Study On Optimal Fermentation Condition For The Expression Of Bioactive Opioid Peptides By Pichia Pastoris

The opioid peptides perform extensive physiologic functions in animals, such as improvingappetite, easing pain, sedating, restraining the motility of gastrointestinal and the secretion ofpeptic, promoting the absorption of electrolyte,regulating the incretion and immunity,cardiovascular function, Its potential availability in medicine,food and animal husbandry madeit become more and more widely used for its advantages of high-effective and nonpoisonouscharacteristics. The main intention of the research was investigated the opioid peptidesexpressed effectively by a recombinant Pichia pastoris strain.The fermentation of a recombinant Pichia pastoris was studied. The growth conditions wasoptimized using orthogonal design and the results as follow: glycerol 24g/L, yeast extract 11g/L,peptone 22g/L, YNB 20ml/L, initial pH 5.0, temperature 30℃,shaking speed 250r/min. Underall above conditions, the final OD₆₀₀ was able to be 2.416 and the weight of dry cell was 18.24g/L.The methods for cell fragmentation of recombinant Pichia pastoris were investigated. Themost efficient way was the ultrasonic method with beadings being added. The optimal conditionfor the cell fragmentation pH is 6.0.After fragmentating, the supernatant was collected bycentrifugation. The supernatant was first treated by 10% TCA for the purpose of sedimentationof large molecule protein, and then treated by Sephadex G-10 chromatography, as a result, 6peaks were acquired. The collections of every peaks were examined via intro in guinea pigileum, and found that the peak 1, 3, 4 which had opiate activities were the object peptides.Most current quantitative analyses of opioid peptide widely used are not able to measure theunknown opioid peptides. Since many opioid peptides having N-terminal tyrosyl residue of theiramino acid sequence, a fluorescence derivatisation reaction for only N-terminal Tyr-containingpeptides was employed in the quantification of opioid peptides.The determination of bioactive of opioid peptides was to compare the amount of theproportion of peak 1, 3, 4. The optimum fermentation conditions of opioid peptides expressedby Pichia pastoris were as follow: initial OD600 2.436, methanol concentration 0.6%(v/v),adding of the methanol 1time/10h, expression time 96h, pH 6.25, temperature 24℃,shakingspeed 250r/min, and from 48h of induction to the end of fermentation at 96h, glycerol 1g/L wasadded. As a result, the N-terminal tyrosine-containing peptides was increased from 196mg/L to537mg/L in 250ml flask.