Science1 publisher2 min readPublished
ANU chemists nearly doubled a PET enzyme's activity by swapping a CH group for nitrogen
A single azatryptophan, which differs from tryptophan by one nitrogen, raised PET breakdown in an already heavily engineered hydrolase while thermal stability held. The team also built a minutes-long activity assay.
The Scientist · Science desk
What happened
- An Australian National University team reports in Angewandte Chemie International Edition that a PET-degrading enzyme carrying one substituted amino acid broke the plastic down nearly twice as efficiently while keeping its thermal stability.
- The substituted residue is azatryptophan, which matches tryptophan except that a carbon-hydrogen group in the side chain is replaced by a nitrogen atom, and it was installed at a single site.
- The PET hydrolases used as the starting point had already been through extensive optimisation with computational design and protein engineering, so the gain came on top of heavy prior tuning.
- The same group built a fluorescence test called PETra that measures PET-degrading activity in minutes, where conventional activity assays need hours or days.
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Why it matters
- capability Fast ranking moves the slow step in this design loop from measurement to protein production, because every candidate now has to have a noncanonical amino acid installed before it can be tested.
- decision A group sitting at its activity-stability ceiling can spend its next round choosing a site and an isostere, and that choice requires expression machinery for noncanonical amino acids that a standard mutagenesis lab may not run.
- constraint Recycling economics turn on enzyme cost per tonne of PET and on how fast real flake releases its monomers, and an activity doubling measured on bench substrates sets neither number.
- precedent If one-atom substitutions pay off in an enzyme already this well tuned, they become a routine axis to screen in other engineered proteins, which is the extension the authors point to for manufacturing, biotechnology and medicine.
The paper presents the work as isosteric engineering of enzymes, getting past activity-stability trade-offs by site-selective CH to N substitutions [16]. Isosteric is literal here: azatryptophan occupies close to the same space as the tryptophan it stands in for, differing by a nitrogen where a carbon-hydrogen group sat [3]. A side chain that keeps its shape leaves the fold unlikely to shift, so stability was expected to survive the change.
"Enzymes can be engineered over and over again to improve their performance, but eventually you reach a point where making them more active can also make them less stable, and vice versa," said Elwy Abdelkader of the Research School of Chemistry at the Australian National University, the study's lead author [5][8]. "Instead of redesigning an enzyme with many mutations, we can make a very small, targeted change and have a significant effect on how it works," Abdelkader said [7].
Taken at face value, a twofold increase gives the same conversion at half the enzyme loading, or in half the time, when rate is linear in the amount of enzyme present [17]. PET does not make that easy to assume, because the polymer is insoluble and structurally complex and the enzyme has to work on a solid surface [11][4]. The phys.org report does not state the assay temperature, the identity of the engineered PETase, or the numerical correlation between the new screen and solid-PET degradation [18].
That screen, PETra, is the part of this work I would expect to change practice first. It puts candidates in the right order, which is what a design cycle needs; yield on bottle flake is beyond it. It substitutes a soluble fluorescent mimic for the polymer, and the authors report that its results correlate strongly with how effectively the enzymes break down solid PET [11][12].
"Proteins are incredibly powerful machines, but we are generally limited to the 20 amino acids found in nature when we engineer them," said Thomas Huber, a co-author [13][20]. The researchers say the same site-selective strategy could be applied to enzymes for sustainable manufacturing, biotechnology and medicine [15].
What to watch
- Whether the full Angewandte Chemie paper reports the assay temperature, the parent enzyme and a correlation coefficient for PETra against solid PET.
- Whether a second azatryptophan site adds further activity or finally costs thermal stability.
- Whether anyone publishes yields and costs for expressing azatryptophan-containing PETases outside a research-scale flask.