scholarly journals Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain

Bioimpacts ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Azam Safary ◽  
Rezvan Moniri ◽  
Maryam Hamzeh-Mivehroud ◽  
Siavoush Dastmalchi
Keyword(s):  
Lymphoblastic Leukemia ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Substrate Affinity ◽  
Bacillus Sp ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Bacillus Strain ◽  
Antineoplastic Effect ◽  
Wide Range

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

scholarly journals Common allotypes of ER aminopeptidase 1 have substrate-dependent and highly variable enzymatic properties

2020 ◽  
Author(s):  
Jonathan P. Hutchinson ◽  
Ioannis Temponeras ◽  
Jonas Kuiper ◽  
Adrian Cortes ◽  
Justyna Korczynska ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Catalytic Efficiency ◽  
Enzymatic Properties ◽  
Human Populations ◽  
Substrate Affinity ◽  
Antigenic Peptides ◽  
Nucleotide Polymorphisms ◽  
Recombinant Enzymes ◽  
Wide Range

AbstractObjectivePolymorphic variation of immune system proteins can drive variability of individual immune responses. ER aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by MHC class I molecules. Coding single nucleotide polymorphisms (SNPs) in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes, has not been thoroughly explored.MethodsWe used phased genotype data to estimate ERAP1 allotype frequency in 2,504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the 10 most common ERAP1 allotypes and systematically characterized their in vitro enzymatic properties.ResultsWe find that ERAP1 allotypes possess a wide range of enzymatic activities, up to 60-fold, whose ranking is substrate-dependent. Strikingly, allotype 10, previously associated with Behçet’s disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a sub-active ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multi-step trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site suggest that allotypic variation influences the communication between the regulatory and the active site.ConclusionOur work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with MHC haplotypes, to immune response variability and to predisposition to chronic inflammatory conditions.

scholarly journals Screening and Optimization of Thermo-Tolerant Bacillus Sp. For Amylase Production and Antifungal Activity

2019 ◽  
Vol 24 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Susmita Sapkota ◽  
Sujan Khadka ◽  
Aava Gautam ◽  
Rojina Maharjan ◽  
Ruby Shah ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Industrial Applications ◽  
Antagonistic Activity ◽  
Soil Samples ◽  
Bacillus Sp ◽  
Biochemical Tests ◽  
Wide Range ◽  
Amylase Production ◽  
Bacillus Spp ◽  
Two Parameters

Amylases are starch degrading enzymes which are produced by plants, animals and microorganisms. Amylases produced by microorganisms have a wide range of industrial applications such as in pharmaceutical, food, textile and paper industries. However, there are still limitations in the isolation of amylase producing microorganisms. The objective of this study was to isolate the potent amylase producing Bacillus sp. from soil samples and evaluate their abilities for inhibiting the aflatoxin producing Aspergillus flavus. In this study, 30 soil samples were used. For the screening and identification of Bacillus strain, morphological and biochemical tests were performed. Iodine assay was done to screen the potent amylase producers. Two parameters (pH and temperature) were used to optimize the cultural conditions for the production of amylase. To determine the total reducing sugar, dinitrosalicylic acid (DNS) assay was used. Altogether 29 colonies were selected and identified as Bacillus spp out of which 16 were selected to determine enzyme activity by cup plate method. Four isolates (DK9, DK10, IM4 and KD7) showing highest amylolytic activities (16 mm, 12 mm, 14 mm and 14 mm zone of hydrolysis) were subjected for further study. Isolate KD7 showed the highest amylolytic activity (0.19 U/mL) compared to other isolates. Maximum amylase production was found at pH 6 and temperature 50° C (0.19 U/mL). Among these 4 isolates, DK9 and KD9 showed strong antagonistic activity against Aspergillus flavus while DK10 and IM4 showed moderate antifungal activities. Thus, the bacterial isolate KD7 was identified as the most potent strain for maximum amylase production.

scholarly journals Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2679 ◽  
Author(s):  
Xi Bai ◽  
Xianjun Yuan ◽  
Aiyou Wen ◽  
Junfeng Li ◽  
Yunfeng Bai ◽  
...  
Keyword(s):  
Symbiotic Bacterium ◽  
Tween 80 ◽  
Relative Activity ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Recombinant Enzyme ◽  
Enzymatic Properties ◽  
Cold Adapted ◽  
E Coli ◽  
Thermal Asymmetric Interlaced Pcr

