Honda Kohsuke, Hara Naoya, Cheng Maria, Nakamura Anna, Mandai Komako, Okano Kenji, Ohtake Hisao
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Metab Eng. 2016 May;35:114-120. doi: 10.1016/j.ymben.2016.02.005. Epub 2016 Feb 18.
Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h.
嗜热酶出色的热稳定性和操作稳定性能够极大地提高生物催化在各个工业领域的适用性。然而,嗜热酶通常与热不稳定的底物、产物及辅因子不相容,因为它们在高温下表现出最大活性。尽管烟酰胺腺嘌呤二核苷酸(磷酸)(NAD(P)(+))和烟酰胺腺嘌呤二核苷酸(磷酸)还原型(NAD(P)H)在广泛的酶促氧化还原反应中发挥着关键作用,但它们在高温下的稳定性相对较低,这往往成为利用嗜热酶进行生物催化化学制造长期运行的主要障碍。在本研究中,我们通过八种嗜热酶的组合构建了一条体外人工代谢途径,用于从其降解产物中补救合成NAD(+)。这些酶在重组大肠杆菌中异源表达,重组细胞经热处理后的粗提物直接用作酶溶液。当在60°C下用实验优化浓度的酶进行孵育时,NAD(+)浓度可在15小时内几乎保持恒定。