Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

Round 1

Reviewer 1 Report

The authors described a detailed  rational method to improve an enzyme activity, such as b-glycosidase LXYL-P1-2.  They identified the key residues to increase the catalytic activity and provided a kinetic analysis of the mutants generated.

 

I would recommend the publication of the paper after minor changes:

• A figure with the chemical structures of the drugs described in the paper would be beneficial for a non-specialized reader, e.g. taxol.
• Line 50-51, The bifunctional 50 β-D-xylosidases/β-D-glucosidase LXYL-P1-2 was identified from Lentinula edodes, which 51 shows very low similarity with other known glycoside hydrolases. Could the authors especify what GH family is the protein?
• Line 57, LXYL-P1-2 has a good application potential. Please, revise the grammar in this sentence.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript described the use of site-directed mutagenesis to mutate three noncatalytic sites of LXYL-P1-2 based on the molecular docking of LXYL-P1-2 and substrate XDT to investigate their roles in β-D-xylosidase/β-D-glucosidase activities. The results indicated that the L220G mutation leads to increased activity that may be due to the enlarged channel for facilitating the entrance of the substrate XDT into the active pocket of the enzyme. However, the Y268E and S466D mutations resulted in significantly decreased activities that may be due to the instability of the substrate in the active pocket of the enzyme or the change of enzyme conformation near the catalytic site. Moreover, the L220G mutation was introduced into a highly active mutant EP2 which harbors the T368E mutation. Both β-D-xylosidase and β-D-glucosidase activities of EP2 mutant were increased. The study demonstrated that both L220G and T368E mutations of LXYL-P1-2 increased the β-glycosidase activity for catalyzing XDT to DT for the efficient semi-synthesis of Taxol. Though several errors were found, it is suggested to accept the manuscript after minor modification.

1. Line 31, delete }.
2. Line 51, delete s of β-D-xylosidases.
3. Line 103-104, 10⁵.
4. In figure 4, the units of enzyme activities were different. Please confirm them.
5. Table 1, please indicate the tests of statistical significance.
6. Line 273, Na₂B₄O₇ 

Author Response

Please see the attachment.

Author Response File: Author Response.docx