菌丝霉素抗菌肽高密度发酵生产及其特性研究

Study on High-density Fermentation Production and Characteristics of Plectasin Antimicrobial Peptide

[目的]开发菌丝霉素抗菌肽,以替代抗生素在饲料生产中添加使用。[方法]以高产菌丝霉素毕赤酵母基因工程菌PPle-BC01为供试菌株,在15 L实验室发酵罐优化发酵工艺的基础上,通过50 L、5 t、60 t发酵罐高密度发酵工艺进一步优化放大试验,实现了60 t罐规模的稳定生产。经层析柱分离、纯化,得到相对纯度≥90%的纯品。纯品水解后的游离氨基酸样品,经PITC衍生化处理后,再经过HPLC检测及氨基酸组成摩尔百分比计算与氨基酸混合标准品比对,分析其氨基酸组成。纯品经多肽蛋白质相对分子质量分析测试,测得plectasin单同位素相对分子量。纯品经蛋白浓度、抗菌效价测定、比活计算以及产品抗菌效价测定,获得产品中plectasin含量,同时测试了解其产品特性。[结果]60 t发酵罐稳定3批次试生产发酵液菌体湿重峰值分别为43.34%、43.78%和43.78%,抗菌效价峰值分别为10566.99、10986.45和10788.56 U/mL;喷雾干燥产品收率分别为12.19%、12.30%和12.50%,抗菌效价分别为100672.10、103561.20和99765.50 U/g。3批次试生产产品中plectasin含量分别为2.26%、2.32%和2.24%。纯品经检测符合plectasin由40个氨基酸组成的多肽的理论氨基酸残基个数值,分子量为4.4407 kD。产品在95℃温度以下具有良好的热稳定性;pH 3.0~9.0具有良好的酸碱稳定性;在0.03%~0.30%猪胆盐浓度具有较好的胆盐耐受性;对胃蛋白酶、胰蛋白酶和蛋白酶K均具有良好的抗降解性。[结论]发酵终产品收率达10%,产品抗菌效价达8万U/g以上,产品中plectasin含量达2%以上,实现了60 t罐规模的产业化稳定生产。

[Objective]To develop plectasin antimicrobial peplide to replace antibiotics in feed production.[Method]In this study,a high-yield plectasin-producing Pichia pastoris strain PPle-BC01 was used as the experimental strain,the steady production of 60-ton fermenters were realized by the scale-up experiments of high-density fermentation process in 50 L,5 t and 60 t fermentor that based on the optimization of fermentation process in 15-liter fermentor.The purified sample of plectasin with relative purity above 90% was obtained by chromatographic column separation and purification.The amino acid composition of the purified sample were analyzed by HPLC determination,calculation of molar percentage of amino acid composition and comparison with reference standards for amino acid mixtures after it’s hydrolyzed free amino acid sample and reference standards for amino acid mixtures were derivatized by PITC.The single-isotope relative molecular weight of plectasin was measured by the relative molecular mass analysis of polypeptide proteins.The content of plectasin in spray-dried products were obtained by determining the concentration,antibacterial titer of pure protein and the antibacterial titer of spray-dried products.The characteristics of spray-dried products were known through the property test.[Result]Peak values of wet weight and antibacterial titer of fermentation broths were 43.34%,43.78%,43.78% and 10566.99,10986.45,10788.56 U/mL,the yield and antibacterial titer of spray-dried products were 12.19%,12.30%,12.50% and 100672.10,103561.20,99765.50 U/g,the content of plectasin in spray-dried products were 2.26%,2.32% and 2.24%,respectively in stable three batchs trial production of 60-ton fermentors.The purified sample was determined to be composed of 40 amino acids and the molecular weight was 4.4407 kD.The spray-dried product had good thermal stability under 95℃,had good acid-base stability in the pH 3.0-9.0 range,had good tolerance at the salt concentration of 0.03%-0.30%in pig bile,had good anti-degradability to pepsin,trypsin and proteinase K.[Conclusion]The yield of the final spray-dried products reached 10%,the antibacterial titer was over 80000 U/g,the content of plectasin in spray-dried products was more than 2%.The industrial and stable production of 60-ton fermenters was realized.