Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. 
Because this property of IBPs can increase the freeze-thaw survival of cells, 
IBPs have attracted the attention from industries for their potential use in 
biotechnological applications. However, their use was largely hampered by the 
lack of the large-scale recombinant production system. In this study, the 
codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and 
subjected to high-level expression in methylotrophic Pichia pastoris system. In 
a laboratory-scale fermentation (7 L), the optimal induction temperature and pH 
were determined to be 25 °C and 6.0, respectively. Further, employing glycerol 
fed-batch phase prior to methanol induction phase enhanced the production of 
recombinant LelBP (rLeIBP) by ∼100 mg/l. The total amount of secreted proteins 
at these conditions (25 °C, pH 6.0, and glycerol fed-batch phase) was ∼443 mg/l, 
60 % of which was rLeIBP, yielding ∼272 mg/l. In the pilot-scale fermentation 
(700 L) under the same conditions, the yield of rLeIBP was 300 mg/l. To our best 
knowledge, this result reports the highest production yield of the recombinant 
IBP. More importantly, the rLeIBP secreted into culture media was stable and 
active for 6 days of fermentation. The thermal hysteresis (TH) activity of 
rLeIBP was about 0.42 °C, which is almost the same to those reported previously. 
The availability of large quantities of rLeIBP may accelerate further 
application studies.