Difference between revisions of "Part:BBa K4275009"

Revision as of 09:56, 10 October 2022

MHETase

As the second enzyme of the PET degradation two-enzyme system found in Ideonella Sakaiensis, the MHETase catalyzes the cleavage of the ester bond in MHET(Monohydroxyethyl terephthalate) and liberates the final products of PET degradation - EG(ethylene glycol) and TPA(terephthalic acid) into the solution. The MHETase amino acid sequence used in this experiment is the same with the wild-type MHETase (GenBank Accession number: GAP38911.1). The enzyme MHETase works in synergy with PETase, the first enzyme in the two-enzyme PET degradation system found in Ideonella Sakaiensis - a PET-degrading and assimilating strain of bacteria isolated from a water treatment plant.

Usage and Biology

The 3D structure of MHETase consists of a catalytic domain and an extensive lid domain. The catalytic domain adopts the α/β-hydrolase fold typical of a serine hydrolase, with an active site consist the catalytic triad Ser225, Asp492, and His528, which shows significant structural conservation compared to PETase. The lid domain partially covers the active site, hinders its capability to bind with PET fiber, and harbors a well-coordinated calcium cation.

Design consideration

1. The first 39 amino acids in the N' terminal (including the leader sequence) is omitted and replaced with a single start codon.

2. 6xHis affinity purification tag (HHHHHH) is added to the C' terminal of the codon sequence.

3. DNA sequence is codon-optimized based on the codon-usage table of E.Coli Strain K12.MG1655.

Sequence and Features