Background Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study, Citrobacter farmeri A1 which was isolated from a wood-inhabiting termite Reticulitermes labralis could secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml). The objective of the present study was to clone and express the C. farmeri EglC gene in Escherichia coli to improve production level and determine the enzymatic properties of the recombinant enzyme. Methods The EglC gene was cloned from C. farmeri A1 by thermal asymmetric interlaced PCR. EglC was transformed into vector pET22b and functionally expressed in E. coli. The recombination protein EglC22b was purified for properties detection. Results SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of the E. coli pET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5–8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30–40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co2+ and Fe3+, but inhibited by Cd2+, Zn2+, Li+, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA. Conclusion These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications.

scholarly journals Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase fromGeobacillussp. CHB1

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xianbo Jia ◽  
Jichen Chen ◽  
Chenqiang Lin ◽  
Xinjian Lin
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Residual Activity ◽  
Industrial Applications ◽  
Activity Level ◽  
High Yield ◽  
E Coli ◽  
Wide Range ◽  
Clone And Express

Catalases are widely used in many scientific areas. A catalase gene (Kat) fromGeobacillussp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed inEscherichia coli(E. coli), which was the first time to clone and express this type of catalase ofgenus Geobacillusstrains as far as we know. ThisKatgene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studiedBacillussp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble inE. coliand made up 30% of the totalE. coliprotein. Fermentation broth of the recombinantE. colishowed a high catalase activity level up to 35,831 U/mL which was only lower than recombinantBacillussp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg andKmof 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

scholarly journals Functional exploration of the GH29 fucosidase family

Glycobiology ◽  
2020 ◽  
Vol 30 (9) ◽  
pp. 735-745 ◽  
Author(s):  
Hendrik Grootaert ◽  
Linde Van Landuyt ◽  
Paco Hulpiau ◽  
Nico Callewaert
Keyword(s):  
Sequence Diversity ◽  
Sialyl Lewis X ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Recombinant Production ◽  
Sialyl Lewis ◽  
Naturally Occurring ◽  
Wide Range ◽  
Deoxy Sugar ◽  
Therapeutic Leads

Abstract The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family’s large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

scholarly journals Exploitation of E. coli for the production of penicillin G amidase: a tool for the synthesis of semisynthetic β-lactam antibiotics

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Krishika Sambyal ◽  
Rahul Vikram Singh
Keyword(s):  
Strain Improvement ◽  
Penicillin G ◽  
Main Body ◽  
Recombinant Enzymes ◽  
Selective Hydrolysis ◽  
E Coli ◽  
Wide Range ◽  
Lactam Ring ◽  
Wild Strains ◽  
Microbial Sources

Abstract Background Penicillin G amidase/acylases from microbial sources is a unique enzyme that belongs to the N-terminal nucleophilic hydrolase structural superfamily. It catalyzes the selective hydrolysis of side chain amide/acyl bond of penicillins and cephalosporins whereas the labile amide/acyl bond in the β-lactam ring remains intact. Main body of abstract This review summarizes the production aspects of PGA from various microbial sources at optimized conditions. The minimal yield from wild strains has been extensively improved using varying strain improvement techniques like recombination and mutagenesis; further applied for the subsequent synthesis of 6-aminopenicillanic acid, which is an intermediate molecule for synthesis of a wide range of novel β-lactam antibiotics. Immobilization of PGA has also been attempted to enhance the durability of enzyme for the industrial purposes. Short conclusion The present review provides an emphasis on exploitation of E. coli to enhance the microbial production of PGA. The latest achievements in the production of recombinant enzymes have also been discussed. Besides E. coli, other potent microbial strains with PGA activity must be explored to enhance the yields. Graphical abstract

scholarly journals AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB

2015 ◽  
Vol 81 (6) ◽  
pp. 1977-1987 ◽  
Author(s):  
Zhoukun Li ◽  
Jiale Wu ◽  
Biying Zhang ◽  
Fei Wang ◽  
Xianfeng Ye ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Specific Activity ◽  
Peptide Mass Fingerprinting ◽  
Soluble Starch ◽  
Industrial Applications ◽  
Product Analysis ◽  
Content Type ◽  
E Coli ◽  
Peptide Mass ◽  
Wide Range

ABSTRACTA novel α-amylase, AmyM, was purified from the culture supernatant ofCorallococcussp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence ofCorallococcuscoralloidesDSM 2259, the AmyM gene was identified and cloned intoEscherichia coli.amyMencodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases.amyMwas expressed inE. coliBL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM inE. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. TheKmandVmaxof recombinant AmyM for soluble starch were 6.61 mg ml−1and 44,301.5 μmol min−1mg−1, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.

