高山被孢霉ATCC 32222中细胞色素b5还原酶I的克隆、表达和功能鉴定

Expression, purification and characterization of cytochrome b5 reductase I from Mortierella alpina ATCC 32222

摘要:

【目的】鉴定产油微生物高山被孢霉ATCC 32222中细胞色素b5还原酶I的功能。【方法】将高山被孢霉ATCC 32222中膜结合细胞色素b5还原酶I基因与人可溶性细胞色素b5还原酶基因序列比对，去除该基因N端穿膜区域后，与人可溶性细胞色素b5基因分别在大肠杆菌中异源表达；通过钴离子亲和层析、离子交换和分子排阻色谱等方法对表达产物进行纯化；以2,6-二氯靛酚钠(DCIP)为底物，测定细胞色素b5还原酶I的体外活性及其对NADH和NADPH的偏好性；在反应体系中存在NADH时，通过全波长扫描方法检测细胞色素b5还原酶I与细胞色素b5的相互作用。【结果】高山被孢霉ATCC 32222中膜结合细胞色素b5还原酶I被成功可溶表达，经纯化后检测到体外活性：使用NADH时酶活为564.57 U，使用NADPH时为51.97 U；在NADH存在时，细胞色素b5还原酶I能够还原细胞色素b5，其吸收峰从411 nm偏移至422 nm，并在521 nm和554 nm处吸光值增加。【结论】细胞色素b5还原酶I N端穿膜区域的去除增加了其可溶性，并保持了蛋白质活性；高山被孢霉ATCC 32222中细胞色素b5还原酶I基因编码的是一种NADH-细胞色素b5还原酶，其在体外能与细胞色素b5相互作用。

Abstract:

[Objective] This study focus on the functional characterization of membrane-bound cytochrome b5 reductase I from Mortierella alpina ATCC 32222. [Methods] We identified the N-terminal transmembrane region in the M. alpina cytochrome b5 reductase I through sequence alignment. The truncated M. alpina cytochrome b5 reductase I gene and human soluble cytochrome b5 gene were expressed in Escherichia coli BL21. The proteins were purified using Cobalt affinity, ion exchange and gel filtration. The activity of purified cytochrome b5 reductase I was determined using DCIP as substrate in the presence of NADH or NADPH. The interaction between purified cytochrome b5 reductase I and cytochrome b5 was monitored by wavelength scan. [Results] The truncated M. alpina cytochrome b5 reductase I was successfully expressed as a soluble protein and purified to homogeneity. The resulting protein was active and able to reduce cytochrome b5 in the presence of NADH. The reduction of cytochrome b5 is characterized by a peak shift from 411 nm to 422 nm and an increase in absorbance at 521 nm and 554 nm. [Conclusion] The deletion of the N-terminal transmembrane region increased the solubility of M. alpina cytochrome b5 reductase I and the activity was maintained. Functional characterization revealed that M. alpina cytochrome b5 reductase I gene encoded a NADH-cytochrome b5 reductase, and it’s able to interact with human cytochrome b5 in vitro.