Modeling
一．Data Analysis Screening Mutants
1. 1 SbPETase protein sequence：
The protein structure of SbPETase based on the secondary structure, super secondary structure and domain is complex, polypeptide chains are further coiled and folded to form more complex spherical molecular structures.The relative position of all atoms in a peptide chain in three-dimensional space.
（MDQTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPYHHHHHH*
1.2 Structure modeling of the PETase and its mutants
PET degrading enzyme (SbPETase) was modified by site directed mutagenesis. Mutant enzyme with improved PET plastic degrading activity was obtained from data analysis.We can use 7dzu as a template ( crystal structure of PETase K169A mutant of R. mucilaginosa ) to get site-directed mutants, including Y60A，L61T，W132A，W132H，V181I，T212S，T212F，R259A，L61T + W132H，L61T + R259A，W132H + R259A，L61T + W132H + R259A(Seq Identity 68.80%). Mutant amino acid sequence
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGALARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYTARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGASMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGHSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTIAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHSCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHFCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSALSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYTARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGHSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSRLSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYTARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGWSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSALSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYLARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGHSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSALSEYRESCPY
QTNPYQRGPDPTTRDLEDSRGPFRYASTNVRSPSGYGAGTIYYPTDVSGSVGAVAVVPGYTARQSSIRWWGPRLASHGFVVITLDTRSTSDQPASRSAQQMAALRQVVALSETRSSPIYGKVDPNRLAVMGHSMGGGGTLISARDNPSLKAAVPFAPWHNTANFSGVQVPTLVIACENDTVAPISRHASSFYNSFSSSLAKAYLEINNGSHTCANTGNSNQALIGKYGVAWIKRFVDNDTRYSPFLCGAPHQADLRSSALSEYRESCPY
1.3 Computational process of mutants and its binding energy
1.3.1. Constructing the spatial structure of BHET protein
Figure 1 The spatial structure of BHET protein
1.3.2 Construction of Binding Space Structure of BHET and R259A
Figure 2 Construction of binding space structure of BHET and R259A
1.3.3 Calculating the binding distance between BHET and R259A protein
Figure 3 Calculating the binding distance between BHET and R259A protein
1.4. Docking using AutoDock
Molecular of 13 protein mutants was docking with PET and BHET molecules.The middle part of Figures 1 and 2 represents the small PET and BHET molecules wrapped in 13 protein mutants.
Figure 4 Docking conformation of BHET in the active site of PETase.
Figure 5 Docking conformation of PET in the active site of PETase.
The binding ability of PET and BHET to 13 protein mutants was determined by calculation and distance.The top 7 by binding distance of BHET to 13 protein mutants：L61T + W132H，L61T + W132H + R259A，L61T，W132H + R259A，W132H + R259A，W132H，R259A；According to the binding distance between PET and 13 protein mutants, the top 4:T212F，L61T + W132H + R259A，W132H and WT.
Table 1 Mutants and its binding energy
Mutants Binding energy (kcal/mol)
BHET PET
1 WT -5.67 -5.02
2 Y60A -5.3 -3.5
3 L61T -5.9 -3.96
4 W132A -5.33 -3.62
5 W132H -5.84 -5.09
6 V181I -5.71 -4.29
7 T212S -5.51 -4.25
8 T212F -5.76 -5.63
9 R259A -5.78 -3.9
10 L61T + W132H -6.02 -3.93
11 L61T + R259A -5.84 -4.07
12 W132H + R259A -5.84 -3.18
13 L61T + W132H + R259A -5.9 -5.31
Figure 6 Binding energies of the PETase and mutants using BHET as substrate.
Figure 6 Binding energies of the PETase and mutants using PET as substrate.
二． Analysis of experimental results
We will combine the distances of PET, BHET and 13 protein mutants, and we will experimentally verify the optimal protein mutants to verification of mutant enzyme activity:WT, L61T, W132H, R259A, L61T + W132H, L61T + R259A, W132H + R259A, L61T + W132H + R259A. Results are as follows：
Figure 7 Verification of mutant enzyme activity experimental results
According to the figure, we found that the binding ability of L61T, W132H, R259A, L61T + R259A, W132H + R259A and L61T + W132H + R259A with BHET was higher than that of WT, while the binding ability of L61T + W132H with BHET was higher than that of WT.Compared with the data calculation, the binding ability of L61T+ W132H with BHET is significantly different.
At the same time, the binding ability of W132H, R259A, W132H + R259A and L61T + W132H + R259A with PET was higher than that of WT. According to the binding distance between PET and 13 protein mutants, L61T + W132H + R259A and W132H were consistent with the verification results. The binding ability of R259A, W132H + R259A with BHET was enhanced.