scholarly journals Biodegradable properties of organophosphorus insecticides by the potential probiotic Lactobacillus plantarum WCP931 with a degrading gene (opdC)

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Jin Hwan Lee ◽  
Hee Yul Lee ◽  
Du Yong Cho ◽  
Min Ju Kim ◽  
Jea Gack Jung ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Minimal Medium ◽  
Methyl Parathion ◽  
Relative Activity ◽  
Maximum Activity ◽  
Traditional Food ◽  
Organophosphorus Insecticides ◽  
Significant Survival ◽  
Sds Page ◽  
Biodegradable Properties

AbstractAn organophosphorus (OP) insecticide-mineralizing strain, Lactobacillus plantarum WCP931, harboring a new OP hydrolase (opdC) gene, was isolated during kimchi (Korean traditional food) fermentation. Strain WCP931 exhibited a significant survival rate of 51 to 96% under artificial gastric acid conditions at pH 2 to 3 after 3 h. The opdC gene, consisting of 831 bp encoding 276 amino acids, was cloned from strain WCP907. Recombinant Escherischia coli harboring the opdC gene depleted 77% chlorpyrifos (CP) in M9 minimal medium after 6 days of incubation. The OpdC enzyme represents a novel member of the GHSQG family of esterolytic enzymes or a new Opd group. The OpdC molecular mass was estimated to be approximately 31 kDa by SDS-PAGE and showed maximum activity at pH 6 and 35 °C. The mutated OpdC (Ser116 → Ala116) enzyme had no activity towards OP insecticides and ρ-nitrophenol-β-butyrate. Importantly, the relative activity of OpdC protein against chlorpyrifos, coumafos, diazinon, fenamifos, methyl parathion, and parathion was higher than that against cadosafos, dyfonate, and ethoprofos insecticides. These results suggested the involvement of OpdC in the biodegradation of OP insecticide-contaminated cabbage during fermentation. The new OpdC enzyme expands the heterogeneity of the lactic acid bacterial Opd enzyme group in nature.

scholarly journals Cloning, expression, and functional characterization of an organophosphates insecticides degrading gene (opdC) from a potential probiotic Lactobacillus plantarum WCP931

2020 ◽  
Author(s):  
Md. Azizul Haque ◽  
Hee Yul Lee ◽  
Du Yong Cho ◽  
Jin Hwan Lee ◽  
Chung Eun Hwang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Survival Rate ◽  
Substrate Specificity ◽  
Functional Characterization ◽  
Relative Activity ◽  
Maximum Activity ◽  
Significant Survival ◽  
Sds Page ◽  
Probiotic Lactobacillus

Abstract An organophosphorus (OP) insecticides degrading strain Lactobacillus plantarum WCP931, harboring OP hydrolase (OpdC) gene, was isolated during kimchi fermentation. The strain WCP931 appeared a significant survival rate of 51 to 96% under the artificial gastric acidic condition at pH 2 to 3 after 3 h. The opdC gene consisting 831 bp encoding 276 amino acids was cloned from the strain WCP907. The recombinant Escherischia coli harboring opdC gene depleted 77% chlorpyrifos (CP) in M9 medium after 6 days of incubation. The OpdC enzyme represents a novel member of GHSQG family of esterolytic enzymes or new Opd group. The OpdC molecular mass was estimated to be approximately 31 kDa in SDS-PAGE and showed maximum activity at 40 οC with pH 6. However, the mutated OpdC (Ser116 → Ala116) enzyme had no activity towards OP insecticides and ρ-nitrophenol-β-butyrate. Importantly, relative activity of OpdC against P-O bond insecticides was higher than P-S bond insecticide, which indicated its broad substrate specificity. It is suggested that opdC gene of strain WCP931 play role for the biodegradation of OP insecticides during kimchi fermentation.

scholarly journals Screening for the Production and Characterization of L-asparaginase from Endophytic Fungi

2021 ◽  
Vol 8 (5) ◽  
pp. 168-173
Author(s):  
Harjeet Singh ◽  
Shweta Sao
Keyword(s):  
Lymphoblastic Leukemia ◽  
Fungal Endophytes ◽  
Erwinia Carotovora ◽  
Maximum Activity ◽  
Phenol Red ◽  
Human Beings ◽  
Ph Indicator ◽  
Carbon And Nitrogen ◽  
Sds Page ◽  
Secondary Neoplasm

L-asparaginase (EC 3.5.11. L-asparagine amidohydrolase) is first enzyme, studied very intensively in human beings with regard to its anti-tumor potential against tumor of lymphoid precursor, acute lymphoblastic leukemia (ALL). The current drugs are suffering from many side effects like immune suppression, infertility, secondary neoplasm. The immunogenic complications associated with its present microbial sources Escherichia coli; Erwinia carotovora limits its medicinal frontier. So there exists a need of switching to novel natural sources to serve as non-immunogenic and better production sources of L-asparaginase. In the present study, four cultures of fungal endophytes viz. TSF-1, TSF-2, TSF-3 and TSF-4 selected on the basis of primary and secondary screening was carried on with L-asparagine as a sole carbon and nitrogen source and phenol red as pH indicator. The maximum protein content was observed to be present in TSF-2 i.e. 2.727 mg /mL and possessed maximum activity of 6.054 Units/ml. Sample was separated by SDS-PAGE, stained by silver staining, showed a single band with molecular weight of approximately ~45kDa.

