[High-level expression of PET depolymerases in Pichia pastoris and construction of a crude enzyme stabilization system].

Authors

Jiaxin Zhou, Haohong Lin, Xiujuan Qian, Anming Xu, Min Jiang

Published in

Sheng wu gong cheng xue bao = Chinese journal of biotechnology. Volume 42. Issue 9. Pages 4289-4304. Sep 25, 2026.

Abstract

Improving the production of polyethylene terephthalate (PET) depolymerases is essential for reducing the cost of enzymatic PET recycling and facilitating its industrial application. To enhance the production of PET hydrolases, the fungal-derived hydrolase HiC and the bacterial-derived hydrolase LCC^ICCG were employed as model enzymes. Strategies including recombinant strain construction, gene copy number optimization, molecular chaperone-assisted expression, fermentation scale-up, and crude enzyme stabilization were systematically investigated. The strains with secretory expression were generated by chromosomal integration, and multicopy transformants were obtained through high-stringency zeocin selection. To further improve protein production, we introduced PDI, ERO1, SEC53, SEC1, HAC1, and GCN4 as helper factors. GCN4 showed the strongest enhancement effect on HiC production, whereas ERO1 and HAC1 were more effective for LCC^ICCG. No significant synergistic effect was observed when ERO1 and HAC1 were co-expressed. In 5-L bioreactors, the optimized strains achieved the titers of 2.36 g/L for HiC and 1.72 g/L for LCC^ICCG. Furthermore, in a 50 L fermenter, the maximum extracellular protein concentration of HiC reached 8.5 g/L. A crude enzyme stabilization system composed of 0.1% Kathon, 10% glycerol, and 0.5% phenoxyethanol was subsequently established. After storage at room temperature for 30 days, HiC and LCC^ICCG retained 56.43% and 78.11% of their initial activities, respectively. The results demonstrate that coordinated optimization of expression and formulation can improve both the production and storage stability of PET depolymerases, thereby supporting their practical application in enzymatic PET recycling.

PMID:
42773676
Bibliographic data and abstract were imported from PubMed on 23 Sep 2026.

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