State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, P.R. China.
J Microbiol Biotechnol. 2021 Apr 28;31(4):570-583. doi: 10.4014/jmb.2101.01039.
α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100°C), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. , a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of α-amylase in through three strategies. Initial experiments showed that co-expression of molecular chaperone peptidyl-prolyl -trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90°C for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.
α-淀粉酶可以在高温条件下（~100°C）水解淀粉和相关碳水化合物中的α-1,4 键，在广泛的工业应用中具有巨大的潜力，但其在异源宿主中的相对较低的生产力限制了其工业应用。作为一种革兰氏阳性菌，由于其非致病性和强大的分泌特性，已被广泛应用于工业生产。本研究通过三种策略来提高在中的α-淀粉酶的产量。初步实验表明，通过基因组整合方式共表达分子伴侣肽基脯氨酰顺反异构酶，使用 CRISPR/Cas9 系统，可以提高可溶性淀粉酶的产量。因此，考虑到天然α-淀粉酶是在高温环境中产生的，并且具有高度的热稳定性，对完整培养物进行 90°C 热处理 15 分钟，从而大大提高了可溶性淀粉酶的产量。在优化培养条件（氮源、碳源、金属离子、温度和 pH）后，在 3-L 发酵罐中的实验产生了 3,806.7 U/ml 的可溶性活性，分别是未经热处理（1,155.1 U/ml）和空载表达载体对照（135.1 U/ml）的 3.3 倍和 28.2 倍。这代表了迄今为止报道的最高α-淀粉酶产量，应该会促进淀粉液化过程和相关工业生产的创新。同时，热处理可能通过传递动能促进聚集的α-淀粉酶折叠成可溶性、活性形式，这对于从嗜热古菌生产蛋白质可能具有普遍意义。
