胆固醇氧化酶PsCO<sub>4</sub>异源表达、纯化及酶学性质分析 <sup>*</sup>

doi:10.13523/j.cb.20180605

中国生物工程杂志

2018, Vol. 38

Issue (6): 34-42 DOI: 10.13523/j.cb.20180605

研究报告

胆固醇氧化酶PsCO₄异源表达、纯化及酶学性质分析 ^*

郭倩倩,高登科,程晓涛,路福平,田之仓优(

),秦慧民(

)

天津科技大学生物工程学院省部共建食品营养与安全国家重点实验室工业发酵微生物教育部重点实验室天津 300457

Heterologous Expression, Purification and Enzymatic Characterization of Cholesterol Oxidase PsCO₄

Qian-qian GUO,Deng-ke GAO,Xiao-tao CHENG,Fu-ping LU,Tanokura Masaru(

),Hui-min QIN(

)

State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology Ministryof Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China

全文: PDF(8321 KB) HTML

摘要：

胆固醇氧化酶专一性催化胆固醇为胆甾-4-烯-3-酮,广泛的应用于临床以及食品加工行业。本论文将来源于Pimelobacter simplex的胆固醇氧化酶PsCO₄,分别转化到大肠杆菌宿主BL21(DE3)、Rosetta(DE3)和C41(DE3)中,在不同温度(15℃、25℃、37℃)及IPTG诱导浓度(0.01mmol/L、0.1mmol/L、0.5mmol/L)下异源表达PsCO₄。结果表明,转入Rosetta(DE3)菌株的PsCO₄蛋白,在IPTG浓度为0.1mmol/L、15℃下经18h诱导表达,PsCO₄可溶性表达量最高(0.63mg/ml)。异源表达的胆固醇氧化酶PsCO₄最适温度为30℃,最适pH为7.5。通过TLC, GC-MS检测出PsCO₄催化胆固醇生成胆甾-4-烯-3-酮。以胆固醇和β-谷甾醇、豆甾醇和孕烯醇酮为底物,测定PsCO₄对四种底物的催化反应动力学参数,胆固醇k_cat/K_m为0.08s ^-1·μM ^-1分别高于β-谷甾醇(0.04s ^-1·μM ^-1)、豆甾醇(0.005s ^-1·μM ^-1)和孕烯醇酮(0.02s ^-1·μM ^-1)。

关键词： 胆固醇氧化酶; 催化活性; 酶学性质; 底物特异性

Abstract:

Cholesterol oxidase, which catalyzes the reaction of cholesterol to cholest-4-en-3-one, is widely used in clinical and food processing industry. Cholesterol oxidase from Pimelobacter simplex (PsCO₄) was transformed into E.coli BL21(DE3), Rosetta(DE3), and C41(DE3). The bacteria were induced at different temperatures (15℃, 25℃, 37℃), and different IPTG concentrations (0.01mmol/L, 0.1mmol/L, 0.5mmol/L). The results showed that PsCO₄ was expressed greatly in supernatant of Rosetta(DE3) (0.63mg/ml), at the conditions of 0.1mmol/L IPTG and 15℃. The optimum temperature and pH of heterologous expressed PsCO₄ was 30℃ and 7.5. The product of cholesterol catalyzed by PsCO₄ was identified by TLC and GC-MS. The substrate specificity of PsCO₄ towards cholesterol, β-sitosterol, stigmasterol and pregnenolone was determined. The K_m/k_cat value of cholesterol (0.08s ^-1·μM ^-1) is higher than β-sitosterol (0.04s ^-1·μM ^-1), stigmasterol (0.005s ^-1·μM ^-1) and pregnenolone (0.02s ^-1·μM ^-1).

Key words: Cholesterol oxidase Catalytic activity Enzymatic property Substrate specificity

收稿日期: 2018-01-08 出版日期: 2018-07-06

ZTFLH:

Q786

基金资助: * 国家自然科学基金资助项目(31771911)

通讯作者: 田之仓优,秦慧民 E-mail: amtanok@mail.ecc.u-tokyo.ac.jp;huiminqin@tust.edu.cn

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引用本文:

郭倩倩,高登科,程晓涛,路福平,田之仓优,秦慧民. 胆固醇氧化酶PsCO₄异源表达、纯化及酶学性质分析 ^*[J]. 中国生物工程杂志, 2018, 38(6): 34-42.

Qian-qian GUO,Deng-ke GAO,Xiao-tao CHENG,Fu-ping LU,Tanokura Masaru,Hui-min QIN. Heterologous Expression, Purification and Enzymatic Characterization of Cholesterol Oxidase PsCO₄. China Biotechnology, 2018, 38(6): 34-42.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180605 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I6/34

图1 胆固醇氧化酶的三步催化反应

图2 PsCO4酶切及菌落PCR验证

图3 不同诱导条件下可溶性蛋白的浓度

图4 亲和层析和离子交换层析

图5 凝胶过滤层析

图6 薄层色谱法和气相色谱-质谱联用法鉴定催化产物

图7 温度(a)和pH(b)对胆固醇氧化酶PsCO4酶活的影响

表1 胆固醇氧化酶的酶学性质

图8 PsCO4活性中心底物结合关键氨基酸比较图

图9 不同来源胆固醇氧化酶底物结合通道

图10 胆固醇氧化酶催化机制(a)[16]和胆固醇氧化酶序列比对(b)

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