In this research, we aimed to develop an overall process suitable for large-scale production by constructing an endoprotease Kex2 expressing strain and further promoting its industrial application.Kex2 gene from Saccharomyces cerevisiae was selected and cloned into plasmid pPICZαA, the resulting plasmid pPICZαA/Kex2 was electrotransformed into strain X33 of Komagataella phaffii.The transformants were screened from agar plates containing antibiotic (zeocin) and further confirmed by PCR.Subsequently, the transformant was cultured in a 5 L bioreactor and induced with methanol, the culture was centrifugated and the supernatant was collected and fractioned by ion-exchange column, purified Kex2 was preserved in 10% (volume fraction) glycerol and lyophilized powder, respectively.PCR results showed that the X33 strain was successfully constructed, and the recombinant X33-Kex2 strain was cultured in a 5 L aeration bioreactor, after induction with methanol for 60 hours protein accumulation in the supernatant was 2.53 g/L.The supernatant was ultrafiltrated, and dialyzed consecutively using hollow fiber columns, the conductivity of the resulting Kex2 solution was low enough to be loaded directly onto the ion-exchange purification system.The specific activity of purified Kex2 was identified as 20.24 U/mg with a total recovery rate of 41.6% from the culture supernatant.Stability results showed the remaining specific activities of Kex2 in 10% glycerol which were preserved at -20 ℃ and 2-8 ℃ were 77.7% and 82.9% respectively after three months, while in lyophilized powder these were 86.2% and 77.8% respectively, demonstrating a relatively excellent stability.The recombinant strain, purification procedure, and the resulting Kex2 in this study are of important significance in reducing the application cost of Kex2.
DOU Peichong
,
DAI Shuchun
,
ZHANG Lianlian
,
SU Jingyun
,
DONG Guoming
,
ZHU Mengdan
,
ZHONG Yuanguang
,
ZHANG Mingyi
. Recombinant expression of Kex2 in Komagataella phaffii and its purification, storage stability[J]. Food and Fermentation Industries, 2025
, 51(19)
: 26
-32
.
DOI: 10.13995/j.cnki.11-1802/ts.041813
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