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 CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

2015 ◽  
Vol 16 (1) ◽  
pp. 31
Author(s):  
Kusdianawati Kusdianawati ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Bugi Ratno Budiarto ◽  
Fatimah Fatimah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Leader Peptide ◽  
Human Consumption ◽  
Mature Peptide ◽  
Expression And Purification ◽  
Food Preservative ◽  
E Coli ◽  
Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from <em>Lactobacillus plantarum</em> S34 in <em>Escherichia coli</em>. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. <em>plantarum</em> S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. <em>plantarum</em> S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. <em>coli</em> BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

Purification and properties of glutamate-phenylpyruvate aminotransferase from the ruminal protozoan Entodinium caudatum

2004 ◽  
Vol 55 (9) ◽  
pp. 991
Author(s):  
Md. Ruhul Amin ◽  
Ryoji Onodera ◽  
R. Islam Khan ◽  
R. John Wallace ◽  
C. Jamie Newbold
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Maximum Activity ◽  
Purification And Properties ◽  
Sds Page ◽  
Wide Range ◽  
S 100 ◽  
A Cell

Entodinium species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify, and investigate properties of one of the enzymes involved in amino acid metabolism by Entodinium caudatum, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 74-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with phenyl-superose, DEAE-Toyopearl 650M, Sephacryl S-100 HR, and Sephadex G-100. The molecular mass of GPA was estimated by SDS–PAGE to be 65.0 kDa. The optimum pH was 6.0 and it was found to be reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, and SDS, EDTA, and some heavy metal ions also inhibited activity. The purification and characterisation of the enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by Entodinium caudatum.

Stress tolerance in Lactobacillus plantarum LMF6 isolated from human breast milk

2020 ◽  
Vol 9 (5) ◽  
pp. 173-184
Author(s):  
Fatima Zohra Baghdad Belhadj ◽  
Faiza Boublenza ◽  
Nour-Eddine Karam
Keyword(s):  
Breast Milk ◽  
Lactobacillus Plantarum ◽  
Food Industry ◽  
Stress Proteins ◽  
Acid Stress ◽  
Human Breast Milk ◽  
Human Breast ◽  
Beneficial Effects ◽  
Sds Page ◽  
Extreme Heat Stress

Lactobacillus plantarum is a lactic acid bacterium widely used in the food industry because of its beneficial effects on human health and its ability of adaptation to different stress conditions, hence the purpose of this work was to study the adaptation abilities of Lactobacillus plantarum LM6 and stress proteins involved during this adapta on. Lb. plantarum LMF6 was isolated from human breast milk and was exposed to acid, alkaline, thermal, oxidative, osmotic, detergent and nutritional stresses in order to determine their effects on growth, viability, tolerance and mortality. SDS-PAGE electrophoresis allowed us to compare the total proteins in the absence and in the presence of stress then the ImageJ® so ware analyzed the obtained pro les. The results show that Lb. plantarum LMF6 is highly tolerant to osmo c (at 9% NaCl, the UFC number is 3.4×1010 UFC/ml), alkaline (4.7×107UFC/ml at pH10), detergent (the UFC number is close to the control), oxydative (3.3×108 UFC/ml), nutri onnal (5.2×107 UFC/ml), acid (pH5, pH4 and pH3) and heat (40°C, 45°C and 50°C with 1.45×1011, 2.78×109 and 2.80×108UFC/ml respec vely) stresses, but sensi ve to extreme acid stress (pH1 and pH2 with mortality rate variable from 5log to 10log) and extreme heat stress (55°C and 60°C when mortality increases to 8log at 60°C). Comparison of proteins profiles allowed us to see quantitative and qualitative differences. Our results allowed to say that Lb. plantarum LMF6 showed interesting characteristics and could be used in food industry as probio c lactobacilli.

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 Laundry Detergent Compatibility of Papain Like Protease Purified From Piper Betel Leaves

2018 ◽  
Vol 7 (3.3) ◽  
pp. 132
Author(s):  
A Mousami Shankar ◽  
Dr G.V.D. Sirisha ◽  
Dr K. Vijaya Rachel
Keyword(s):  
Laundry Detergent ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Native Page ◽  
Chromatographic Techniques ◽  
Detergent Compatibility ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
The Stability ◽  
Better Than

Enzymes have wide applications in detergent industry from early 1900’s. Mostly, clothes are soiled by protein based grime. Most of the detergents have either amylase / protease. Various sources were scrutinized for potent protease activity and Betel leaves were selected, the enzyme was then isolated, purified to homogeneity by ammonium sulphate precipitation, DEAE-Cellulose and gel permeation chromatographic techniques. The enzyme was monomeric in nature with a molecular mass of 38kDa as determined by native PAGE and SDS-PAGE. The enzyme shows maximum activity at 60oC and pH 4.0. The Km and Vmax of the enzyme were 4x10-3M and 54µmol/min/mg respectively. The enzyme was categorically inhibited by PCMB and iodo-acetamide suggesting it to have papain like nature. The stability of the enzyme is assessed over the stretch of alkaline pH and temperature. This evaluation validates the stability of the enzyme and its use in detergent formulations. It was evident that after adding the enzyme preparation the stains (tea, chocolate, blood) were removed much better than that of the controls, which affirms that papain like enzyme from betel leaves, enhances detergent activity.  

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.

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