Fermentation Optimization For The Production Of β-glucosidase In Picha Pastoris

β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21, BGL) is a kind of hydrolase that is capable of catalyzing the cleavage of non-reductiveβ-D-glucoside bonds at the end of the sugar chains and it has been widely used in cellulose degradation. With in-depth research,β-glucosidase can also display transglycosylation activity as catalyze the transfer of sugar residues from one activated donor substrate to another accepter molecule and it is used in enzymatic synthesis of gentiooligosaccharide, salidroside and some other products.Some significant results have been got in our previous study: theβ-glucosidase gene from Aspergillus Niger CMI CC324626 has been cloned and expressed in Picha pastoris and the recombinant enzyme was utilized for the enzymatic synthesis of gentiooligosaccharide. In the present study, the engineered P. pastoris was cultivated in a 3L bioreactor and fermentation conditions were optimized in detail to improveβ-glucosidase production. At the same time, protein disulfide isomerase (PDI) was co-expressed to improve the nascent chain folding andβ-glucosidase production. In the following experiment, mixed carbon sources of sorbitol and methanol were fed and the induction temperature was reduced in the induction phase to improve the cell activity and reduce cell mortality. The major findings are as follows:(1) The fermentation optimization of the engineered P. pastoris was performed in a 3L bioreactor to improve the production ofβ-glucosidase. The results showed that: properly reduced initial cell density could effectively cut down the fermentation period and attain the maximum yield ofβ-glucosidase production; the expression of alcohol oxidase (AOX) was induced and methanol metabolism started when the carbon source was switched from glycerol to methanol, proper methanol concentration controlled in the induction phase could effectively reduce the toxicity to cells and improve foreign protein expression; at the same time, the control over the oxygen concentration during the induction process, not only prevented damage to cells by acetic acid and ethanol acid accumulated due to a lacking of dissolved oxygen, but also avoided possible impairments to the cells caused by an overdose of dissolved oxygen which could lead to a accumulation of ROS intracellular, influencing the expression of foreign proteins.Finally, the optimized condition was obtained as following: when the initial cell concentration reached DCW of 38.4g/L, 2% (v/v) of methanol was added and methanol concentration was controlled at 0.5%(v/v) when 2% of metnanol consumed, dissolved oxygen concentration was maintained between 20% and 30% throughout the induction phase. In this condition,β-glucosidase activity and productivity reached 1854 U/mL and 17.2 U/ (mL·h).(2) However, the results showed that, with the improved expression of target protein, the specific activity ofβ-glucosidase in the culture medium decreased, indicating a probable mis-folding of protein. In order to improve the protein folding environment, protein disulfide isomerase (PDI) was co-expressed in the engineered P. pastoris. The pdi gene (NCBI accession number AJ302014) was amplified using the genome of the P. pastoris as the template and cloned into the expression vector pPICZαA; the recombinant plasmid was lineared and electroporated into the P. pastoris withβ-glucosidase gene. After the identification of pdi instert into the genome, the recombinant P. pastoris was cultivated in shake-flasks and 3L bioreactor. The results showed that, finalβ-glucosidase activity was 2-fold to the control in shake-flask cultivation; alsoβ-glucosidase activity reached 2523 U/mL in the 3L bioreactor fermentation which was 1.36-fold to that without PDI co-expression, the specific activity achieved 712 U/mg which was 1.25-fold to that of control when cultivated in the same condition.(3) With the successful co-expression of protein disulfide isomerase, the production ofβ-glucosidase was effectively improved; however, cell growth was inhibited after 60h induction of methanol, which, to some extent, suppressed the expression ofβ-glucosidase during the later period. In order to improve cell activity and cut down the cell mortality, mixed carbon sources of sorbitol and methanol were fed in the induction phase and induction temperature was reduced. The results suggested that, co-feded mixed carbon sources and reduced induction temperature were benefit for cell growth but not for the improvement ofβ-glucosidase production.