毕赤酵母工程菌高密度发酵中氨态氮含量的测定

Determination of Ammonia Nitrogen in High Density Fermentation of Pichia Engineered Strain

首次尝试将钠氏法应用于毕赤酵母高密度发酵中,并与靛酚蓝检测法进行了比较,证实钠氏法在0-10 mg/L的范围内,相关性好（r^2=0.996 5）,精确度（RSD=2.14%-5.32%）和精密度（Prec ision=97%-105%）高,更适合于毕赤酵母发酵中氨态氮含量的检测。并对其检测条件进行优化,确定检测波长为400 nm,最佳的显色反应为10 min。该方法用于毕赤酵母高密度发酵表达重组人血清白蛋白胸腺肽的结果表明,在发酵液氨基氮含量低于1.5 mg/kg,重组目的蛋白的表达出现了明显的降解,改善并控制发酵液中氨态氮的含量后,可以明显控制目的蛋白的降解,提高产品的产率。结果证实该方法用于发酵液中氨态氮的检测时,准确可靠,切实可行。

This is the first attempt to use Nessler reagent in high density fermentation by Pichia and compare with determination method of indophenol blue, and confirmed that in the range of 0 - 10 mg/L, Nessler method was more suitable for determining ammonia nitrogen in fermentation by Pichia with good reciprocity （ r^2 = 0. 996 5 ） , accuracy （RSD =2.14% -5.32% ）, and precision （ = 97% -105% ）. The determination conditions were also optimized, and confirmed the wavelength was at 400 nm, and the best color developing reaction was 10 minutes. The results when this method was used in high density fermentation by Pichia to express recombinant human serum albumin thymic peptide indicated that when ammonia nitrogen in fermentation broth was below 1.5 mg/kg, the expression of target recombinant protein was apparently degraded. After improve and control ammonia nitrogen in fermentation broth, the degradation of target protein could be apparently controlled to improve the production rate of the product. The results confirmed that using this method to determine ammonia nitrogen in fermentation broth accurate, reliable, and practically feasible.