scholarly journals Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Syed Shoaib Ahmed ◽  
Mohsina Akhter ◽  
Muhammad Sajjad ◽  
Roquyya Gul ◽  
Sana Khurshid
Keyword(s):  
Escherichia Coli ◽  
Clostridium Thermocellum ◽  
Expression System ◽  
Alpha Helix ◽  
Cellular Protein ◽  
Industrial Applications ◽  
Heat Inactivation ◽  
Potential Candidate ◽  
Cellulolytic Activity ◽  
High Level

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from Clostridium thermocellum (C. thermocellum) by cloning in an Escherichia coli (E. coli) expression system. BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction. Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively. The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a Km of 19.83 mM and a Vmax of 0.12 μmol/min. The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7. The optimized maximum expression level was attained in M9NG medium with lactose as an inducer. Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%). Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from C. thermocellum a potential candidate in industrial applications.

Endoglucanase E, produced at high level in Escherichia coli as a lacZ′ fusion protein, is part of the Clostridium thermocellum cellulosome

1990 ◽  
Vol 12 (9) ◽  
pp. 656-662 ◽  
Author(s):  
Geoffrey P. Hazlewood ◽  
Keith Davidson ◽  
Jonathan H. Clarke ◽  
Alastair J. Durrant ◽  
Judith Hall ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Clostridium Thermocellum ◽  
Lacz Fusion ◽  
High Level

scholarly journals Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization

2019 ◽  
Vol 21 (1) ◽  
pp. 103 ◽  
Author(s):  
Byung Hoon Jo ◽  
In Seong Hwang
Keyword(s):  
Escherichia Coli ◽  
Carbonic Anhydrase ◽  
Co2 Capture ◽  
Industrial Applications ◽  
Expression Level ◽  
High Level Expression ◽  
Whole Cell ◽  
Operational Conditions ◽  
Periplasmic Expression ◽  
High Level

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.

scholarly journals High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme

1993 ◽  
Vol 294 (1) ◽  
pp. 69-77 ◽  
Author(s):  
C Wang ◽  
M R Lee
Keyword(s):  
Escherichia Coli ◽  
Dissociation Constants ◽  
Expression System ◽  
Deae Cellulose ◽  
Basic Amino Acid ◽  
Coated Vesicle ◽  
Amino Acid Residues ◽  
High Level ◽  
Binding Component

We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.

scholarly journals Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7142
Author(s):  
Junhao Cheng ◽  
Marhaba Ahmat ◽  
Henan Guo ◽  
Xubiao Wei ◽  
Lulu Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Bacterial Infections ◽  
Expression System ◽  
Enterotoxigenic Escherichia Coli ◽  
Proteinase K ◽  
Hybrid Peptide ◽  
Tev Protease ◽  
High Level

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

scholarly journals Complete Genome Sequence of Escherichia coli BL21-AI

2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Niketa Bhawsinghka ◽  
Katie F. Glenn ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Expression System ◽  
High Yield ◽  
Content Type ◽  
System A ◽  
Phage T7 ◽  
High Level

Escherichia coli BL21-AI is a commercially available strain possessing a phage T7-based protein-expression system. A combination of tight regulation and high yield makes it widely used for high-level expression of toxic recombinant proteins. Here, we present the complete genome sequence of BL21-AI and provide insights on its genome.

scholarly journals EXPRESSION OF CELLULOSE-DEGRADING ENDOGLUCANASE FROM BACILLUS SUBTILIS USING PTOLT EXPRESSION SYSTEM IN ESCHERICHIA COLI

2021 ◽  
Vol 55 (5-6) ◽  
pp. 619-627
Author(s):  
HÜLYA KUDUĞ CEYLAN ◽  
YAKUP ULUSU ◽  
SEMA BILGIN ◽  
İSA GÖKÇE
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Expression System ◽  
Industrial Applications ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
E Coli ◽  
Glycosidic Bonds ◽  
Sds Page ◽  
Dna Fragment

Endoglucanases randomly hydrolyse the cellulose chains by acting upon internal β-1,4-D-glycosidic bonds and are used extensively in industrial applications. In this study, bacterial endoglucanase gene yhfE was obtained by PCR, using primers based on genomic sequences of Bacillus subtilis strains. 1041 bp DNA fragment of yhfE was cloned into Escherichia coli DH5α through the use of pTolT expression plasmid. PCR, restriction enzyme analysis and DNA sequencing were performed in order to confirm the cloning. E. coli BL21-AI cells expressed the yhfE after induction at 0.04% of arabinose concentration for 4 h. The expected 38.7 kDa size yhfE protein after digestion with thrombin of the His-tagged fusion protein (yhfE-TolAIII) was visualized by SDS-PAGE. The yhfE-TolAIII production yield was approximately 82 mg/L. The recombinant yhfE was characterized by MALDI-TOF mass spectrometry and CD analysis.

scholarly journals Using cellulose binding module of CBM3A as the purification tag for secreted Endoglucanase A (CelA) in Bacillus subtilis

2016 ◽  
Vol 19 (2) ◽  
pp. 5-14
Author(s):  
Phuong Hoang Ngoc Nguyen ◽  
Phuoc Thanh Nguyen ◽  
Thang Luong Pham ◽  
Hoang Duc Nguyen ◽  
Thuoc Linh Tran ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Protein Purification ◽  
Clostridium Thermocellum ◽  
Expression System ◽  
Secreted Proteins ◽  
Amorphous Cellulose ◽  
Recombinant Protein Purification ◽  
Bacillus Subtilis Wb800n ◽  
Cellulose Binding

Traditional methods of recombinant protein purification are uneconomic and inconvenient to the secreted proteins at large-volume. CBM3a, a module from cellulosome’s scaffoldin of Clostridium thermocellum, directs the binding of the cellulase complex on the cheap cellulose substrate. Most of previous studies about CBM3a fused with cellulases as the purification tag were conducted in intracellular Escherichia coli system. In this research, we used the extracellular Bacillus subtilis WB800N expression system to investigate the CBM3a-tag fused with endoglucanase CelA into plasmid pHT. The results indicated that protein CelA was secrected and purified by CBM3a-tag binding on the Regenerated Amorphous Cellulose (RAC) subtrate. This can be used for further improvement in protein purification tag designing.

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