The strain 4 with the highest enzyme activity was selected to optimize the culture conditions. As showcased in Fig. 3（a）， the enzyme activity of Abf62A was ow with 36 h， implying that P. pastoris was in the cell growth period， with less protein expression and low enzyme activity. Subsequently， the enzyme activity increased continuously， and the enzyme activity reached the highest at 66 h. However， the enzyme activity began to decline instead of rising for 72 h. The decline in enzyme activity beyond 66 h is attributed to proteolytic degradation during prolonged fermentation， a well-documented phenomenon in Pichia pastoris systems ［21］. While protein aggregation may contribute to activity loss， protease-mediated hydrolysis is recognized as the primary factor for heterologous protein instability in late-phase cultures ［21］. Therefore， 66 h was established as the optimal cultivation period to maximize functional enzyme yield.

The cell growth and cell metabolism of P. pastoris will be substantially influenced by temperature. As showcased in Fig. 3（b）， the enzyme activity of Abf62A expressed at 24-36 ℃ basically increased gradually， acquiring the highest at 30 ℃， and then declined with the increase of reaction temperature， but the enzyme activity of Abf62A expressed at 26 ℃ was also very high as 94.3%， which was due to the inconsistency between the optimum growth temperature of P. pastoris and the optimum temperature for protein expression. The optimum temperature for yeast growth was generally 30 ℃， while the suitable temperature for protein expression was lower， between 20 ℃ and 28 ℃. At 30 ℃， the expression of natural protein was high. While the protein expression of single yeast was high at 26 ℃， but the cell concentration was low， which led to a small difference in enzyme activity of Abf62A cultured at these two temperatures. Accordingly， the appropriate culture temperature was 30 ℃.