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 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.

Biochemical properties of the alkaline lipase of Bacillus flexus XJU-1 and its detergent compatibility

Biologia ◽  
2014 ◽  
Vol 69 (9) ◽  
Author(s):  
Francois Niyonzima ◽  
Sunil More
Keyword(s):  
Sodium Dodecyl ◽  
Tween 80 ◽  
Relative Activity ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Liquid Chromatography Mass Spectrometry ◽  
Initial Activity ◽  
Detergent Compatibility ◽  
Bacillus Flexus ◽  
Stain Removal

AbstractThe possibility of using Bacillus flexus XJU-1 lipase in detergent preparations was studied. The enzyme was monomeric protein as confirmed by liquid chromatography-mass spectrometry and its molecular weight was 15.95 kDa. The lipase showed optimum activity at pH 10.0 and was 100% stable for 24 h at pH 10.0 and 11.0. It exhibited maximum activity at 70°C and retained more than 70% of the initial activity at 60, 70 and 80°C for 24 h. The activity was stimulated by Ca2+, Ba2+, Mg2+ and Co2+, whereas 50% of the initial activity was lost with Fe3+ and Hg2+. The activity was inhibited by 10 mM N-bromosuccinimide and tosyl-L-lysylchloromethylketone, while N-ethylmaleimide, phenylmethylsulphonylfluoride and urea did not show any effect. The enzyme significantly hydrolysed olive, cottonseed, sunflower, groundnut, and gingelly oils. With p-nitrophenyl palmitate, Vmax and Km were 62.5 U/mL and 2.25 mM, respectively. The lipase maintained its stability in Tween-80, Triton-100 and H2O2 at 1%, but an activation of 10% and a reduction of 15% in relative activity were observed with NaClO and sodium dodecyl sulphate, respectively. The enzyme retained maximum storage stability for 20 days at −20, 4 and 30°C. In the presence of 0.7% (w/v) Ariel, Henko, Super wheel, Tide plus and Rin, a retention of more than 84.90% initial activity was recorded after 24 h at 60°C. The supplementation of the lipase to the detergents improved the olive oil stain removal. These properties suggested the present enzyme as a potential additive for detergent preparations.

scholarly journals Screening of Novel Laccase Producers—Isolation and Characterization of Cold-Adapted Laccase from Kabatiella bupleuri G3 Capable of Synthetic Dye Decolorization

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 828
Author(s):  
Katarzyna M. Wiśniewska ◽  
Aleksandra Twarda-Clapa ◽  
Aneta M. Białkowska
Keyword(s):  
Morphological Characteristics ◽  
Tween 80 ◽  
Dye Decolorization ◽  
Defined Medium ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Its2 Region ◽  
Synthetic Dyes ◽  
Cold Adapted ◽  
Isolation And Characterization

Psychrophilic laccases catalyzing the bond formation in mild, environmentally friendly conditions are one of the biocatalysts at the focus of green chemistry. Screening of 41 cold-adapted strains of yeast and yeast-like fungi revealed a new laccase-producing strain, which was identified as Kabatiella bupleuri G3 IBMiP according to the morphological characteristics and analysis of sequences of the D1/D2 regions of 26S rDNA domain and the ITS1–5,8S–ITS2 region. The extracellular activity of laccase in reaction with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at the optimal pH 3.5 was 215 U/L after 15 days of growth in a medium with waste material and 126 U/L after 25 days of cultivation in a defined medium. Copper (II) ions (0.4 mM), Tween 80 (1.0 mM) and ascorbic acid (5.0 mM) increased the production of laccase. The optimum temperature for enzyme operation is in the range of 30–40 °C and retains over 60% of the maximum activity at 10 °C. New laccase shows high thermolability—half-life at 40 °C was only 60 min. Enzyme degradation of synthetic dyes was the highest for crystal violet, i.e., 48.6% after 1-h reaction with ABTS as a mediator. Outcomes of this study present the K. bupleuri laccase as a potential psychrozyme for environmental and industrial applications.

scholarly journals Identification and molecular characterization of a psychrophilic GH1 β-glucosidase from the subtropical soil microorganism Exiguobacterium sp. GXG2

