College of Food Science and Technology, Nanjing Agriculture University, 1 Weigang, Nanjing, 210095, China.
College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, 21003, China.
Appl Biochem Biotechnol. 2021 Jan;193(1):296-306. doi: 10.1007/s12010-020-03419-2. Epub 2020 Sep 21.
Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.
普鲁兰酶是一种能够特异性切割α-1,6 糖苷键的支链酶，被广泛应用于淀粉糖化、葡萄糖、麦芽糖和生物乙醇的生产。耐热普鲁兰酶是从各种微生物中分离出来的；然而，由于缺乏普鲁兰酶的工业生产，阻碍了实验室向工业的转化。在本研究中，通过改变 C/N 值和培养基的总能量，用廉价的玉米淀粉和玉米浆代替昂贵的麦芽糖糖浆和豆粕，在摇瓶中展示了 440 U/mL 的普鲁兰酶。随后，在 50-L 和 50-m 生物反应器中探索了培养条件。在分批培养中，普鲁兰酶的活性达到 896 U/mL，而通过控制溶解氧、pH 值、还原糖含量和温度，在补料分批培养中增加到 1743 U/mL。值得注意的是，根据补料分批培养的技术参数，将培养体积扩大到 50 m。普鲁兰酶的工业化生产取得了成功，活性达到 1546 U/mL。当产品在室温（25°C）下储存 6 个月时，普鲁兰酶的活性仍保持在 90%以上。在 60 和 80°C 下的半衰期分别为 119.45 h 和 51.18 h，满足了普鲁兰酶工业应用的要求。
