Protein-glutaminase (PG) can hydrolyze glutamine residues of protein side chains to produce ammonia and protein-L-glutamate, thus increasing negative charge and decreasing isoelectric point of protein to improve functional properties of protein. However, the original strain Chryseobacterium proteolyticum has low yield of PG (only 0.258 U/mL) and poor capability for genetic manipulation, so heterologous expression has been adopted in recent years to improve yield of PG. In this study, we successfully produced heterologous Pro-PG from Pichia pastoris GS115.Beyond heterologous expression, further experiments were launched to optimize the culture medium and identify the characteristics of recombinant PG. Results showed that trypsin incubated PG activity from the recombinant P. pastoris pPIC9K-Pro-PG /GS115 was promoted to 0.878 U/mL induced with additional 1% methanol for 120 h in shaking flasks. Moreover, the results of the enzyme characteristics study confirmed that the optimal temperature of the recombinant PG was 60℃, and the relative enzyme activity remained more than 80% under the condition of less than 60℃ for 1h. The optimal pH of PG was 6.0, and the relative enzyme activity remained above 70% at pH 3.0-8.0 for 1h.
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