Sun Pengjie, Li Changgeng, Gong Yu, Wang Jinduo, Xu Qingyang
National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin, China.
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.
Front Bioeng Biotechnol. 2022 Aug 10;10:969668. doi: 10.3389/fbioe.2022.969668. eCollection 2022.
In order to solve the problems of high complexity, many by-products, high pollution and difficult extraction of the existing adenine production process, in this study, ceramic membrane-coupled mixed cell fermentation was used to produce adenine while reducing the synthesis of by-products and simplifying the production process of adenine. Nucleoside hydrolase (encoded by the gene) was used to produce adenine by coordinated fermentation with the adenosine-producing bacterium XGL. The adenosine hydrolase (AdHy)-expressing strain BL21-AdHy was successfully employed and the highest activity of the crude enzyme solution was found by orthogonal experiments at 170 W power, 42% duty cycle, and 8 min of sonication. The highest AdHy activity was found after 18 h of induction incubation. BL21-AdHy was induced for 18 h and sonicated under the above ultrasonic conditions and the resulting crude enzyme solution was used for co-fermentation of the strain and enzyme. Moreover, 15% (v/v) of the AdHy crude enzyme solution was added to fermentation of XGL after 35 h. Finally, the whole fermentation system was dialyzed using coupled ceramic membranes for 45 and 75 h, followed by the addition of fresh medium. In contrast, the AdHy crude enzyme solution was added after 35, 65, and 90 h of fermentation, with three additions of 15, 15, and 10% of the XGL fermentation system. The process was validated in a 5 L fermenter and 14 ± 0.25 g/L of adenine was obtained, with no accumulation of adenosine and d-ribose as by-products. The enzymatic activity of the AdHy crude solution treated with ultrasound was greatly improved. It also reduced the cellular activity of BL21-AdHy and reduced effects on bacterial co-fermentation. Membrane-coupled dialysis solved the problem of decreased yield due to poor bacterial survival and decreased viability, and eliminated inhibition of the product synthesis pathway by adenosine. The batch addition of crude enzyme broth allowed the continuous conversion of adenosine to adenine. This production method provides the highest yield of biologically produced adenine reported to date, reduces the cost of adenine production, and has positive implications for the industrial production of adenine by fermentation. And it provides a reference for producing other high-value-added products made by fermentation.
为了解决现有腺嘌呤生产工艺复杂度高、副产物多、污染大及提取困难等问题，本研究采用陶瓷膜耦合混合细胞发酵法生产腺嘌呤，同时减少副产物合成并简化腺嘌呤生产工艺。利用核苷水解酶（由基因编码）与产腺苷菌XGL协同发酵生产腺嘌呤。成功构建了表达腺苷水解酶（AdHy）的菌株BL21-AdHy，并通过正交实验确定在功率170W、占空比42%、超声处理8min条件下粗酶液活性最高。诱导培养18h后AdHy活性最高。将BL21-AdHy诱导培养18h后在上述超声条件下进行超声处理，所得粗酶液用于菌株与酶的共发酵。此外，在35h后向XGL发酵体系中添加15%（v/v）的AdHy粗酶液。最后，整个发酵体系用耦合陶瓷膜透析45h和75h，随后添加新鲜培养基。相比之下，在XGL发酵35、65和90h后添加AdHy粗酶液，分三次添加，每次添加量分别为XGL发酵体系的15%、15%和10%。该工艺在5L发酵罐中得到验证，获得了14±0.25g/L的腺嘌呤，且无副产物腺苷和d-核糖的积累。超声处理后的AdHy粗酶液酶活性得到极大提高。同时降低了BL21-AdHy的细胞活性及对细菌共发酵的影响。膜耦合透析解决了因细菌存活率低和活力下降导致产量降低的问题，并消除了腺苷对产物合成途径的抑制。分批添加粗酶液可使腺苷持续转化为腺嘌呤。该生产方法提供了迄今为止报道的生物法生产腺嘌呤的最高产量，降低了腺嘌呤生产成本，对腺嘌呤的发酵工业化生产具有积极意义。并且为发酵生产其他高附加值产品提供了参考。
