Analysis of intracellular amino acids in the fermentation of Pichia pastoris X-33 and KM71H

Analysis of extra- and intra- cellular metabolites is very important to study cell metabolish. Intracellular amino acids in yeast are of great interest as precursors of desired products in biomass production of the feed industries and the production of glutamate. In this study, the fermentations of P. pastoris X-33 and KM71H were carried out in shake flasks. After centrifugation, the harvested cells were mechanically disrupted by using glass bead. The supernatants were used to analyze intracellular amino acid by HPLC system. For HPLC analysis, Resolve C18 column was used with Fluorescence detector. The OPA(o-phthalaldehyde) derivation reaction was employed for analysing amino acids at 30 $^{\circ}C$, 1 ml/min and gradient mode. The concentration of intracellular protein was measured by spectrophotometer.

10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose가 첨가된 YPD 배지를 이용하여 200 rpm, 30 $^{\circ}C$에서 24시간 배양한 Pichia pastoris X-33과 KM71H를 각각 대수 증식기에 시료를 채취하여 4000 g, 4 $^{\circ}C$에서 5분간 원심분리하여 펠릿만을 얻어 멸균된 증류수를 500 ${\mu}{\ell}$ 넣고 4000 g, 4 $^{\circ}C$에서 5분간 원심분리하여 2-3회 세포 내 단백질과 아미노산 분석에 이용하였다. HPLC는 OPA 시약을 만들어 4 $^{\circ}C$에서 24시간 안정화시켜 사용하였고, 용액 A와 B를 미리 준비하여 기울기용리로 30 $^{\circ}C$에서 1ml/min, Ex 330/ Em 420 nm에서 80분간 분석하였다.