scholarly journals Expression and Biochemical Characterization of a Novel Marine Chitosanase from Streptomyces niveus Suitable for Preparation of Chitobiose

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 300
Author(s):  
Tong Chen ◽  
Gong Cheng ◽  
Siming Jiao ◽  
Lishi Ren ◽  
Chuanfang Zhao ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Kinetic Parameters ◽  
Sequence Alignment ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Degree Of Polymerization ◽  
E Coli ◽  
Hydrolysis Products ◽  
Apparent Kinetic Parameters

It is known that bioactivities of chitooligosaccharide (COS) are closely related to the degree of polymerization (DP); therefore, it is essential to prepare COS with controllable DP, such as chitobiose showing high antioxidant and antihyperlipidemia activities. In this study, BLAST, sequence alignment and phylogenetic analysis of characterized glycoside hydrolase (GH) 46 endo-chitosanases revealed that a chitosanase Sn1-CSN from Streptomyces niveus was different from others. Sn1-CSN was overexpressed in E. coli, purified and characterized in detail. It showed the highest activity at pH 6.0 and exhibited superior stability between pH 4.0 and pH 11.0. Sn1-CSN displayed the highest activity at 50 °C and was fairly stable at ≤45 °C. Its apparent kinetic parameters against chitosan (DDA: degree of deacetylation, >94%) were determined, with Km and kcat values of 1.8 mg/mL and 88.3 s−1, respectively. Cu2+ enhanced the activity of Sn1-CSN by 54.2%, whereas Fe3+ inhibited activity by 15.1%. Hydrolysis products of chitosan (DDA > 94%) by Sn1-CSN were mainly composed of chitobiose (87.3%), whereas partially acetylated chitosan with DDA 69% was mainly converted into partially acetylated COS with DP 2-13. This endo-chitosanase has great potential to be used for the preparation of chitobiose and partially acetylated COS with different DPs.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

2018 ◽  
Author(s):  
Krithika Rajagopalan ◽  
Jonathan Dworkin
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Bacterial Genomes ◽  
Phosphatase Inhibitors ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

2021 ◽  
Author(s):  
Gregory S Bulmer ◽  
Fang Wei Yuen ◽  
Naimah Begum ◽  
Bethan S Jones ◽  
Sabine S Flitsch ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Enzyme Specificity ◽  
Fungal Enzymes ◽  
Glycoside Hydrolase Family 43 ◽  
Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

2018 ◽  
Vol 43 (6) ◽  
pp. 638-650
Author(s):  
Ruth Ololade Amiola ◽  
Adedeji Nelson Ademakinwa ◽  
Zainab Adenike Ayinla ◽  
Esther Nkechi Ezima ◽  
Femi Kayode Agboola
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Biochemical Properties ◽  
Subunit Molecular Weight ◽  
Cyanoalanine Synthase ◽  
Germinating Seeds ◽  
Sorghum Bicolor L

Abstract Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Presence of NDM in non-E. coli Enterobacteriaceae in the poultry production environment

2019 ◽  
Vol 74 (8) ◽  
pp. 2209-2213 ◽  
Author(s):  
Rongmin Zhang ◽  
Jiyun Li ◽  
Yang Wang ◽  
Jianzhong Shen ◽  
Zhangqi Shen ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Antibiotic Resistance Genes ◽  
Comparative Genomic ◽  
Poultry Production ◽  
Production Environment ◽  
Genomic Context ◽  
E Coli ◽  
Commercial Chicken ◽  
Chicken Farm

Abstract Objectives Characterization of non-Escherichia coli NDM-carrying Enterobacteriaceae in the poultry production environment. Methods A total of 36 NDM-positive Enterobacteriaceae (22 Klebsiella pneumoniae, 13 Enterobacter cloacae and 1 Salmonella enterica) were isolated from a chicken farm and WGS was conducted using Illumina Hiseq2500. The genomic characterization of the isolates acquired through WGS analysis included the genomic context-flanking blaNDM genes, MLST, the antibiotic resistance genes (ARGs) and replicon types of plasmids. WGS information for another 73 K. pneumoniae isolates from different sources was retrieved from GenBank and then combined with isolates in this study for comparative genomic and phylogenetic analysis. Results Three types of genetic environment carrying blaNDM were identified in 36 non-E. coli Enterobacteriaceae isolates. Sequence comparison analysis indicated these genetic environments were completely identical to our previous findings. WGS further revealed three major types of plasmids (IncFIB, IncX3 and IncFII) from these isolates and the phylogenetic analysis suggested several K. pneumoniae isolates with ST11, ST37 and ST147 from the commercial chicken farm that were closely related to isolates of human origin. Conclusions The blaNDM-harbouring genetic contexts were identified not only in E. coli, but also in K. pneumoniae, E. cloacae and S. enterica, which may indicate that blaNDM has been widely disseminated to non-E. coli Enterobacteriaceae species in animal farms. The close relationship of K. pneumoniae isolates from different origins suggests they could serve as a key vehicle for the transfer of ARGs between humans and food animal production environments.

