Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil

AbstractAs an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4–30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5–35 °C and pH 6.0–12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

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

Journal of Apicultural Science ◽

10.2478/jas-2020-0015 ◽

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.

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Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

Applied and Environmental Microbiology ◽

10.1128/aem.00074-09 ◽

2009 ◽

Vol 75 (15) ◽

pp. 5161-5166 ◽

Cited By ~ 26

Author(s):

I-Chien Chen ◽

Wei-De Lin ◽

Shin-Kuang Hsu ◽

Venkatesan Thiruvengadam ◽

Wen-Hwei Hsu

Keyword(s):

Specific Activity ◽

Metagenomic Library ◽

Functional Complementation ◽

Isolation And Characterization ◽

Soil Metagenome ◽

Sequence Encoding ◽

First Time ◽

Selection Agent ◽

Lysine Racemase

ABSTRACT A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.

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Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Applied and Environmental Microbiology ◽

10.1128/aem.68.9.4390-4398.2002 ◽

2002 ◽

Vol 68 (9) ◽

pp. 4390-4398 ◽

Cited By ~ 104

Author(s):

S. A. F. T. van Hijum ◽

G. H. van Geel-Schutten ◽

H. Rahaoui ◽

M. J. E. C. van der Maarel ◽

L. Dijkhuizen

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Lactobacillus Reuteri ◽

Bacterial Strains ◽

Potential Health ◽

Isolation And Characterization ◽

A Cell ◽

Encoding Gene ◽

First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

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

Marine Drugs ◽

10.3390/md18110546 ◽

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.

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Isolation and Characterization of Crotosparsamide, a New Cyclic Nonapeptide from Croton sparsiflorus

Natural Product Communications ◽

10.1177/1934578x1000501209 ◽

2010 ◽

Vol 5 (12) ◽

pp. 1934578X1000501 ◽

Cited By ~ 1

Author(s):

Rashad Mehmood ◽

Abdul Malik

Keyword(s):

Spectroscopic Data ◽

2D Nmr ◽

Spectral Studies ◽

Isolation And Characterization ◽

First Time

Crotosparsamide (1), a new cyclic nonapeptide, has been isolated from the n-butanol soluble sub-fraction of Croton sparsiflorus along with p-hydroxy methylcinnamate and kaempferol, which are reported for the first time from this species. Their structures were determined by chemical and spectral studies including ESIMS, and 1D and 2D NMR spectroscopic data.

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Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

Biochemical Journal ◽

10.1042/bj2690013 ◽

1990 ◽

Vol 269 (1) ◽

pp. 13-18 ◽

Cited By ~ 39

Author(s):

Y Homma ◽

Y Emori ◽

F Shibasaki ◽

K Suzuki ◽

T Takenawa

Keyword(s):

Phospholipase C ◽

Column Chromatography ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Deae Cellulose ◽

Isolation And Characterization ◽

Delta Type ◽

Hydrolysis Of ◽

Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

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Purification and characterization of a cold-active protease from psychrotrophicSerratia marcescensAP3801

Journal of the American Oil Chemists Society ◽

10.1007/s11746-997-0240-8 ◽

1997 ◽

Vol 74 (11) ◽

pp. 1377-1383 ◽

Cited By ~ 10

Author(s):

Yasutaka Morita ◽

Kenji Kondoh ◽

Quamrul Hasan ◽

Toshifumi Sakaguchi ◽

Yuji Murakami ◽

...

Keyword(s):

Purification And Characterization ◽

Cold Active ◽

Active Protease

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The Quest for Phenolic Compounds from Macroalgae: A Review of Extraction and Identification Methodologies

Biomolecules ◽

10.3390/biom9120847 ◽

2019 ◽

Vol 9 (12) ◽

pp. 847 ◽

Cited By ~ 5

Author(s):

Sónia A. O. Santos ◽

Rafael Félix ◽

Adriana C. S. Pais ◽

Sílvia M. Rocha ◽

Armando J. D. Silvestre

Keyword(s):

Phenolic Compounds ◽

Biological Activities ◽

Mass Spectrometry Data ◽

Brown Macroalgae ◽

Isolation And Characterization ◽

Analytical Strategies ◽

Critical Revision ◽

Identification Techniques ◽

First Time

The current interest of the scientific community for the exploitation of high-value compounds from macroalgae is related to the increasing knowledge of their biological activities and health benefits. Macroalgae phenolic compounds, particularly phlorotannins, have gained particular attention due to their specific bioactivities, including antioxidant, antiproliferative, or antidiabetic. Notwithstanding, the characterization of macroalgae phenolic compounds is a multi-step task, with high challenges associated with their isolation and characterization, due to the highly complex and polysaccharide-rich matrix of macroalgae. Therefore, this fraction is far from being fully explored. In fact, a critical revision of the extraction and characterization methodologies already used in the analysis of phenolic compounds from macroalgae is lacking in the literature, and it is of uttermost importance to compile validated methodologies and discourage misleading practices. The aim of this review is to discuss the state-of-the-art of phenolic compounds already identified in green, red, and brown macroalgae, reviewing their structural classification, as well as critically discussing extraction methodologies, chromatographic separation techniques, and the analytical strategies for their characterization, including information about structural identification techniques and key spectroscopic profiles. For the first time, mass spectrometry data of phlorotannins, a chemical family quite exclusive of macroalgae, is compiled and discussed.

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Isolation and characterization of two lytic cold-active bacteriophages infecting Pseudomonas fluorescens from the Napahai plateau wetland

Canadian Journal of Microbiology ◽

10.1139/cjm-2017-0572 ◽

2018 ◽

Vol 64 (3) ◽

pp. 183-190 ◽

Cited By ~ 3

Author(s):

Yingying Xiang ◽

Shuang Wang ◽

Jiankai Li ◽

Yunlin Wei ◽

Qi Zhang ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Flood Control ◽

Long Tail ◽

Ph Values ◽

Plateau Wetland ◽

Isolation And Characterization ◽

Adaptation Mechanisms ◽

The Earth ◽

Cold Active

As the “kidneys of the Earth”, wetlands play important roles as biodiversity reservoirs, in water purification, and in flood control. In this study, 2 lytic cold-active bacteriophages, named VW-6S and VW-6B, infecting Pseudomonas fluorescens W-6 cells from the Napahai plateau wetland in China were isolated and characterized. Electron microscopy showed that both VW-6S and VW-6B had an icosahedral head (66.7 and 61.1 nm, respectively) and a long tail (8.3 nm width × 233.3 nm length and 11.1 nm width × 166.7 nm length, respectively). The bacteriophages VW-6S and VW-6B were classified as Siphoviridae and had an approximate genome size of 30–40 kb. The latent and burst periods of VW-6S were 60 and 30 min, whereas those of VW-6B were 30 and 30 min, respectively. The optimal pH values for the bacteriophages VW-6S and VW-6B were 8.0 and 10.0, respectively, and their activity decreased rapidly at temperatures higher than 60 °C. These cold-active bacteriophages provide good materials for further study of cold-adaptation mechanisms and interaction with the host P. fluorescens.

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Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

Eukaryotic Cell ◽

10.1128/ec.00352-08 ◽

2009 ◽

Vol 8 (4) ◽

pp. 595-605 ◽

Cited By ~ 60

Author(s):

Michael R. Botts ◽

Steven S. Giles ◽

Marcellene A. Gates ◽

Thomas R. Kozel ◽

Christina M. Hull

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Critical Role ◽

Spore Formation ◽

Yeast Cells ◽

Isolation And Characterization ◽

Specific Configuration ◽

First Time

ABSTRACT Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

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