scholarly journals Characterization of Thermostable Amylase From Halophilic Lactobacillus Plantarum TS1

2021 ◽  
Author(s):  
Sania Riaz ◽  
Anum Fareed ◽  
Habiba Zaffar ◽  
Shafique Ur Rehman ◽  
Muhammad Jamil ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Saline Soil ◽  
Salt Range ◽  
Sds Page ◽  
Wide Range ◽  
Two Temperatures ◽  
Harsh Conditions ◽  
Thermostable Amylase ◽  
Important Enzyme

Abstract Amylase is an important enzyme use extensively in various industrial processes. It is mainly involved in the catalysis of starch that requires harsh conditions; therefore, it is required to isolate amylases with unique properties that makes it more applicable. Extremophiles are the major resource of such enzymes; therefore, amylase positive strains were isolated from the saline soil where the temperature is also exceptionally high. Five amylase positive strains were isolated from the Karak salt range, Kohat Pakistan and were identified by phylogenetic analysis. DNS based assay was employed to compare the activities of different amylases obtained from five strains while using the starch as a substrate. The amylase obtained from Lactobacillus plantarum TS1 was found to be more efficient, which was purified and characterized. The SDS-PAGE of purified amylase showed a single band with an estimated size of 10 kDa. The kinetic parameters were measured at two temperatures i.e. at 37 °C and 50 °C. The Kcat as well as the Kcat/KM were found to be high when temperature increased from 37 °C to 50 °C. Amylase was active at wide range of temperature as well as pH and work efficiently in the presence of salts and various organic solvents.

scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

scholarly journals Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)

2019 ◽  
Vol 1 (4) ◽  
pp. 8-18
Author(s):  
Samir A.M. Zaahkouk ◽  
Doaa A. Darwish ◽  
Hassan M.M. Masoud ◽  
Mohamed M. Abdel-Monsef ◽  
Mohamed S. Helmy ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Ovis Aries ◽  
Major Band ◽  
Sheep Liver ◽  
Sds Page ◽  
Commercially Important ◽  
Important Enzyme

Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.

scholarly journals Isolation and Biochemical Characterization of Cellulase Produced by Bacterial Isolates from Sugarcane Waste Soil

2021 ◽  
pp. 28-35
Author(s):  
A. U. Hassan ◽  
R. Jafaru ◽  
I. B. Mato ◽  
E. Kereakede ◽  
A. H. Galadanci ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Biochemical Characterization ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Enzyme Concentration ◽  
Microbiological Analysis ◽  
Cellulase Enzyme ◽  
Sds Page ◽  
Important Enzyme

Cellulase is one of the most economically important enzyme, which aids in catalyzing cellulolysis, the decomposition of cellulose and other related polysaccharides. So the demand/importance of this enzyme in both domestic and commercial sectors cannot be over emphasized. In this research cellulase-producing bacteria were isolated from soil around sugarcane waste dumping area, which was identified to be P. fulorescens after numerous biochemical and microbiological analysis. The bacteria were then grown and used to ferment certain biomass, with the aim of using the organisms to produce the cellulase enzyme. The total protein/cellulase enzyme activity of the medium was ascertained. Optimization/characterization for maximum cellulase activity was done by varying the temperature, pH, enzyme concentration and substrate concentration, in which the optimum condition for cellulase production was ascertain to be at a temperature and pH of 40˚C and pH 7 respectively. SDS-PAGE electrophoresis was carried out to determine and reconfirm the presence and molecular weight of the isolated enzyme. The estimated extrapolated molecular weight of the enzyme was found to be 13.5KDa.

scholarly journals Screening and Characterization of Thermostable Amylase-Producing Bacteria Isolated from Soil Samples of Afdera, Afar Region, and Molecular Detection of Amylase-Coding Gene

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Semira Nureddin Yassin ◽  
Tamene Milkessa Jiru ◽  
Meera Indracanti
Keyword(s):  
Maximum Activity ◽  
Soil Samples ◽  
Effect Of Temperature ◽  
Bacterial Isolates ◽  
Original Activity ◽  
Zone Formation ◽  
Afar Region ◽  
Wide Range ◽  
Thermostable Amylase