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bangqiao Yin ◽  
Hengsen Gu ◽  
Xueyan Mo ◽  
Yue Xu ◽  
Bing Yan ◽  
...  
Keyword(s):  
Metal Ion ◽  
Functional Characterization ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Screening Strategy ◽  
Soil Microorganism ◽  
Potential Candidate ◽  
Cold Adapted ◽  
Subtropical Soil

Abstract The products of bacterial β-glucosidases with favorable cold-adapted properties have industrial applications. A psychrophilic β-glucosidase gene named bglG from subtropical soil microorganism Exiguobacterium sp. GXG2 was isolated and characterized by function-based screening strategy. Results of multiple alignments showed that the derived protein BglG shared 45.7% identities with reviewed β-glucosidases in the UniProtKB/Swiss-Prot database. Functional characterization of the β-glucosidase BglG indicated that BglG was a 468 aa protein with a molecular weight of 53.2 kDa. The BglG showed the highest activity in pH 7.0 at 35 °C and exhibited consistently high levels of activity within low temperatures ranging from 5 to 35 °C. The BglG appeared to be a psychrophilic enzyme. The values of Km, Vmax, kcat, and kcat/Km of recombinant BglG toward ρNPG were 1.1 mM, 1.4 µg/mL/min, 12.7 s−1, and 11.5 mM/s, respectively. The specific enzyme activity of BglG was 12.14 U/mg. The metal ion of Ca2+ and Fe3+ could stimulate the activity of BglG, whereas Mn2+ inhibited the activity. The cold-adapted β-glucosidase BglG displayed remarkable biochemical properties, making it a potential candidate for future industrial applications.

scholarly journals Partial purification and biochemical characterization of an alkaline esterase from Sorghum bicolor

2020 ◽  
Vol 42 ◽  
pp. e52115
Author(s):  
Fabiana Guillen Moreira Gasparin ◽  
Marcio de Barros ◽  
Gabriela Alves Macedo
Keyword(s):  
Biochemical Characterization ◽  
Low Cost ◽  
Reaction Medium ◽  
Short Chain Fatty Acids ◽  
Relative Activity ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Water Soluble ◽  
Partial Purification ◽  
Alternative Source

Esterases are enzymes that present good potential for industrial applications since they catalyze the formation or cleavage of ester bonds in water-soluble substrates, and sorghum seeds can represent an alternative source of this enzyme. The extraction of esterase from sorghum seeds is an economical alternative to obtain an enzyme of great interest. Esterases may improve the quality or accelerate the maturation of cheeses, cured bacon and fermented sausages and may also resolve racemic mixtures. Recently, seed esterases have been the focus of much attention as biocatalysts. In some cases, these enzymes present advantages over animal and microbial lipases due to some quite interesting features such as specificity and low cost, being a great alternative for their commercial exploitation as industrial enzymes The esterase studied here was extracted from sorghum seeds and some of its biochemical properties determined using synthetic substrates (p-nitrophenyl butyrate, caprylate, laurate and palmitate). The enzyme presented optimum activity at pH 8.0 and was stable in all the pH ranges studied. The optimum temperature for its activity was 40ºC but it showed low stability at this temperature (40% relative activity). The values derived for Km and Vmax were 0.67mM and 125 U.mg-1, respectively, obtained using p-nitrophenyl butyrate as the substrate. The enzyme showed an increase in activity when K2HPO4 was added to the reaction medium, but the ions Mn2+, CO+, Hg+ and Fe2+ strongly inhibited the enzyme activity. This enzyme showed a preference for the hydrolysis of short chain fatty acids. The characteristics of sorghum esterase are very similar to those of the microbial esterases used in detergent processing.

Analysis of the monitoring study of the antibiotic-resistance of the agents of purulent-inflammatory processes of soft tissue

2018 ◽  
Vol 22 (2) ◽  
pp. 285-288
Author(s):  
A.P. Prevar ◽  
A.V. Kryzshanovskaya ◽  
V.A. Radionov ◽  
V.M. Mrug
Keyword(s):  
Soft Tissues ◽  
High Sensitivity ◽  
Biochemical Properties ◽  
Antimicrobial Drugs ◽  
Strict Adherence ◽  
E Coli ◽  
Monitoring Study ◽  
Treatment Measures ◽  
Inflammatory Processes ◽  
Main Factor