Asparaginase administration in children suffering from acute lymphoblastic leukemia

2000 ◽  
Vol 20 (03) ◽  
pp. 151-153
Author(s):  
C. Wermes ◽  
R. Schobess ◽  
S. Halimeh ◽  
W. Nürnberger ◽  
A. Kosch ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Childhood Leukemia ◽  
Case Reports ◽  
Lymphoblastic Leukemia ◽  
Variable Number ◽  
E Coli ◽  
Number Of Patients ◽  
Wide Range ◽  
Prothrombotic Risk Factors ◽  
Vascular Accidents

Summary Background: Previous studies focusing on asparaginase(ASP)-induced acquired alterations of the hemostatic system reported in childhood acute lymphoblastic leukemia (ALL) could not sufficiently explain the sporadic manifestations of thrombotic events in the studied populations, reported as between 0.8% and 11%. Therefore the present retrospective overview was conducted to unravel the possible interaction between prothrombotic risk factors, leukemia protocols and the concomitant use of different anti-leukemic agents with respect to the presence of symptomatic venous vascular accidents. Methods: Published reports on childhood leukemia and hemostatic alterations (no case-reports) obtained from a Medline-based search (1980-2000) were included in the present overview. Conclusions: Based on the data presented here, evidence is given that the wide range of thrombotic events reported is due mainly to different study designs, prospective versus retrospective data, E. coli ASP sources of different origin (Erwinia, E. coli A, E. coli B) and dosages, administered alone or in combination with steroids, as well as a variable number of patients included in the studies reported.

Substituent Effects on the Thermal Decomposition of Phosphate Esters on Ferrous Surfaces

2019 ◽  
Author(s):  
James Ewen ◽  
Carlos Ayestaran Latorre ◽  
Arash Khajeh ◽  
Joshua Moore ◽  
Joseph Remias ◽  
...  
Keyword(s):  
Thermal Decomposition ◽  
Film Formation ◽  
Substituent Effects ◽  
Vapour Phase ◽  
Industrial Applications ◽  
Phosphate Esters ◽  
Antiwear Additives ◽  
Formation Mechanisms ◽  
Phosphate Ester ◽  
Wide Range

<p>Phosphate esters have a wide range of industrial applications, for example in tribology where they are used as vapour phase lubricants and antiwear additives. To rationally design phosphate esters with improved tribological performance, an atomic-level understanding of their film formation mechanisms is required. One important aspect is the thermal decomposition of phosphate esters on steel surfaces, since this initiates film formation. In this study, ReaxFF molecular dynamics simulations are used to study the thermal decomposition of phosphate esters with different substituents on several ferrous surfaces. On Fe<sub>3</sub>O<sub>4</sub>(001) and α-Fe(110), chemisorption interactions between the phosphate esters and the surfaces occur even at room temperature, and the number of molecule-surface bonds increases as the temperature is increased from 300 to 1000 K. Conversely, on hydroxylated, amorphous Fe<sub>3</sub>O<sub>4</sub>, most of the molecules are physisorbed, even at high temperature. Thermal decomposition rates were much higher on Fe<sub>3</sub>O<sub>4</sub>(001) and particularly α-Fe(110) compared to hydroxylated, amorphous Fe<sub>3</sub>O<sub>4</sub>. This suggests that water passivates ferrous surfaces and inhibits phosphate ester chemisorption, decomposition, and ultimately film formation. On Fe<sub>3</sub>O<sub>4</sub>(001), thermal decomposition proceeds mainly through C-O cleavage (to form surface alkyl and aryl groups) and C-H cleavage (to form surface hydroxyls). The onset temperature for C-O cleavage on Fe<sub>3</sub>O<sub>4</sub>(001) increases in the order: tertiary alkyl < secondary alkyl < primary linear alkyl ≈ primary branched alkyl < aryl. This order is in agreement with experimental observations for the thermal stability of antiwear additives with similar substituents. The results highlight surface and substituent effects on the thermal decomposition of phosphate esters which should be helpful for the design of new molecules with improved performance.</p>

Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

2018 ◽  
Author(s):  
Jiajun Wang ◽  
Jayesh Arun Bafna ◽  
Satya Prathyusha Bhamidimarri ◽  
Mathias Winterhalter
Keyword(s):  
Dwell Time ◽  
Covalent Binding ◽  
Channel Structure ◽  
Ion Current ◽  
E Coli ◽  
Current Fluctuation ◽  
Wide Range ◽  
Outer Membrane Channel ◽  
Biological Channels ◽  
Constriction Region

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from <i>E. coli</i>. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpF<sub>wt</sub>. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.

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