A kind of rhamnosidase tperha mutant and its preparation method and application
A rhamnosidase, tperha-k579a technology, applied in the field of biomedicine, can solve the problems of unknown content of icariside I and inability to efficiently catalyze conversion, and achieve the effect of short enzymatic hydrolysis time and high conversion rate
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Embodiment 1
[0048] Establishment of TpeRha enzyme tertiary structure model and determination of mutation sites:
[0049] (1) Establish a three-dimensional structural model of TpeRha
[0050] The homology modeling tool MODELLER was used to carry out homology modeling on TpeRha. After the model was evaluated, a reliable 3D structural model was obtained according to the scoring criteria. The 3D structural model is as follows: figure 1 shown.
[0051] (2) Determination of mutation sites
[0052] Such as figure 2 As shown, the three-dimensional structure model of TpeRha obtained by homology modeling was established based on the three-dimensional structure model of Dictyoglomusthermophilum α-L-rhamnosidase DtRha (PDB ID: 6i60). Studies have shown that in the crystal structure model of DtRha enzyme and glycoside ligand, there are 11 amino acid residues that interact with rhamnosyl. The conformational comparison of the TpeRha enzyme and the three-dimensional structure model of the DtRha enzy...
Embodiment 2
[0054] Obtaining of recombinant wild enzyme strain BL21(DE3) / pET-28a-TpeRha:
[0055] Optimize the α-L-rhamnosidase gene from Thermotoga petroleum DSM 13995, its nucleotide sequence is as SEQ ID NO: 2 (GenBank: ABQ47687.1), connect it to the pET-28a plasmid, and obtain the recombinant The plasmid was named pET-28a-TpeRha, such as image 3 As indicated, the plasmid was transformed into Escherichia coli BL21 (DE3), and the recombinant strain was named BL21 (DE3) / pET-28a-TpeRha. The amino acid sequence of the wild type TpeRha enzyme expressed by the recombinant wild enzyme strain is shown in SEQ ID NO:1.
Embodiment 3
[0057] Obtaining of TpeRha mutant strains:
[0058] (1) Construction of mutant vectors by whole plasmid PCR
[0059] 1. A small amount of plasmid extraction of the recombinant plasmid pET-28a-TpeRha;
[0060] 2. Design mutation primers. The primers have a 15 bp overlapping region and a 15 bp extension region, and the mutation site is designed in the overlapping region. The plasmid pET-28a-TpeRha was used as a template for PCR amplification of the whole plasmid, and the PCR system is shown in Table 1:
[0061]
[0062] The primers Primer-F and Primer-R are PCR upstream primers and downstream primers designed according to different mutation sites, and the specific primer information is shown in Table 2.
[0063]
[0064] The primer pair H130A-F and H130A-R are used to obtain the mutant TpeRha-H130A, the amino acid sequence of the mutant TpeRha-H130A is SEQ ID NO:9.
[0065] The primer pair R369A-F and R369A-R are used to obtain mutant TpeRha-R369A, the amino acid sequen...
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