Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum

Sajjad, Muhammad; Khan, Muhammad Imran; Zafar, Rehan; Ahmad, Sajjad; Niazi, Umar; Akhtar, Muhammad

doi:10.1016/j.jbiotec.2012.05.023

Cited by 26 publications

(20 citation statements)

References 27 publications

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“…However, previously (Table 2). These activities are higher as compared to those of the engineered variants of CelA of C. thermocellum, we reported previously (Sajjad et al, 2012). Removal of non-catalytic domain from C-terminal of celCCD from C. cellulolyticum showed 2-to 3.5-fold increase in activity as compared to that of the wild-type enzyme (Shima, Igarashi, & Kodama, 1993).…”

Section: Enzyme Characteristicsmentioning

confidence: 50%

“…The RAC was prepared by the previously described method (Jia et al, ). Pretreatment of bagasse, wheat and rice straw were done as described previously (Sajjad et al, ). For the assays using pretreated substrates, the reaction mixtures consisting of 80 mg of the substrate resuspended in 10 ml of the enzyme solution containing 45 CMC units of Cel5A, tCel5A1, or tCel5A2 were incubated for 8 hr at 60 °C.…”

Section: Methodsmentioning

confidence: 99%

“…The RAC was prepared by the previously described method (Jia et al, 2013). Pretreatment of bagasse, wheat and rice straw were done as described previously (Sajjad et al, 2012). For the assays using…”

Section: Enzyme Characteristicsmentioning

confidence: 99%

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Truncation of the processive Cel5A of Thermotoga maritima results in soluble expression and several fold increase in activity

Basit

Akhtar

2018

Biotech & Bioengineering

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Cel5A of Thermotoga maritima, a 37 kDa cellulase of the family GH5, was expressed in partially soluble state in E. coli. However, the truncated version tCel5A1, produced by removing ten residues from the C-terminal of Cel5A, was expressed in a completely soluble form. tCel5A1 showed 7.3- fold increased specific activity against carboxy methyl cellulose while the increase in activities against regenerated amorphous cellulose and Avicel were 21 and 16 fold, respectively. tCel5A1 is stable at 60 °C for more than 2 hr and it showed temperature and pH optima 70 °C and 6.0, respectively, under the assay conditions used. These characteristics are similar to those of the native enzyme. As expected, CD spectral analysis showed that C-terminal truncation has little effect on the secondary structure of the molecule. tCel5A1 showed higher binding to pretreated rice straw (84%) as compared to the native form (46%). Molecular modelling analysis of tCel5A1 showed that the removal of C-terminal residues exposed the active site residues Glu253, Trp286, and Phe292, which are located in the catalytic cavity close to the C-terminus. Making these residues more accessible to the substrate would result in increased activity. The ratio of 10.01 between the soluble to the insoluble reducing groups produced from RAC on treatment with tCel5A1, and the presence of cellobiose as the major end product in the hydrolysate showed that tCel5A1 is a processive cellulase. Although other processive cellulases belonging to the family GH5, mainly of the fungal origin, have been reported, but tCel5A1, to our knowledge, is the first processive cellulase from an extreme thermophile reported so far.

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Section: Enzyme Characteristicsmentioning

confidence: 50%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Truncation of the processive Cel5A of Thermotoga maritima results in soluble expression and several fold increase in activity

Basit

Akhtar

2018

Biotech & Bioengineering

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“…Structural alignment between the query and template sequences was performed using 3D-Coffee (Poirot et al, 2004). The three dimensional structures of fusion proteins were generated as described previously (Sajjad et al, 2012). Briefly, models were generated using MODELLER (Eswar et al, 2006), and were clustered by NMRCLUST (Kelley et al, 1996).…”

Section: Molecular Modelingmentioning

confidence: 99%

Fusion of selected regions of mycobacterial antigens for enhancing sensitivity in serodiagnosis of tuberculosis

Afzal

Khurshid

Khalid

et al. 2015

Journal of Microbiological Methods

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“…To evaluate the possible domain-domain interactions of fusion proteins, we employed the same strategy as used previously [ 40 ]. Firstly, the protein binding sites were determined using CPORT [ 41 ].…”

Section: Methodsmentioning

confidence: 99%

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis

et al. 2016

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Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.

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Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum

Cited by 26 publications

References 27 publications

Truncation of the processive Cel5A of Thermotoga maritima results in soluble expression and several fold increase in activity

Truncation of the processive Cel5A of Thermotoga maritima results in soluble expression and several fold increase in activity

Fusion of selected regions of mycobacterial antigens for enhancing sensitivity in serodiagnosis of tuberculosis

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis

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