scholarly journals Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

2016 ◽  
Vol 11s1 ◽  
pp. ACI.S40292
Author(s):  
Tom Manczak ◽  
Henrik Toft Simonsen
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Radioactive Isotopes ◽  
Published Data ◽  
Enzymatic Characterization ◽  
Turnover Number ◽  
Terpene Synthases ◽  
Thapsia Garganica ◽  
Insight Into

A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/ KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data.

scholarly journals Resistance Profiling and Molecular Characterization of Extended-Spectrum/Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli Isolated from Healthy Broiler Chickens in South Korea

2020 ◽  
Vol 8 (9) ◽  
pp. 1434 ◽  
Author(s):  
Hyun-Ju Song ◽  
Dong Chan Moon ◽  
Abraham Fikru Mechesso ◽  
Hee Young Kang ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phylogenetic Analysis ◽  
Resistance Gene ◽  
Broiler Chickens ◽  
Human Consumption ◽  
E Coli ◽  
Extended Spectrum ◽  
First Time ◽  
Pfge Profiles

We aimed to identify and characterize extended-spectrum β-lactamase (ESBL)-and/or plasmid-mediated AmpC β-lactamase (pAmpC)-producing Escherichia coli isolated from healthy broiler chickens slaughtered for human consumption in Korea. A total of 332 E. coli isolates were identified from 339 cloacal swabs in 2019. More than 90% of the isolates were resistant to multiple antimicrobials. ESBL/pAmpC-production was noted in 14% (46/332) of the isolates. Six of the CTX-M-β-lactamase-producing isolates were found to co-harbor at least one plasmid-mediated quinolone resistance gene. We observed the co-existence of blaCMY-2 and mcr-1 genes in the same isolate for the first time in Korea. Phylogenetic analysis demonstrated that the majority of blaCMY-2-carrying isolates belonged to subgroup D. Conjugation confirmed the transferability of blaCTX-M and blaCMY-2 genes, as well as non-β-lactam resistance traits from 60.9% (28/46) of the ESBL/pAmpC-producing isolates to a recipient E. coli J53. The ISECP, IS903, and orf477 elements were detected in the upstream or downstream regions. The blaCTX-M and blaCMY-2 genes mainly belonged to the IncI1, IncHI2, and/or IncFII plasmids. Additionally, the majority of ESBL/pAmpC-producing isolates exhibited heterogeneous PFGE profiles. This study showed that healthy chickens act as reservoirs of ESBL/pAmpC-producing E. coli that can potentially be transmitted to humans.

scholarly journals Dissection of the Hydrogen Metabolism of the EnterobacteriumTrabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Ute Lindenstrauß ◽  
Constanze Pinske
Keyword(s):  
Fossil Fuels ◽  
Biochemical Characterization ◽  
Model Organism ◽  
Attractive Alternative ◽  
Hydrogen Metabolism ◽  
Content Type ◽  
E Coli ◽  
Formate Hydrogenlyase ◽  
Alternative Carbon Source

ABSTRACTTrabulsiella guamensisis a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterizedEscherichia coliformate hydrogenlyase (FHL-2Ec) complex. TheT. guamensisFHL-2 (FHL-2Tg) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2Tgcomplex catalyzes the disproportionation of formate to CO2and H2. FHL-2Tghas activity similar to that of theE. coliFHL-1Eccomplex in H2evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2Tgoperon in the heterologousE. colihost has now enabled us to unambiguously prove FHL-2Tgactivity, and it allowed us to characterize the FHL-2Tgcomplex biochemically. Although the formate dehydrogenase (FdhH) genefdhFis not contained in the operon, the FdhH is part of the complex, and FHL-2Tgactivity was dependent on the presence ofE. coliFdhH. Also, in contrast toE. coli,T. guamensiscan ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2Tgand the H2-forming FHL-2Tgunder these conditions.IMPORTANCEBiological H2production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2producers, and the prototype FHL-1Eccomplex inE. coliis well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2Eccomplex, allow a first biochemical characterization ofT. guamensis’s fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

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