was activated by Na(+) and inhibited by mannose-6-P but not mannose-1-P. Genetic analysis revealed high homology with bacterial isomerases. GlcNAc-6-P 2-epimerase from E. coli K92 is a ManNAc-inducible protein and is detected from the early logarithmic phase of growth. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes the biosynthesis of sialic acid, GlcNAc-6-P 2-epimerase plays a catabolic role. When E. coli grows using ManNAc as a carbon source, this enzyme converts the intracellular ManNAc-6-P generated into GlcNAc-6-P, diverting the metabolic flux of ManNAc to GlcNAc.

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