Studying thermostable amylase-producing bacteria in extreme environments has a crucial role to overcome different industrial challenges. Afar Region is one of the hottest and salty areas, making it the home of extremophiles. This study aimed at screening and characterizing amylase-producing bacteria isolated from soil samples of Afdera, Afar Region, and detection of their amylase-coding genes. Thus, a total of 49 bacterial isolates were obtained from the collected soil samples. Out of these, three isolates (M2, M8, and M13) were selected on the basis of diameter of the average clear zone formation and time taken to decolorize iodine solution. Based on their morphological and biochemical characteristics, the isolates were identified as genus Bacillus. PCR amplification and detection of the amylase-coding gene confirmed the presence of the amylase gene in the three bacterial isolates. Optimum amylase production time for these isolates was 48 hrs (M13 and M8) and 72 hrs (M2) corresponding to the amylase activity of 0.67 U/mL for M13, 0.74 U/mL for M8, and 0.73 U/mL for M2 with an optimum temperature of 55°C. Studies on the effect of temperature revealed that the crude enzyme had a maximum activity and stability at 75°C, 70°C, and 65°C for isolates M13, M8, and M2, respectively. Additionally, amylase produced from all isolates retained more than 66.41% of their original activity after incubating them at a temperature range from 55 to 80°C for 50 min. Optimum pH for the activity of all crude amylases was in the range from 5 to 9 with a peak activity at pH 8. Their activity decreased significantly by the presence of Zn+2 and Mg2+; however, their activity increased by the presence of Ca+2. Moreover, the three crude amylases were stable (0–3 M) with NaCl concentration. Amylases of this finding with thermophilic and halophilic characteristics offer a wide range of applications in food, brewing, textile, starch, paper, and deterrent industries. Thus, identification of these Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of amylases are recommended for effective utilization in different industries.

scholarly journals Purification and Characterization of α-amylase from a Novel Thermoalkalophilic Strain of Bacillus sonorensis GV2 Isolated from Mushroom Compost

2019 ◽  
pp. 1-14
Author(s):  
Gitanjali Vyas ◽  
Nivedita Sharma ◽  
Nisha Sharma
Keyword(s):  
Corn Starch ◽  
Gel Filtration ◽  
Mushroom Compost ◽  
Raw Starch ◽  
Peptide Mass ◽  
Sds Page ◽  
Wide Range ◽  
Bacillus Sonorensis

A novel thermoalkalophilic α-amylase producing bacterial strain Bacillus sonorensis GV2 |KJ775811.1| was isolated from mushroom compost. The purification of α-amylase was performed through different chromatography techniques. After purification with SDS-PAGE and Sephadex G-75 gel filtration, the molecular weight of monomeric α-amylase was found to be 45 kDa. The enzyme showed an optimal activity over a wide range of temperature and pH of 35-60oC and 7-11, respectively. The effect of divalent ions i.e. Mg2+ and Ca2+ showed a positive increase in enzyme activity. This enzyme is unique in a sense that it also exhibited a considerable raw corn starch hydrolyzing activity at 55oC. The end products when subjected to TLC which were identified as main maltooligosaccharides, proving the endo action of an enzyme. The Vmax and Km values of Bacillus sonorensis GV2 α-amylase were found to be 1347 μmol/mg/min and 3.46  mMol/ml. The MALDI peptide mass fingerprint analysis of the reduced and carboxymethylated amylase digested with chymotrypsin indicated that this partial amino acid sequence was homologous by a score of 6 with UDP transferalyase. All these findings suggests about the potential role of this α-amylase for raw starch degrading applications in the relevant industry.

scholarly journals ANALYSING THE BIOCATALYTIC ASPECT OF AMYLASE FROM PLANT AND MICROBIAL SOURCES FOR INDUSTRIAL APPLICATION

2020 ◽  
pp. 85-87
Author(s):  
DIGUMARTHY NIHARIKA ◽  
DIPTI SONI JAIPURIAR ◽  
VIRENDRA VAISHNAV
Keyword(s):  
Bacillus Subtilis ◽  
Enzymatic Activity ◽  
Enzyme Assay ◽  
Exchange Chromatography ◽  
Sds Page ◽  
Wide Range ◽  
Green Pea ◽  
Pharmaceutical Industries ◽  
Plant Sources ◽  
Important Enzyme

Objective: Biocatalysts have a wide role in industries and amylases are one of the best enzymes used in various industries as they stay stable physically and chemically even under environmental or physical stress. Extraction of amylase from plant sources like chickpea and green pea and also the microbial source of Bacillus subtilis. Methods: Enzymatic activity was determined by enzyme assay and purified through the following steps such as salt precipitation, dialysis, Ion-exchange chromatography and characterized using SDS PAGE. Results: Enzymatic activity of amylase produced by bacillus subtilis was determined the highest (4600 U/ml) activity and molecular weight 48.9kDa was recorded. Conclusion: Amylase is a highly important enzyme that breaks down the starch molecules into simple sugars. It has potential in a wide range of industrial processes such as food, fermentation and pharmaceutical industries. All the sources used in this study proves that the bacteria have the highest concentration of enzyme in the form of protein.

