Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/12400
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dc.contributor.authorTajwar, Razia-
dc.date.accessioned2020-04-28T06:57:12Z-
dc.date.available2020-04-28T06:57:12Z-
dc.date.issued2019-
dc.identifier.govdoc19582-
dc.identifier.urihttp://prr.hec.gov.pk/jspui/handle/123456789/12400-
dc.description.abstractXylanases cleave β-1,4-glycosidic bond in xylan backbone and produce xylooligosaccharides. Xylan is the second most abundant carbohydrate polysaccharide and it is a major component of hemicellulose found in plant cell wall. Xylanases from the extremophile sources are of great importance because they are active and stable at wide range of temperature and pH. Xylanases have various applications such as they are used in the production of biofuels, paper and pulp industry, food industry and animal feed. Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused separately to the N- and C-terminal of the catalytic domain of XynB (XynB-C) to determine the effects on activity, thermostability, pH stability, substrate binding and 3-Dimensional (3D) structure of XynB. For this purpose, the genes xynB-C, CBM6-linker and CBM22-linker were synthesized by Genscript and provided in the cloning vector pUC57. The fusion proteins were created by sequential cloning using the appropriate restriction sites into the expression vector pET22b (+). CBM6 and CBM22 were separately fused to the both 5ʹ- and 3ʹ-end of xynB-C. After confirmation by colony PCR and restriction digestion analysis of the positive clones, E. coli BL21 CodonPlus (DE3)-RIPL cells were transformed with the recombinant plasmids pxynB-C, pxynB-B6C, pxynB-CB6, pxynB-B22C and pxynB-CB22 for the expression of proteins. All of the enzyme variants XynB-C, XynB-B6C, XynB-CB6, XynB-B22C and XynB-CB22 were successfully expressed in a soluble form. Partial purification was done by the heat treatment at 60 °C and further purification was done by fractionation of the enzymes using the ion exchange column QFF. Purified enzymes were assayed against the soluble birchwood xylan and oat spelts xylan as well as against the insoluble birchwood xylan and oat spelts xylan. Enzymatic activities of XynB-C and its variants were also performed against the pre-treated wheat straw. XynB-B22C and XynB-CB22 showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively, whereas activity of XynB-CB22 was also increased 2.76-fold against the soluble birchwood xylan. Like XynB-B22C, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity only against the insoluble birchwood xylan, whereas its attachment to the N-terminal did not show any increase of activity against the soluble and the insoluble birchwood xylan. Almost similar trend in activity profiles was observed when the soluble and the insoluble oat spelts xylan were used as substrate. XynB-CB22 showed 2.5- and 3.10-fold increase in activity against the soluble and the insoluble oat spelts xylan, respectively, whereas XynB-B22C and XynB-CB6 showed 1.6- and 1.9-fold increase in activity, respectively, only against the insoluble oat spelts xylan. Again, XynB-B6C did not show any increase in activity against the soluble and the insoluble oat spelts xylan. XynB-CB22, XynB-B22C and XynB-CB6 also showed increase in activity against the pre-treated wheat straw by 60%, 35% and 20%, respectively, whereas XynB-B6C showed no increase in activity as compared to that of the native XynB-C. Substrate binding studies with the insoluble substrate showed that fusion of CBM22 to either N- or C-terminal and CBM6 fusion to the C-terminal of XynB-C increased its binding with the insoluble substrate, whereas XynB-B6C showed little increase in substrate binding. XynB-CB22 also has lower Km values for the soluble and the insoluble substrate than that of XynB-C, whereas XynB-B22C and XynB-CB6 have lower Km values only for the insoluble substrate. Km values of XynB-B6C for the soluble and the insoluble substrate showed that fusion of CBM6 did not increase the affinity of XynB-C with the soluble and the insoluble substrate. The data of substrate binding studies and Km values for the variants of XynB-C are in agreement with the results of their activities. Thermostability studies showed that the variants carrying CBM22 were more thermostable than the variants carrying CBM6, though thermostability of XynB-C decreased with fusion of CBMs. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60 °C for 4 hours. After the incubation of 4 hours at 70 °C, the activities of XynB-B6C and XynB-CB6 remained 21% and 69%, respectively, whereas XynB-B22C and XynB-CB22 retained 87% and 94% activities, respectively. At 80 °C after 4 hours of incubation, XynB-B6C and XynB-CB6 lost almost all their activity while the activities of XynB-B22C and XynB-CB22 remained 56% and 78%, respectively, after this treatment. The native enzyme XynB-C is very stable as after incubation at 80 °C for 4 hours, it lost very little activity. All the variants showed the same optimum pH and temperature for the activity as that for the native XynB-C. pH stability of XynB-C and all its variants was determined by incubating the enzymes for 2 hours in different pH buffer ranging from 4.0-10.0 and results showed that all variants are quite stable at broad range of pH (4.0-10.0) with only little loss of activity. Secondary structural analysis and temperature ramping studies were done through circular dichroism (CD) spectroscopy for XynB-C and all its variants. CD results showed that all the XynB variants had the particular α/β mix structure of xylanase belonging to the family GH10 with a single broad negative peak around 210–220 nm and a positive peak around 195–196 nm. Comparison of secondary structure contents obtained by molecular modelling were found to be in agreement with the data from circular dichroism analysis. Temperature ramping studies showed that the secondary structure contents of the XynB-C variants carrying CBMs retained their integrity at 60 °C. But unfolding of the structure was observed at 80 °C, as the secondary structure contents changed and this change was more pronounced in the case of variants carrying CBM6. However, the secondary structure contents of the native enzyme XynB-C were only slightly changed even at 80 °C, which showed that the 3D structure of XynB-C remained intact with increasing temperature. Structural studies of XynB variants XynB-B6C, XynB-CB6, XynB-B22C and XynB-CB22 were done by creating their 3D structures using homology modelling and docked with the ligand (xylan) molecule. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule in the case of XynB-CB6, XynB-B22C and XynB-CB22, whereas in XynB-B6C, the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity than any other variant of XynB-C. This study shows that a favorable orientation of the catalytic domain and the CBM would allow arrangement of the active site residues of the catalytic domain and the binding residues of the CBM in a unique fashion, to obtain the maximum activity.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.language.isoen_USen_US
dc.publisherUniversity of the Punjab , Lahoreen_US
dc.subjectBiological Sciencesen_US
dc.subjectBiological Sciencesen_US
dc.subjectBiologyen_US
dc.titleProtein engineering and characterization of xylanases from Thermotoga maritimaen_US
dc.typeThesisen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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