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首页> 外文期刊>Biotechnology Progress >Liposomal Encapsulation of Yeast Alcohol Dehydrogenase with Cofactor for Stabilization of the Enzyme Structure and Activity
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Liposomal Encapsulation of Yeast Alcohol Dehydrogenase with Cofactor for Stabilization of the Enzyme Structure and Activity

机译：酵母乙醇脱氢酶与辅助因子的脂质体封装，用于稳定酶的结构和活性

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Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD~+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD~+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD~+ concentrations were 2.3 mg/ mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD~+ was examined on the thermal stability of YADH at 45 and 50 °C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD~+ as well as the free YADH with and without NAD~+. Free YADH was increasingly deactivated during its incubation at 45 °C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD~+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD~+ was lowered at 50 °C. The turbidity measurements for the above free YADH solution with and without NAD~+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 °C than at 45 °C even in the presence of NAD~+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD~+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 °C much higher than those of the above free enzyme systems, YADH/NAD~+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD~+ at 50 °C than at 45 °C. This was considered to be because even at 50 °C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD~+ in liposomes.
机译：酵母乙醇脱氢酶（YADH）及其辅因子烟酰胺腺嘌呤二核苷酸（NAD〜+）可以稳定地包裹在由POPC（1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱）组成的脂质体中。含YADH和NAD +的脂质体（YADH-NADL）的平均直径为100 nm。脂质体的YADH和NAD〜+浓度分别为2.3 mg / mL和3.9 mM。研究了脂质体包封和NAD〜+的存在对YADH在45和50°C下的热稳定性的协同作用。将YADH-NADL的酶稳定性与含有3.3 mg / mL YADH（不含NAD〜+）和游离YADH（含和不含NAD〜+）的脂质体YADH（YADHL）的稳定性进行了比较。由于四聚体YADH分解成亚基，游离YADH在45°C孵育2 h期间逐渐失活，酶浓度从3.3 mg / mL降低至0.01 mg / mL。在该温度下，3.9 NM的游离NAD〜+的共存通过形成其热稳定复合物而提高了2.3 mg / mL的游离YADH的稳定性，尽管在50°C下NAD〜+的稳定作用降低了。在有和没有NAD〜+的情况下，上述游离YADH溶液的浊度测量表明，即使在存在NAD〜+的情况下，酶三级结构的变化在50°C比在45°C更明显。这表明由于YADH与NAD +的固有亲和力降低，因此YADH易于在游离溶液中失活。另一方面，两种脂质体酶系统YADH-NADL和YADHL在45和50°C时都显示出比上述游离酶系统YADH / NAD〜+和YADH更高的稳定性。这些结果暗示脂质体膜稳定了酶的三级和四级结构。此外，YADH-NADL的酶活性显示出比YADHL更高的稳定性，在50°C时的NAD〜+作用比在45°C时更为显着。认为这是因为即使在50°C时，脂质膜对脂质体YADH的三级和四级结构的稳定作用也使该酶与脂质体中的NAD〜+形成了热稳定的复合物。

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