Cbes-AXE2 Lab Report
Results and Discussions
Cbes-AXE2 encodes a novel acetyl xylan esterase
The CAZy database has grouped esterases acting on carbohydrates into 16 different families of carbohydrate esterases (CEs). AXEs (EC 3.1.1.72) acting on acetylated xylan are distributed in only 8 families – CE1-7 and 16. Among these, other than CE4, all other CEs belong to serine esterase and have the prototype catalytic triad Ser-His-Asp (Glu). Genralyy, the catalytic serine of the triad lies in a conserved motif of either GxSxG or GDSL. The conserved domain analysis of Cbes-AXE2 predicts a catalytic triad containing SGNH hydrolase type esterase domain. This domain is present in a variety of hydrolases for example esterase domain of viral haemagglutinin-esterase surface …show more content…
The optimum pH was at 7.5, and the temperature optimum was at 70°C (Fig. 2b and 2c). The optimum conditions of Cbes-AXE2 are very close to the optimum growth conditions of the organism which is around at pH 7.0 and in the temperature range of 75-90°C. Theoretically, the Cbes-AXE2 is expected to show the optimal temperature around 78°C, which is the optimal growth temperature of the organism, but we observed a significant difference of approximately 8°C. This could be explained because of the absence of certain posttranslational modifications in recombinant protein in comparison of native Cbes-AXE2. A similar change in the optimum temperature has also been seen in the recombinant version of CelA of C. bescii, lacking glycosylation in comparison of native …show more content…
The model has GMQE (Global mean quality estimate) score of 0.82 which denotes a good model. The model superimposed very well to evolutionary related protein AXE2 with an RMSD (Root Mean Square Deviation) of 0.217 Å. In Fig. 5a, the predicted model, has been shown with overlaid catalytic triad residues and the loop involved in catalysis. The position of Ser- His- Asp (catalytic triad) was quite similar to the predicted residue position of 15, 192 and195 respectively, as described earlier in Fig 1b. Notably, In the Fig 5a, the orientation of the active side loop was significantly altered that could be an important factor to effect the observed difference in catalytic efficiency and Km between Cbes-AXE2 and AXE2. The geometry of the residues forming the catalytic triad was also analyzed and shown in Fig 5b. The catalytic serine was positioned at a distance of 2.9 Å, and Aspartic acid was located at 3.8 Å away from Histidine residue. These observations suggest that the residues comprising the catalytic triad are apparently located at an optimum distance to facilitate the
Cbes-AXE2 encodes a novel acetyl xylan esterase
The CAZy database has grouped esterases acting on carbohydrates into 16 different families of carbohydrate esterases (CEs). AXEs (EC 3.1.1.72) acting on acetylated xylan are distributed in only 8 families – CE1-7 and 16. Among these, other than CE4, all other CEs belong to serine esterase and have the prototype catalytic triad Ser-His-Asp (Glu). Genralyy, the catalytic serine of the triad lies in a conserved motif of either GxSxG or GDSL. The conserved domain analysis of Cbes-AXE2 predicts a catalytic triad containing SGNH hydrolase type esterase domain. This domain is present in a variety of hydrolases for example esterase domain of viral haemagglutinin-esterase surface …show more content…
The optimum pH was at 7.5, and the temperature optimum was at 70°C (Fig. 2b and 2c). The optimum conditions of Cbes-AXE2 are very close to the optimum growth conditions of the organism which is around at pH 7.0 and in the temperature range of 75-90°C. Theoretically, the Cbes-AXE2 is expected to show the optimal temperature around 78°C, which is the optimal growth temperature of the organism, but we observed a significant difference of approximately 8°C. This could be explained because of the absence of certain posttranslational modifications in recombinant protein in comparison of native Cbes-AXE2. A similar change in the optimum temperature has also been seen in the recombinant version of CelA of C. bescii, lacking glycosylation in comparison of native …show more content…
The model has GMQE (Global mean quality estimate) score of 0.82 which denotes a good model. The model superimposed very well to evolutionary related protein AXE2 with an RMSD (Root Mean Square Deviation) of 0.217 Å. In Fig. 5a, the predicted model, has been shown with overlaid catalytic triad residues and the loop involved in catalysis. The position of Ser- His- Asp (catalytic triad) was quite similar to the predicted residue position of 15, 192 and195 respectively, as described earlier in Fig 1b. Notably, In the Fig 5a, the orientation of the active side loop was significantly altered that could be an important factor to effect the observed difference in catalytic efficiency and Km between Cbes-AXE2 and AXE2. The geometry of the residues forming the catalytic triad was also analyzed and shown in Fig 5b. The catalytic serine was positioned at a distance of 2.9 Å, and Aspartic acid was located at 3.8 Å away from Histidine residue. These observations suggest that the residues comprising the catalytic triad are apparently located at an optimum distance to facilitate the