Abstract：
Prenyltransferases catalyze the synthesis of prenylated flavonoids, providing these with greater lipid solubility, biological activity, and availability. In this study, a thermostable prenyltransferase (AfPT) from Aspergillus fumigatiaffinis was cloned and expressed in Escherichia coli. By optimizing induction conditions, the expression level of AfPT reached 39.3 mU/mL, which was approximately 200 % of that before optimization. Additionally, we determined the enzymatic properties of AfPT. Subsequently, AfPT was immobilized on carboxymethyl cellulose magnetic nanoparticles (CMN) at a maximum load of 0.6 mg/mg. Optimal activity of CMN-AfPT was achieved at pH 8.0 and 55 °C. Thermostability assays showed that the residual activity of CMN-AfPT was greater than 50 % after incubation at 55 °C for 4 h. Km and Vmax of CMN-AfPT for naringenin were 0.082 mM and 5.57 nmol/min/mg, respectively. The Kcat/Km ratio of CMN-AfPT was higher than that of AfPT. Residual prenyltransferase activity of CMN-AfPT remained higher than 70 % even after 30 days of storage. Further, CMN-AfPT retained 68 % of its original activity after 10 cycles of reuse. Compared with free AfPT, CMN-AfPT showed higher catalytic efficiency, thermostability, metal ion tolerance, substrate affinity, storage stability, and reusability. Our study presents a thermostable prenyltransferase and its immobilized form for the production of prenylated flavonoids in vitro.
摘要：
戊烯酰转移酶催化戊烯化黄酮类化合物的合成，为这些提供更大的脂溶性，生物活性,和可用性。在这项研究中,克隆了来自烟曲霉的热稳定的异戊烯基转移酶（AfPT），并在大肠杆菌中表达。通过优化诱导条件,AfPT的表达水平达到39.3mU/mL,大约是优化前的200%。此外,我们确定了AfPT的酶学性质。随后，将AfPT以0.6mg/mg的最大负载固定在羧甲基纤维素磁性纳米颗粒(CMN)上。CMN-AfPT的最佳活性在pH8.0和55°C下实现。热稳定性分析表明，在55℃孵育4小时后，CMN-AfPT的残留活性大于50％。CMN-AfPT对柚皮素的Km和Vmax分别为0.082mM和5.57nmol/min/mg，分别。CMN-AfPT的Kcat/Km比值高于AfPT。即使在储存30天后，CMN-AfPT的残留异戊二烯基转移酶活性仍高于70％。Further,CMN-AfPT在重复使用10个循环后保留了其原始活性的68％。与免费AfPT相比，CMN-AfPT显示出更高的催化效率，热稳定性,金属离子耐受性，底物亲和力，储存稳定性，和可重用性。我们的研究提出了一种热稳定的异戊烯基转移酶及其固定化形式，用于体外生产异戊烯化类黄酮。