The main factor in the treatment of suppurative and inflammatory processes is the timely optimization of treatment measures taking into account the nature of the microflora and its susceptibility to antimicrobial drugs. The purpose of the study is to monitor the spectrum of microorganisms – pathogens of purulent-inflammatory processes of soft tissues in surgical patients; study of the sensitivity of isolated strains to antibiotics. The material was collected in accordance with aseptic rules. The identification of a pure culture of bacteria was carried out according to morphological, culture, biochemical properties, and the presence of virulence enzymes. Sensitivity of bacteria to antibiotics was determined by the standard disks method (by Kirby-Bauer’s). 255 patients with purulent-inflammatory processes of soft tissues were examined for the period from 2014 to 2017. 229 strains of isolated bacteria were included to Escherichia coli, Citrobacer freundii, Enterobacter cloacae, E.aerogenes, Proteus vulgaris, Staphylococcus aureus, S.epidermidis, Streptococcus pyogenes, S.viridians, S.agalactiae, Pseudomonas aeruginosa. The main cause of purulent-inflammatory processes of soft tissues is Staphylococci (67,2%). Compared to previous studies, the number of P.aeruginosa isolated cultures increased (7.9%). In monoculture and in association with other microorganisms, E. coli (9.6% of cases), E.cloacae et aerogenes (3.9% of cases), P.vulgaris (3.9% of cases), C.freundi (2.5% of cases), S.agalactiae, S.pyogenes, S.viridans (3.5%). The number of associated sows reaches 12%. Clinical strains of microorganisms remain most sensitive to fluoroquinolones, cephalosporins, and also retains high sensitivity to gentamicin, lincomycin, rifampicin, which is important for empirical antibiotic therapy. To increase the effectiveness of antibacterial therapy, strict adherence to the mode of appointment of antibiotics, justification of indications, a combination of antibiotics of different spectrum of action, mandatory correction after determining the sensitivity of the pathogen.

scholarly journals Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease

Catalysts ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 55 ◽  
Author(s):  
Muhammad Syafiq Mohd Razib ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Fairolniza Mohd Shariff ◽  
Mohd Shukuri Mohamad Ali
Keyword(s):  
Structural Analysis ◽  
Organic Solvent ◽  
Average Diameter ◽  
Relative Activity ◽  
Biochemical Properties ◽  
Biochemical Interaction ◽  
Organic Solvent Tolerant ◽  
Random Aggregate ◽  
Solvent Tolerant ◽  
The Stability

Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm.

scholarly journals CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics

The Analyst ◽  
2014 ◽  
Vol 139 (13) ◽  
pp. 3314-3323 ◽  
Author(s):  
Thomas Beneyton ◽  
Faith Coldren ◽  
Jean-Christophe Baret ◽  
Andrew D. Griffiths ◽  
Valérie Taly
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Recombinant Enzyme ◽  
Cell Analysis ◽  
E Coli ◽  
Cota Laccase

A high-throughput cell analysis and sorting platform using droplet-based microfluidics is introduced for directed evolution of recombinant CotA laccase expressed in E. coli.

P. vortex-mediated strategies for polysaccharides decomposition

TECHNOLOGY ◽  
2015 ◽  
Vol 03 (02n03) ◽  
pp. 80-83
Author(s):  
Mark Polikovsky ◽  
Eshel Ben-Jacob ◽  
Alin Finkelshtein
Keyword(s):  
Degradation Products ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Industrial Applications ◽  
Biofuel Production ◽  
E Coli ◽  
Novel Approach ◽  
Textile Manufacture ◽  
Hydrolysis Of Cellulose ◽  
The Relationship

Cellulose hydrolysis has many industrial applications such as biofuel production, food, paper and textile manufacture. Here, we present a novel approach to cellulose hydrolysis using a consortium of motile bacteria, Paenibacillus vortex, that can swarm on solid medium carrying a non-motile recombinant E. coli cargo strain expressing the β-glucosidase and cellulase genes that facilitate the hydrolysis of cellulose. These two species cooperate; the relationship is mutually beneficial: the E. coli is dispersed over long distances, while the P. vortex bacteria gain from the supply of cellulose degradation products. This enables the use of such consortia in this area of biotechnology.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

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