Isolation and characterization of a rare waterborne lytic phage of Salmonella enterica serovar Paratyphi B

2013 ◽  
Vol 59 (5) ◽  
pp. 318-323 ◽  
Author(s):  
Sangeeta S. Ahiwale ◽  
Ashok V. Bankar ◽  
Sujata N. Tagunde ◽  
Smita Zinjarde ◽  
Hans W. Ackermann ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Burst Size ◽  
Lytic Phage ◽  
Isolation And Characterization ◽  
Bacterial Contaminants ◽  
Sds Page ◽  
Wide Range ◽  
Paratyphi B ◽  
Drug Resistant Strains

A lytic phage of Salmonella serovar Paratyphi B, named φSPB, was isolated from surface waters of the Pavana River in India. Phage φSPB is a member of the Podoviridae family and is morphologically similar to the 7-11 phages of the C3 morphotype of tailed phages, characterized by a very long, cigar-shaped head. The head measured approximately 153 × 57 nm, and the tail size was 12 × 7 nm. The phage was stable over a wide range of pH (4–9) and temperature (4–40 °C). The adsorption rate constant was 4.7 × 10−10. Latent and eclipse periods were 10 and 15 min, respectively, and the burst size was 100 plaque-forming units/infected cell after 25 min at 37 °C. The phage DNA was 59 kb in size. Ten major proteins were observed on SDS–PAGE, although some of these proteins could be bacterial contaminants. This is the first report of Salmonella enterica subsp. enterica serovar Paratyphi B phage of C3 morphotype from India that has many unique features, such as high replication potential, short replication time, and stability over a wide range of pH and temperature, making it a promising biocontrol agent against the drug-resistant strains of Salmonella Paratyphi B.

scholarly journals Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays)

1996 ◽  
Vol 315 (3) ◽  
pp. 965-970 ◽  
Author(s):  
Philip S. SHELDON ◽  
Michael A. VENIS
Keyword(s):  
Cyclosporin A ◽  
Sequence Similarity ◽  
Terminal Sequence ◽  
Purification And Characterization ◽  
Cyclophilin B ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses ◽  
High Level

Methods for the purification and separation of peptidyl prolyl cis–trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appear as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).

scholarly journals Screening and Characterization of a Mutant Fungal Aspartic Proteinase from Mucor pusillus

2015 ◽  
Vol 9 (1) ◽  
pp. 119-126
Author(s):  
Li Yuqiu ◽  
Tan Hua ◽  
Li Da ◽  
Li Zhoulin ◽  
Chi Yanping ◽  
...  
Keyword(s):  
Cheddar Cheese ◽  
Aspartic Proteinase ◽  
Site Directed Mutagenesis ◽  
Soluble Nitrogen ◽  
Milk Clotting ◽  
Sds Page ◽  
Wide Range ◽  
Hydrolysis Of ◽  
Pepstatin A

In this study, site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet (MPR) in order to find a potential substitution of commercial chymosin. Mutant G186D/E13D screened from thousands of mutants showed a significant milk-clotting activity (MCA). Mutant G186D/E13D rennet was purified and characterized. The molecular weight was estimated to be 44 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pH (5.0-7.0) and 60ºC. The enzyme was inhibited by metal ions (Fe2+, Fe3+, Cu+ and Zn2+), 1.10-Phenantrolin and pepstatin A. Further texture analysis of types of cheddar cheese made by non-mutant rennet, mutant (G186D/E13D) rennet and commercial rennet suggested that the soluble nitrogen content and hardness of cheddar cheese made by chimeric mutant rennet was decreased without any significant change in flavor between these cheeses. The result implicated that, to some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably as a candidate for a useful milk coagulant.

scholarly journals First Characterization of Sphingomyeline Phosphodiesterase Expression in the Bumblebee, Bombus lantschouensis

Sociobiology ◽  
2017 ◽  
Vol 64 (1) ◽  
pp. 85 ◽  
Author(s):  
Lei Han ◽  
Shaoyu He ◽  
Jie Dong ◽  
Ye Wang ◽  
Jiaxing Huang ◽  
...  
Keyword(s):  
Polyclonal Antibody ◽  
Venom Gland ◽  
Polyclonal Antiserum ◽  
Wild Plants ◽  
Sphingolipid Metabolism ◽  
Larval Stages ◽  
Sds Page ◽  
High Level ◽  
Important Enzyme

  The bumblebee (Bombus lantschouensis Vogt) is an important pollinator of wild plants. Sphingomyelin phosphodiesterase (SMPD) is a hydrolase that plays a major role in sphingolipid metabolism reactions. We report the preparation and characterization of a polyclonal antibody for bumblebee SMPD. We then use the polyclonal antiserum to detect the SMPD protein at different development stages and in different tissues. Our results showed that a 1228bp fragment homologous with the B. terrestris SMPD gene was successfully amplified. The molecular weight of the fusion protein was about 70 kDa by SDS-PAGE. An effective polyclonal antibody against SMPD was also obtained from mice and found to have a higher specificity for bumblebee SMPD. Western blotting detection showed that SMPD was expressed at a high level in queen ovaries, although expression was lower in the midgut and venom gland. SMPD expression decreased from the egg stage until the pdd stage. We interpret our results as showing that the development of an effective polyclonal antiserum for the SMPD protein of a bumblebee, which provides a tool for exploring the function of the SMPD gene. In addition, the work has confirmed that SMPD should be considered as an important enzyme during bumblebee egg and larval stages.

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